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Hindawi Publishing CorporationJournal of Computational MedicineVolume 2013 Article ID 312183 5 pageshttpdxdoiorg1011552013312183
Research ArticleMolecular Docking Study on the Interaction of Riboflavin(Vitamin B2) and Cyanocobalamin (Vitamin B12) Coenzymes
Ambreen Hafeez1 Zafar Saied Saify2 Afshan Naz1 Farzana Yasmin3 and Naheed Akhtar1
1 Biophysics Research Unit Department of Biochemistry University of Karachi Karachi 75270 Pakistan2 International Center for Biological and Chemical Sciences HEJ Research Institute of Chemistry University of KarachiKarachi 75270 Pakistan
3 Biomedical Engineering Department NED University of Engineering and Technology Karachi 75270 Pakistan
Correspondence should be addressed to Ambreen Hafeez ambreenfvhotmailcom
Received 14 November 2012 Revised 25 February 2013 Accepted 13 March 2013
Academic Editor Rocky Goldsmith
Copyright copy 2013 Ambreen Hafeez et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Cobalamins are the largest and structurally complex cofactors found in biological systems and have attracted considerable attentiondue to their participation in the metabolic reactions taking place in humans animals andmicroorganisms Riboflavin (vitamin B
2)
is a micronutrient and is the precursor of coenzymes FMN and FAD required for a wide variety of cellular processes with a keyrole in energy-based metabolic reactions As coenzymes of both vitamins are the part of enzyme systems the possibility of theirmutual interaction in the body cannot be overruled A molecular docking study was conducted on riboflavin molecule with B
12
coenzymes present in the enzymes glutamate mutase diol dehydratase and methionine synthase by using ArgusLab 401 softwareto understand the possible mode of interaction between these vitaminsThe results fromArgusLab showed the best binding affinityof riboflavin with the enzyme glutamate mutase for which the calculated least binding energy has been found to be minus713 kcalmolThe results indicate a significant inhibitory effect of riboflavin on the catalysis of B
12-dependent enzymes This information can be
utilized to design potent therapeutic drugs having structural similarity to that of riboflavin
1 Introduction
B12
cofactors play important roles in the metabolism ofmicroorganisms animals and humans They are involvedin the metabolism of almost every cell of the body specif-ically the DNA synthesis and regulation The structureand reactivity of B
12derivatives and structural aspects of
their interactions with proteins and nucleotides are crucialfor the efficient catalysis by the important B
12-dependent
enzymes [1] Biologically active cobalamins adenosylcobal-amin (AdoCbl) and methylcobalamin (MeCbl) are cofactorsfor many enzyme systems containing a metal carbon bondinvolved in enzyme catalyzed reactions [2] They catalyzeenzymatic reactions which involve the making and breakingof the CndashCo bond of these cofactors The X-ray structuresof B12-enzyme complexes revealed that the B
12-cofactor
undergoes a major conformational change on binding to theapoenzyme in AdoCbl and MeCbl containing enzymes such
as isomerases eliminases and methyltransferases [3] A keystep in the catalytic mechanism of coenzyme-B
12containing
enzymes is the homolysis of CondashC organometallic bond thatleads to the intricate pathways of B
12metabolic functions and
the catalysis of related chemical reactions [4]TheCondashCbondundergoes homolytic cleavage in B
12-dependent enzymes
more quickly as compared to that of the isolated cofactor inaqueous solution [5] which is a clue to the catalytic role ofvitamin B
12
Coenzyme B12-dependent enzymes may bind to their
cofactors in two possible modes ldquobase-onrdquo or ldquobase-offrdquo modes In base-on mode the original 56-dimeth-ylbenzimidazole base coordinates cobalt as the 120572-ligand atthe lower side in the enzyme-coenzyme complex whilein base-off binding mode the 56-dimethylbenzimidazolemoiety is displaced and substituted by an exogenous ligandsuch as histidine residue of the protein which coordinatesthe Co-atom as 120572-ligand that is base-offHis-on mode [6]
2 Journal of Computational Medicine
Cobalamins in the +3 oxidation state exist usually in thebase-on form with the axial ligand X (Ado CH
3 or CN)
coordinated on the 120573 side (upper side) of the octahedralcobalt compound while in the +2 oxidation state it has no120573 ligand and is called cob(II)alamin or B12r and in the +1oxidation state it has been assigned a name co(I)alamin orB12s in the base-off form along with the absence of 120573 ligand[7]
The objective of the current study was to evaluate thebinding affinity of riboflavin with B
12coenzymes by molecu-
lar docking technique to find out its effect on the inhibitionor acceleration of enzyme activity This depends on theinteraction of the functional groups of riboflavin with thoseamino acid residues of B
12enzymes that are present in the
active site cavity or take part in enzyme catalysis indirectlyMolecular docking techniques are used to predict how
a protein interacts with small vitamin-like molecules Thisability governs a significant part of the proteinrsquos dynamicswhich may enhanceinhibit its biological function [8] Twofactors are of paramount importance in molecular dockingstudies optimizing the candidate ligand for the correct nativeconformation in the presence of which it can achieve a bestfit orientation to bind with a protein of interest and theconformational flexibility of ligand and protein [9] Thusthe accurate prediction of the binding modes between theligand and protein is of fundamental importance in mod-ern structure-based drug design Computer-based molecularmodeling aims to speed up drug discoveries by predictingpotential effectiveness of ligand-protein interactions prior tolaborious experiments and costly preclinical trials
Numerous software packages have been developed withthe implementation of various molecular docking algorithmsbased on different search methods [10] The present workof molecular docking has been done using commerciallyavailable software ArgusLab 401 It is amolecularmodelinggraphics and drug designing program based on geneticalgorithm It is implementedwith exhaustive searchmethodsthe Argus Dock docking engine and AScore scoring function[11] It is also capable of performing molecular geometrycalculations and molecular structure visualization
2 Materials and Methods
21 Computational Methodology
211 Data Set Three-dimensional (3D) experimentallyknown protein-ligand complexes were obtained fromBrookhaven Protein Data Bank (PDB) [12] (httpwwwrcsborg) These were the structures of enzymes from threemajor enzyme families with bound coenzyme B
12 glutamate
mutase from isomerases [13] diol dehydratase from lyases[14] and methionine synthase from transferases [15] havingPDB codes 1CCW 1EGM and 3IV9 respectively
212 Input File Preparations for Energy Minimization ofProtein For each of the protein-ligand complexes chosenfor the study a ldquoclean input filerdquo was generated by removingwater molecules ions ligands and subunits not involved
in ligand binding from the original structure file Watermolecules were removed because ArgusLab sometimes failedto dock the compounds having water molecules at theirbinding sites [16] All hydrogen atoms in the protein wereallowed to optimizeThe hydrogen locations are not specifiedby the X-ray structure but these are necessary to improvethe hydrogen bond geometries at the same time maintainingthe protein conformation very close to that observed in thecrystallographic model The resulting receptor model wassaved to a PDB file Minimization was performed by geom-etry convergence function of ArgusLab software performedaccording to Hartree-Fock calculation method
213 Ligand Input File Preparation andOptimization Ligandinput structure was drawn using Marvin Sketch softwareThe structure was cleaned in 3D format and energy wasminimized using Marvin sketch software The resultingstructure was then saved in ldquomdl molrdquo and ldquosdf rdquo file formatsfor molecular docking studies
22 Docking Methodology After the preparation of the pro-tein and ligand molecular docking studies were performedby ArgusLab 401 to evaluate the interactions
221 ArgusLab 401 ArgusLab is implemented with shape-based search algorithm Docking has been done using ldquoArgusDockrdquo exhaustive search docking function of ArgusLab withgrid resolution of 040 A Docking precision was set toldquoRegular precisionrdquo and ldquoFlexiblerdquo ligand docking mode wasemployed for each docking run The stability of each dockedpose was evaluated using ArgusLab energy calculations andthe number of hydrogen bonds formed [17]
222 Molecular Docking Study To perform docking onefirst needs to define atoms that make up the ligand and thebinding sites of the protein where the ligand should bindTheprepared 3D structures of 1ccw 1egm and 3iv9 proteins weredownloaded into the ArgusLab program and binding siteswere made by choosing ldquoMake binding site for this proteinrdquooption The ligand (cleaned riboflavin molecule) was thenintroduced and docking calculation was allowed to run usingshape-based search algorithm and AScore scoring functionThe scoring function is responsible for evaluating the energybetween the ligand and the protein target Flexible dockingwas allowed by constructing grids over the binding sites ofthe protein and energy-based rotation is set for that ligandrsquosgroup of atoms that do not have rotatable bonds For eachrotation torsions are created and poses (conformations) aregenerated during the docking process [11] For each complex10 independent runs were conducted and one pose wasreturned for each run The best docking model was selectedaccording to the lowest AScore calculated by ArgusLab andthe most suitable binding conformation was selected on thebasis of hydrogen bond interactions between the ligand andprotein near the substrate binding site The lowest energyposes indicate the highest binding affinity as high energyproduces the unstable conformations
Journal of Computational Medicine 3
Table 1 Binding energies of riboflavin coenzyme B12 containingenzyme complex by AScore scoring function of ArgusLab
S no Enzyme name (pdb code) AScore (kcalmol)1 Glutamate mutase (1ccw) minus7132 Diol dehydratase (1egm) minus6983 Methionine synthase (3iv9) minus607
Figure 1 Minimized structure of riboflavin Atoms in red areoxygen blue are nitrogen grey are carbon and white are hydrogenThe number of hydrogen atoms is indicated in light green color
3 Results and Discussion
Minimized structure of riboflavin is given in Figure 1Docking studies of the compound riboflavin with each ofthe three enzymes having PDB codes 1ccw 1egm and 3iv9were carried out by ArgusLab 401 The least binding energyexhibits the highest activity which has been observed by theranking of poses generated by AScore scoring function ofArgusLab and is given in Table 1
List of hydrogen bonds between riboflavin and coenzymeB12-dependent enzymes is given in Table 2 The best fitted
poses adopted by riboflavin docked into enzymes 1ccw 1egmand 3iv9 are shown in Figures 2 3 and 4 respectively
In the present study cyanocobalamin coenzyme wastaken as an active ligand instead of cocrystallized inhibitorD-tartaric acid to bind with the compound riboflavin in orderto examine a possible mode of interaction between these twovitamins in an enzyme system The docked binding mode ofriboflavin was manually inspected in order to verify that iteffectively binds to the catalytic site The docking results ofriboflavin with each of the individual enzymes are as follows
31 Glutamate Mutase The compound riboflavin interactedwith enzyme glutamate mutase (in complex with coenzymeB12
and the inhibitor D-tartaric acid) by least bindingenergy of minus713 kcalmol In Figure 2 riboflavin seemed tobind at the lower axial end of the coenzyme B
12with the
base 56-dimethylbenzimidazole (DMB) by replacing water
357Thr
234Ala
260Asn
90Val
117Tyr
252
607Ile
120Gly
272121Thr
Figure 2 Docking of riboflavin into enzyme glutamate mutase[13] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink stick model Inhibitor TARis seen in green color in thick stick
molecules The oxygen (O2) of carbonyl group of riboflavinbinds with nitrogen atom (NH) of amino acid residue Gly120The hydrogen bond distance between these groups is 252 AAnother hydrogen bond was formed between the nitrogenatom (N1) of riboflavin and NH of amino acid Thr121 ata distance of 272 A No interaction was observed betweenthe riboflavin and cocrystallized inhibitor D-tartaric acidIn glutamate mutase the elongation of CondashN bond probablycontributes to the weakening of CondashC bond in the coenzyme[13] Therefore the docked position and conformation ofriboflavin revealed that the compound may inhibit the usualcatalytic process of the enzymeby altering the conformationalchange in the nucleotide base which plays an important rolein initiating the CondashC bond cleavage and thus hinders thecontinuous progress of radical reactions The relative confor-mation and arrangement of cofactor and substrate also play apart in this aspect The calculated AScore shows a significantaffinity of riboflavin towards the enzyme glutamate mutase
32 Diol Dehydratase In Figure 3 deep analysis of thedocked structure of riboflavin revealed that the moleculeseemed to bind in between the enzymersquos active site that isthe E-subunit and B
12-cofactor binding domain as indicated
by the presence of amino acid residues Thr222 Val300Phe374 and Gln336 These amino acid residues have beenfound to form the hydrophobic contacts with the inhibitor1 2-propanediol and play an important role in holdingthe substrate in the active site [14] Three hydrogen bondswere formed with this enzyme the nitrogen atom (N5) ofriboflavin interacted with the sulphur (SH) of amino acidresidue Cys302 The hydrogen bond distance between thesegroups was found to be 250 A Another two hydrogen bonds
4 Journal of Computational Medicine
Table 2 List of hydrogen bonds between riboflavin and coenzyme B12 containing enzymes
S no Enzyme name No of H-bonds Amino acid residue atom Ligand atom H-bond distance (A)
1 Glutamate mutase (1ccw) 2 Gly120 (NH) (O2) 252Thr121 (NH) (N1) 272Cys302 (SH) (N5) 250
2 Diol dehydratase (1egm) 3 Ser301 (O) (N1) 296Arg699 (N) (O2) 282
3 Methionine synthase (3iv9) 2 His759 (NH) (O4) 268His1145 (NH) (O2) 272
300Val 336Gln 374Phe
302Cys301Ser
222Thr282 373Met
699Arg296
250
226Tyr
654Leu 656Asn
659Leu
662Gln
Figure 3 Docking of riboflavin into enzyme diol dehydratase[14] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink color in thick stick
were formed between the nitrogen (N1) of riboflavin andoxygen atom of Ser301 at a distance of 296 A and betweennitrogen of Arg699 and oxygen atom (O2) of riboflavin ata distance of 282 A The E-subunit comprises of active siteof the enzyme which has been found to pack against theupper face of B
12cofactor and may facilitate the transfer of
51015840-adenosyl radical from cofactor to substrate Therefore itmay be assumed that the riboflavin may interfere with thecatalytic process of the enzyme by binding at the interfaceof cofactor B
12and the enzymersquos active site and thus inhibit
the radical shuttling mechanism The AScore value obtainedfor this complex was minus698 kcalmol which indicates thatriboflavin significantly binds with the enzyme diol dehy-dratase
33 Methionine Synthase Figure 3 shows the binding ofriboflavin near the cofactor making contact with amino acidresidue His 759 The oxygen atom (O4) of the carbonylgroup of riboflavin was hydrogen bonded to N-atom of theHis 759 at a distance of 268 A Another hydrogen bonding
1148Lys
1145His
1176Ser
1039Asp
268
759His
272
753Thr
836Thr
834Gly
831Leu
Figure 4 Docking of riboflavin into enzyme methionine synthase[15] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink color in thick stick
was seen between the N-atom of His1145 and the carbonyloxygen (O2) of riboflavin at a distance of 272 AThe dockingAScore of the ligand-protein complex was minus607 kcalmolThe crystal structure of methionine synthase revealed thatthe enzyme is present in ldquobase-offrdquo or ldquoHis-onrdquo form withHis759 as the lower axial ligand His759 has been found toact as transient intermediate in the reductive reactivation andconformational transition of cobalt following methylationin methionine synthase-dependent enzyme catalysis [15]Therefore we may assume that riboflavin may inhibit theimportant catalytic step of the enzyme that is reactivationand methylation probably by making contact with the sameamino acid residue (His759) that is involved in the reactionThe binding energy also shows a significant ligand-receptorcomplex with this enzyme
4 Conclusion
By analyzing the docking results we hypothesized thatriboflavin might have inhibitory activity against cobalamincoenzymesThe enzyme glutamate mutase has been found tobe the most susceptible protein target for the studied ligandand the riboflavin showed the best binding affinity with thisenzyme having least binding energy of minus713 kcalmol
Journal of Computational Medicine 5
Glutamate mutase and diol dehydratase can be consid-ered as important targets for the development of antibioticsOther useful therapeutic drugs can also be synthesized toenhance the function of enzyme methionine synthase as itconverts homocystein into methionine and requires methyl-cobalamin coenzyme for its function Inactivation of thiscoenzyme leads to the elevated levels of homocystein in bloodand urine andmay result in clotting and in long-term damageto the arteries (stroke and heart attack) Therefore theinteraction of vitamin B
2and B
12and their role in important
metabolic reactions in the body should be considered beforepreparing multivitamin complexes
The fact that riboflavin participates in energy-basedmetabolic reactions may play an important role in enzymecatalysis which depends on a delicate energy balance fordifferent reaction pathways The local amino acids andsubstrates in the active sites of the enzymes may function inthis respect
Further experimental approaches can be adopted toprobe the effect of structural alterations of flavin groupof compounds in the catalytic properties of coenzymeB12-dependent enzymes
References
[1] K Gruber B Puffer and B Krautler ldquoVitamin B12-derivatives-
enzyme cofactors and ligands of proteins and nucleic acidsrdquoChemical Society Reviews vol 40 no 8 pp 4346ndash4363 2011
[2] L Randaccio S Geremia N Demitri and JWuerges ldquoVitaminB12 unique metalorganic compounds and the most complex
vitaminsrdquoMolecules vol 15 no 5 pp 3228ndash3259 2010[3] R Banerjee and SW Ragsdale ldquoThemany faces of vitamin B
12
catalysis by cobalamin-dependent enzymesrdquo Annual Review ofBiochemistry vol 72 pp 209ndash247 2003
[4] R G Matthews ldquoCobalamin and corrinoid dependentenzymesrdquo in Metal Ions in Life Sciences A Sigel H Sigeland R K O Sigel Eds vol 6 pp 53ndash114 Royal Society ofChemistry Cambridge UK 2009
[5] P J Kasper and U Ryde ldquoHow the Co-C bond is cleavedin coenzyme B
12enzymes a theoretical studyrdquo Journal of the
American Chemical Society vol 127 no 25 pp 9117ndash9128 2005[6] L Randaccio S Geremia and J Wuerges ldquoCrystallography of
vitamin B12proteinsrdquo Journal of Organometallic Chemistry vol
692 no 6 pp 1198ndash1215 2007[7] B Krautler ldquoOrganometallic chemistry of B
12coenzymesrdquo in
Metal Ions in Life Sciences A Sigel H Sigel and R K O SigelEds vol 6 pp 1ndash51 Royal Society of Chemistry CambridgeUK 2009
[8] A Kahraman R JMorris R A Laskowski and JMThorntonldquoShape variation in protein binding pockets and their ligandsrdquoJournal of Molecular Biology vol 368 no 1 pp 283ndash301 2007
[9] S F Sousa P A Fernandes and M J Ramos ldquoProtein-ligand docking current status and future challengesrdquo ProteinsStructure Function and Genetics vol 65 no 1 pp 15ndash26 2006
[10] R D Taylor P J Jewsbury and JW Essex ldquoA review of protein-small molecule docking methodsrdquo Journal of Computer-AidedMolecular Design vol 16 no 3 pp 151ndash166 2002
[11] M A Thompson ldquoMolecular docking using ArgusLab anefficient shape-based search algorithm and AScore scoring
functionrdquo in Proceedings of the ACS Meeting Philadelphia PaUSA March-April 2004 172 CINF 42
[12] R Wang X Fang Y Lu and S Wang ldquoThe PDBbind databasecollection of binding affinities for protein-ligand complexeswith known three-dimensional structuresrdquo Journal ofMedicinalChemistry vol 47 no 12 pp 2977ndash2980 2004
[13] R Reitzer K Gruber G Jogl et al ldquoGlutamate mutase fromClostridium cochlearium the structure of a coenzyme B
12-
dependent enzyme provides new mechanistic insightsrdquo Struc-ture vol 7 no 8 pp 891ndash902 1999
[14] N Shibata J Masuda T Tobimatsu et al ldquoA new modeof B12
binding and the direct participation of a potassiumion in enzyme catalysis X-ray structure of diol dehydrataserdquoStructure vol 7 no 8 pp 997ndash1008 1999
[15] M Koutmos S Datta K A Pattridge J L Smith and RG Matthews ldquoInsights into the reactivation of cobalamin-dependent methionine synthaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no44 pp 18527ndash18532 2009
[16] S Joy P S Nair R Hariharan and M R Pillai ldquoDetailedcomparison of the protein-ligand docking efficiencies ofGOLDa commercial package and arguslab a licensable freewarerdquo InSilico Biology vol 6 no 6 pp 601ndash605 2006
[17] M Thompson ArgusLab 401 Planaria software LLC SeattleWash USA 2004
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![Page 2: Research Article Molecular Docking Study on the …downloads.hindawi.com/archive/2013/312183.pdfResearch Article Molecular Docking Study on the Interaction of Riboflavin (Vitamin B](https://reader033.vdocuments.site/reader033/viewer/2022042116/5e93aafdc60b2918c71e72c8/html5/thumbnails/2.jpg)
2 Journal of Computational Medicine
Cobalamins in the +3 oxidation state exist usually in thebase-on form with the axial ligand X (Ado CH
3 or CN)
coordinated on the 120573 side (upper side) of the octahedralcobalt compound while in the +2 oxidation state it has no120573 ligand and is called cob(II)alamin or B12r and in the +1oxidation state it has been assigned a name co(I)alamin orB12s in the base-off form along with the absence of 120573 ligand[7]
The objective of the current study was to evaluate thebinding affinity of riboflavin with B
12coenzymes by molecu-
lar docking technique to find out its effect on the inhibitionor acceleration of enzyme activity This depends on theinteraction of the functional groups of riboflavin with thoseamino acid residues of B
12enzymes that are present in the
active site cavity or take part in enzyme catalysis indirectlyMolecular docking techniques are used to predict how
a protein interacts with small vitamin-like molecules Thisability governs a significant part of the proteinrsquos dynamicswhich may enhanceinhibit its biological function [8] Twofactors are of paramount importance in molecular dockingstudies optimizing the candidate ligand for the correct nativeconformation in the presence of which it can achieve a bestfit orientation to bind with a protein of interest and theconformational flexibility of ligand and protein [9] Thusthe accurate prediction of the binding modes between theligand and protein is of fundamental importance in mod-ern structure-based drug design Computer-based molecularmodeling aims to speed up drug discoveries by predictingpotential effectiveness of ligand-protein interactions prior tolaborious experiments and costly preclinical trials
Numerous software packages have been developed withthe implementation of various molecular docking algorithmsbased on different search methods [10] The present workof molecular docking has been done using commerciallyavailable software ArgusLab 401 It is amolecularmodelinggraphics and drug designing program based on geneticalgorithm It is implementedwith exhaustive searchmethodsthe Argus Dock docking engine and AScore scoring function[11] It is also capable of performing molecular geometrycalculations and molecular structure visualization
2 Materials and Methods
21 Computational Methodology
211 Data Set Three-dimensional (3D) experimentallyknown protein-ligand complexes were obtained fromBrookhaven Protein Data Bank (PDB) [12] (httpwwwrcsborg) These were the structures of enzymes from threemajor enzyme families with bound coenzyme B
12 glutamate
mutase from isomerases [13] diol dehydratase from lyases[14] and methionine synthase from transferases [15] havingPDB codes 1CCW 1EGM and 3IV9 respectively
212 Input File Preparations for Energy Minimization ofProtein For each of the protein-ligand complexes chosenfor the study a ldquoclean input filerdquo was generated by removingwater molecules ions ligands and subunits not involved
in ligand binding from the original structure file Watermolecules were removed because ArgusLab sometimes failedto dock the compounds having water molecules at theirbinding sites [16] All hydrogen atoms in the protein wereallowed to optimizeThe hydrogen locations are not specifiedby the X-ray structure but these are necessary to improvethe hydrogen bond geometries at the same time maintainingthe protein conformation very close to that observed in thecrystallographic model The resulting receptor model wassaved to a PDB file Minimization was performed by geom-etry convergence function of ArgusLab software performedaccording to Hartree-Fock calculation method
213 Ligand Input File Preparation andOptimization Ligandinput structure was drawn using Marvin Sketch softwareThe structure was cleaned in 3D format and energy wasminimized using Marvin sketch software The resultingstructure was then saved in ldquomdl molrdquo and ldquosdf rdquo file formatsfor molecular docking studies
22 Docking Methodology After the preparation of the pro-tein and ligand molecular docking studies were performedby ArgusLab 401 to evaluate the interactions
221 ArgusLab 401 ArgusLab is implemented with shape-based search algorithm Docking has been done using ldquoArgusDockrdquo exhaustive search docking function of ArgusLab withgrid resolution of 040 A Docking precision was set toldquoRegular precisionrdquo and ldquoFlexiblerdquo ligand docking mode wasemployed for each docking run The stability of each dockedpose was evaluated using ArgusLab energy calculations andthe number of hydrogen bonds formed [17]
222 Molecular Docking Study To perform docking onefirst needs to define atoms that make up the ligand and thebinding sites of the protein where the ligand should bindTheprepared 3D structures of 1ccw 1egm and 3iv9 proteins weredownloaded into the ArgusLab program and binding siteswere made by choosing ldquoMake binding site for this proteinrdquooption The ligand (cleaned riboflavin molecule) was thenintroduced and docking calculation was allowed to run usingshape-based search algorithm and AScore scoring functionThe scoring function is responsible for evaluating the energybetween the ligand and the protein target Flexible dockingwas allowed by constructing grids over the binding sites ofthe protein and energy-based rotation is set for that ligandrsquosgroup of atoms that do not have rotatable bonds For eachrotation torsions are created and poses (conformations) aregenerated during the docking process [11] For each complex10 independent runs were conducted and one pose wasreturned for each run The best docking model was selectedaccording to the lowest AScore calculated by ArgusLab andthe most suitable binding conformation was selected on thebasis of hydrogen bond interactions between the ligand andprotein near the substrate binding site The lowest energyposes indicate the highest binding affinity as high energyproduces the unstable conformations
Journal of Computational Medicine 3
Table 1 Binding energies of riboflavin coenzyme B12 containingenzyme complex by AScore scoring function of ArgusLab
S no Enzyme name (pdb code) AScore (kcalmol)1 Glutamate mutase (1ccw) minus7132 Diol dehydratase (1egm) minus6983 Methionine synthase (3iv9) minus607
Figure 1 Minimized structure of riboflavin Atoms in red areoxygen blue are nitrogen grey are carbon and white are hydrogenThe number of hydrogen atoms is indicated in light green color
3 Results and Discussion
Minimized structure of riboflavin is given in Figure 1Docking studies of the compound riboflavin with each ofthe three enzymes having PDB codes 1ccw 1egm and 3iv9were carried out by ArgusLab 401 The least binding energyexhibits the highest activity which has been observed by theranking of poses generated by AScore scoring function ofArgusLab and is given in Table 1
List of hydrogen bonds between riboflavin and coenzymeB12-dependent enzymes is given in Table 2 The best fitted
poses adopted by riboflavin docked into enzymes 1ccw 1egmand 3iv9 are shown in Figures 2 3 and 4 respectively
In the present study cyanocobalamin coenzyme wastaken as an active ligand instead of cocrystallized inhibitorD-tartaric acid to bind with the compound riboflavin in orderto examine a possible mode of interaction between these twovitamins in an enzyme system The docked binding mode ofriboflavin was manually inspected in order to verify that iteffectively binds to the catalytic site The docking results ofriboflavin with each of the individual enzymes are as follows
31 Glutamate Mutase The compound riboflavin interactedwith enzyme glutamate mutase (in complex with coenzymeB12
and the inhibitor D-tartaric acid) by least bindingenergy of minus713 kcalmol In Figure 2 riboflavin seemed tobind at the lower axial end of the coenzyme B
12with the
base 56-dimethylbenzimidazole (DMB) by replacing water
357Thr
234Ala
260Asn
90Val
117Tyr
252
607Ile
120Gly
272121Thr
Figure 2 Docking of riboflavin into enzyme glutamate mutase[13] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink stick model Inhibitor TARis seen in green color in thick stick
molecules The oxygen (O2) of carbonyl group of riboflavinbinds with nitrogen atom (NH) of amino acid residue Gly120The hydrogen bond distance between these groups is 252 AAnother hydrogen bond was formed between the nitrogenatom (N1) of riboflavin and NH of amino acid Thr121 ata distance of 272 A No interaction was observed betweenthe riboflavin and cocrystallized inhibitor D-tartaric acidIn glutamate mutase the elongation of CondashN bond probablycontributes to the weakening of CondashC bond in the coenzyme[13] Therefore the docked position and conformation ofriboflavin revealed that the compound may inhibit the usualcatalytic process of the enzymeby altering the conformationalchange in the nucleotide base which plays an important rolein initiating the CondashC bond cleavage and thus hinders thecontinuous progress of radical reactions The relative confor-mation and arrangement of cofactor and substrate also play apart in this aspect The calculated AScore shows a significantaffinity of riboflavin towards the enzyme glutamate mutase
32 Diol Dehydratase In Figure 3 deep analysis of thedocked structure of riboflavin revealed that the moleculeseemed to bind in between the enzymersquos active site that isthe E-subunit and B
12-cofactor binding domain as indicated
by the presence of amino acid residues Thr222 Val300Phe374 and Gln336 These amino acid residues have beenfound to form the hydrophobic contacts with the inhibitor1 2-propanediol and play an important role in holdingthe substrate in the active site [14] Three hydrogen bondswere formed with this enzyme the nitrogen atom (N5) ofriboflavin interacted with the sulphur (SH) of amino acidresidue Cys302 The hydrogen bond distance between thesegroups was found to be 250 A Another two hydrogen bonds
4 Journal of Computational Medicine
Table 2 List of hydrogen bonds between riboflavin and coenzyme B12 containing enzymes
S no Enzyme name No of H-bonds Amino acid residue atom Ligand atom H-bond distance (A)
1 Glutamate mutase (1ccw) 2 Gly120 (NH) (O2) 252Thr121 (NH) (N1) 272Cys302 (SH) (N5) 250
2 Diol dehydratase (1egm) 3 Ser301 (O) (N1) 296Arg699 (N) (O2) 282
3 Methionine synthase (3iv9) 2 His759 (NH) (O4) 268His1145 (NH) (O2) 272
300Val 336Gln 374Phe
302Cys301Ser
222Thr282 373Met
699Arg296
250
226Tyr
654Leu 656Asn
659Leu
662Gln
Figure 3 Docking of riboflavin into enzyme diol dehydratase[14] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink color in thick stick
were formed between the nitrogen (N1) of riboflavin andoxygen atom of Ser301 at a distance of 296 A and betweennitrogen of Arg699 and oxygen atom (O2) of riboflavin ata distance of 282 A The E-subunit comprises of active siteof the enzyme which has been found to pack against theupper face of B
12cofactor and may facilitate the transfer of
51015840-adenosyl radical from cofactor to substrate Therefore itmay be assumed that the riboflavin may interfere with thecatalytic process of the enzyme by binding at the interfaceof cofactor B
12and the enzymersquos active site and thus inhibit
the radical shuttling mechanism The AScore value obtainedfor this complex was minus698 kcalmol which indicates thatriboflavin significantly binds with the enzyme diol dehy-dratase
33 Methionine Synthase Figure 3 shows the binding ofriboflavin near the cofactor making contact with amino acidresidue His 759 The oxygen atom (O4) of the carbonylgroup of riboflavin was hydrogen bonded to N-atom of theHis 759 at a distance of 268 A Another hydrogen bonding
1148Lys
1145His
1176Ser
1039Asp
268
759His
272
753Thr
836Thr
834Gly
831Leu
Figure 4 Docking of riboflavin into enzyme methionine synthase[15] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink color in thick stick
was seen between the N-atom of His1145 and the carbonyloxygen (O2) of riboflavin at a distance of 272 AThe dockingAScore of the ligand-protein complex was minus607 kcalmolThe crystal structure of methionine synthase revealed thatthe enzyme is present in ldquobase-offrdquo or ldquoHis-onrdquo form withHis759 as the lower axial ligand His759 has been found toact as transient intermediate in the reductive reactivation andconformational transition of cobalt following methylationin methionine synthase-dependent enzyme catalysis [15]Therefore we may assume that riboflavin may inhibit theimportant catalytic step of the enzyme that is reactivationand methylation probably by making contact with the sameamino acid residue (His759) that is involved in the reactionThe binding energy also shows a significant ligand-receptorcomplex with this enzyme
4 Conclusion
By analyzing the docking results we hypothesized thatriboflavin might have inhibitory activity against cobalamincoenzymesThe enzyme glutamate mutase has been found tobe the most susceptible protein target for the studied ligandand the riboflavin showed the best binding affinity with thisenzyme having least binding energy of minus713 kcalmol
Journal of Computational Medicine 5
Glutamate mutase and diol dehydratase can be consid-ered as important targets for the development of antibioticsOther useful therapeutic drugs can also be synthesized toenhance the function of enzyme methionine synthase as itconverts homocystein into methionine and requires methyl-cobalamin coenzyme for its function Inactivation of thiscoenzyme leads to the elevated levels of homocystein in bloodand urine andmay result in clotting and in long-term damageto the arteries (stroke and heart attack) Therefore theinteraction of vitamin B
2and B
12and their role in important
metabolic reactions in the body should be considered beforepreparing multivitamin complexes
The fact that riboflavin participates in energy-basedmetabolic reactions may play an important role in enzymecatalysis which depends on a delicate energy balance fordifferent reaction pathways The local amino acids andsubstrates in the active sites of the enzymes may function inthis respect
Further experimental approaches can be adopted toprobe the effect of structural alterations of flavin groupof compounds in the catalytic properties of coenzymeB12-dependent enzymes
References
[1] K Gruber B Puffer and B Krautler ldquoVitamin B12-derivatives-
enzyme cofactors and ligands of proteins and nucleic acidsrdquoChemical Society Reviews vol 40 no 8 pp 4346ndash4363 2011
[2] L Randaccio S Geremia N Demitri and JWuerges ldquoVitaminB12 unique metalorganic compounds and the most complex
vitaminsrdquoMolecules vol 15 no 5 pp 3228ndash3259 2010[3] R Banerjee and SW Ragsdale ldquoThemany faces of vitamin B
12
catalysis by cobalamin-dependent enzymesrdquo Annual Review ofBiochemistry vol 72 pp 209ndash247 2003
[4] R G Matthews ldquoCobalamin and corrinoid dependentenzymesrdquo in Metal Ions in Life Sciences A Sigel H Sigeland R K O Sigel Eds vol 6 pp 53ndash114 Royal Society ofChemistry Cambridge UK 2009
[5] P J Kasper and U Ryde ldquoHow the Co-C bond is cleavedin coenzyme B
12enzymes a theoretical studyrdquo Journal of the
American Chemical Society vol 127 no 25 pp 9117ndash9128 2005[6] L Randaccio S Geremia and J Wuerges ldquoCrystallography of
vitamin B12proteinsrdquo Journal of Organometallic Chemistry vol
692 no 6 pp 1198ndash1215 2007[7] B Krautler ldquoOrganometallic chemistry of B
12coenzymesrdquo in
Metal Ions in Life Sciences A Sigel H Sigel and R K O SigelEds vol 6 pp 1ndash51 Royal Society of Chemistry CambridgeUK 2009
[8] A Kahraman R JMorris R A Laskowski and JMThorntonldquoShape variation in protein binding pockets and their ligandsrdquoJournal of Molecular Biology vol 368 no 1 pp 283ndash301 2007
[9] S F Sousa P A Fernandes and M J Ramos ldquoProtein-ligand docking current status and future challengesrdquo ProteinsStructure Function and Genetics vol 65 no 1 pp 15ndash26 2006
[10] R D Taylor P J Jewsbury and JW Essex ldquoA review of protein-small molecule docking methodsrdquo Journal of Computer-AidedMolecular Design vol 16 no 3 pp 151ndash166 2002
[11] M A Thompson ldquoMolecular docking using ArgusLab anefficient shape-based search algorithm and AScore scoring
functionrdquo in Proceedings of the ACS Meeting Philadelphia PaUSA March-April 2004 172 CINF 42
[12] R Wang X Fang Y Lu and S Wang ldquoThe PDBbind databasecollection of binding affinities for protein-ligand complexeswith known three-dimensional structuresrdquo Journal ofMedicinalChemistry vol 47 no 12 pp 2977ndash2980 2004
[13] R Reitzer K Gruber G Jogl et al ldquoGlutamate mutase fromClostridium cochlearium the structure of a coenzyme B
12-
dependent enzyme provides new mechanistic insightsrdquo Struc-ture vol 7 no 8 pp 891ndash902 1999
[14] N Shibata J Masuda T Tobimatsu et al ldquoA new modeof B12
binding and the direct participation of a potassiumion in enzyme catalysis X-ray structure of diol dehydrataserdquoStructure vol 7 no 8 pp 997ndash1008 1999
[15] M Koutmos S Datta K A Pattridge J L Smith and RG Matthews ldquoInsights into the reactivation of cobalamin-dependent methionine synthaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no44 pp 18527ndash18532 2009
[16] S Joy P S Nair R Hariharan and M R Pillai ldquoDetailedcomparison of the protein-ligand docking efficiencies ofGOLDa commercial package and arguslab a licensable freewarerdquo InSilico Biology vol 6 no 6 pp 601ndash605 2006
[17] M Thompson ArgusLab 401 Planaria software LLC SeattleWash USA 2004
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
![Page 3: Research Article Molecular Docking Study on the …downloads.hindawi.com/archive/2013/312183.pdfResearch Article Molecular Docking Study on the Interaction of Riboflavin (Vitamin B](https://reader033.vdocuments.site/reader033/viewer/2022042116/5e93aafdc60b2918c71e72c8/html5/thumbnails/3.jpg)
Journal of Computational Medicine 3
Table 1 Binding energies of riboflavin coenzyme B12 containingenzyme complex by AScore scoring function of ArgusLab
S no Enzyme name (pdb code) AScore (kcalmol)1 Glutamate mutase (1ccw) minus7132 Diol dehydratase (1egm) minus6983 Methionine synthase (3iv9) minus607
Figure 1 Minimized structure of riboflavin Atoms in red areoxygen blue are nitrogen grey are carbon and white are hydrogenThe number of hydrogen atoms is indicated in light green color
3 Results and Discussion
Minimized structure of riboflavin is given in Figure 1Docking studies of the compound riboflavin with each ofthe three enzymes having PDB codes 1ccw 1egm and 3iv9were carried out by ArgusLab 401 The least binding energyexhibits the highest activity which has been observed by theranking of poses generated by AScore scoring function ofArgusLab and is given in Table 1
List of hydrogen bonds between riboflavin and coenzymeB12-dependent enzymes is given in Table 2 The best fitted
poses adopted by riboflavin docked into enzymes 1ccw 1egmand 3iv9 are shown in Figures 2 3 and 4 respectively
In the present study cyanocobalamin coenzyme wastaken as an active ligand instead of cocrystallized inhibitorD-tartaric acid to bind with the compound riboflavin in orderto examine a possible mode of interaction between these twovitamins in an enzyme system The docked binding mode ofriboflavin was manually inspected in order to verify that iteffectively binds to the catalytic site The docking results ofriboflavin with each of the individual enzymes are as follows
31 Glutamate Mutase The compound riboflavin interactedwith enzyme glutamate mutase (in complex with coenzymeB12
and the inhibitor D-tartaric acid) by least bindingenergy of minus713 kcalmol In Figure 2 riboflavin seemed tobind at the lower axial end of the coenzyme B
12with the
base 56-dimethylbenzimidazole (DMB) by replacing water
357Thr
234Ala
260Asn
90Val
117Tyr
252
607Ile
120Gly
272121Thr
Figure 2 Docking of riboflavin into enzyme glutamate mutase[13] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink stick model Inhibitor TARis seen in green color in thick stick
molecules The oxygen (O2) of carbonyl group of riboflavinbinds with nitrogen atom (NH) of amino acid residue Gly120The hydrogen bond distance between these groups is 252 AAnother hydrogen bond was formed between the nitrogenatom (N1) of riboflavin and NH of amino acid Thr121 ata distance of 272 A No interaction was observed betweenthe riboflavin and cocrystallized inhibitor D-tartaric acidIn glutamate mutase the elongation of CondashN bond probablycontributes to the weakening of CondashC bond in the coenzyme[13] Therefore the docked position and conformation ofriboflavin revealed that the compound may inhibit the usualcatalytic process of the enzymeby altering the conformationalchange in the nucleotide base which plays an important rolein initiating the CondashC bond cleavage and thus hinders thecontinuous progress of radical reactions The relative confor-mation and arrangement of cofactor and substrate also play apart in this aspect The calculated AScore shows a significantaffinity of riboflavin towards the enzyme glutamate mutase
32 Diol Dehydratase In Figure 3 deep analysis of thedocked structure of riboflavin revealed that the moleculeseemed to bind in between the enzymersquos active site that isthe E-subunit and B
12-cofactor binding domain as indicated
by the presence of amino acid residues Thr222 Val300Phe374 and Gln336 These amino acid residues have beenfound to form the hydrophobic contacts with the inhibitor1 2-propanediol and play an important role in holdingthe substrate in the active site [14] Three hydrogen bondswere formed with this enzyme the nitrogen atom (N5) ofriboflavin interacted with the sulphur (SH) of amino acidresidue Cys302 The hydrogen bond distance between thesegroups was found to be 250 A Another two hydrogen bonds
4 Journal of Computational Medicine
Table 2 List of hydrogen bonds between riboflavin and coenzyme B12 containing enzymes
S no Enzyme name No of H-bonds Amino acid residue atom Ligand atom H-bond distance (A)
1 Glutamate mutase (1ccw) 2 Gly120 (NH) (O2) 252Thr121 (NH) (N1) 272Cys302 (SH) (N5) 250
2 Diol dehydratase (1egm) 3 Ser301 (O) (N1) 296Arg699 (N) (O2) 282
3 Methionine synthase (3iv9) 2 His759 (NH) (O4) 268His1145 (NH) (O2) 272
300Val 336Gln 374Phe
302Cys301Ser
222Thr282 373Met
699Arg296
250
226Tyr
654Leu 656Asn
659Leu
662Gln
Figure 3 Docking of riboflavin into enzyme diol dehydratase[14] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink color in thick stick
were formed between the nitrogen (N1) of riboflavin andoxygen atom of Ser301 at a distance of 296 A and betweennitrogen of Arg699 and oxygen atom (O2) of riboflavin ata distance of 282 A The E-subunit comprises of active siteof the enzyme which has been found to pack against theupper face of B
12cofactor and may facilitate the transfer of
51015840-adenosyl radical from cofactor to substrate Therefore itmay be assumed that the riboflavin may interfere with thecatalytic process of the enzyme by binding at the interfaceof cofactor B
12and the enzymersquos active site and thus inhibit
the radical shuttling mechanism The AScore value obtainedfor this complex was minus698 kcalmol which indicates thatriboflavin significantly binds with the enzyme diol dehy-dratase
33 Methionine Synthase Figure 3 shows the binding ofriboflavin near the cofactor making contact with amino acidresidue His 759 The oxygen atom (O4) of the carbonylgroup of riboflavin was hydrogen bonded to N-atom of theHis 759 at a distance of 268 A Another hydrogen bonding
1148Lys
1145His
1176Ser
1039Asp
268
759His
272
753Thr
836Thr
834Gly
831Leu
Figure 4 Docking of riboflavin into enzyme methionine synthase[15] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink color in thick stick
was seen between the N-atom of His1145 and the carbonyloxygen (O2) of riboflavin at a distance of 272 AThe dockingAScore of the ligand-protein complex was minus607 kcalmolThe crystal structure of methionine synthase revealed thatthe enzyme is present in ldquobase-offrdquo or ldquoHis-onrdquo form withHis759 as the lower axial ligand His759 has been found toact as transient intermediate in the reductive reactivation andconformational transition of cobalt following methylationin methionine synthase-dependent enzyme catalysis [15]Therefore we may assume that riboflavin may inhibit theimportant catalytic step of the enzyme that is reactivationand methylation probably by making contact with the sameamino acid residue (His759) that is involved in the reactionThe binding energy also shows a significant ligand-receptorcomplex with this enzyme
4 Conclusion
By analyzing the docking results we hypothesized thatriboflavin might have inhibitory activity against cobalamincoenzymesThe enzyme glutamate mutase has been found tobe the most susceptible protein target for the studied ligandand the riboflavin showed the best binding affinity with thisenzyme having least binding energy of minus713 kcalmol
Journal of Computational Medicine 5
Glutamate mutase and diol dehydratase can be consid-ered as important targets for the development of antibioticsOther useful therapeutic drugs can also be synthesized toenhance the function of enzyme methionine synthase as itconverts homocystein into methionine and requires methyl-cobalamin coenzyme for its function Inactivation of thiscoenzyme leads to the elevated levels of homocystein in bloodand urine andmay result in clotting and in long-term damageto the arteries (stroke and heart attack) Therefore theinteraction of vitamin B
2and B
12and their role in important
metabolic reactions in the body should be considered beforepreparing multivitamin complexes
The fact that riboflavin participates in energy-basedmetabolic reactions may play an important role in enzymecatalysis which depends on a delicate energy balance fordifferent reaction pathways The local amino acids andsubstrates in the active sites of the enzymes may function inthis respect
Further experimental approaches can be adopted toprobe the effect of structural alterations of flavin groupof compounds in the catalytic properties of coenzymeB12-dependent enzymes
References
[1] K Gruber B Puffer and B Krautler ldquoVitamin B12-derivatives-
enzyme cofactors and ligands of proteins and nucleic acidsrdquoChemical Society Reviews vol 40 no 8 pp 4346ndash4363 2011
[2] L Randaccio S Geremia N Demitri and JWuerges ldquoVitaminB12 unique metalorganic compounds and the most complex
vitaminsrdquoMolecules vol 15 no 5 pp 3228ndash3259 2010[3] R Banerjee and SW Ragsdale ldquoThemany faces of vitamin B
12
catalysis by cobalamin-dependent enzymesrdquo Annual Review ofBiochemistry vol 72 pp 209ndash247 2003
[4] R G Matthews ldquoCobalamin and corrinoid dependentenzymesrdquo in Metal Ions in Life Sciences A Sigel H Sigeland R K O Sigel Eds vol 6 pp 53ndash114 Royal Society ofChemistry Cambridge UK 2009
[5] P J Kasper and U Ryde ldquoHow the Co-C bond is cleavedin coenzyme B
12enzymes a theoretical studyrdquo Journal of the
American Chemical Society vol 127 no 25 pp 9117ndash9128 2005[6] L Randaccio S Geremia and J Wuerges ldquoCrystallography of
vitamin B12proteinsrdquo Journal of Organometallic Chemistry vol
692 no 6 pp 1198ndash1215 2007[7] B Krautler ldquoOrganometallic chemistry of B
12coenzymesrdquo in
Metal Ions in Life Sciences A Sigel H Sigel and R K O SigelEds vol 6 pp 1ndash51 Royal Society of Chemistry CambridgeUK 2009
[8] A Kahraman R JMorris R A Laskowski and JMThorntonldquoShape variation in protein binding pockets and their ligandsrdquoJournal of Molecular Biology vol 368 no 1 pp 283ndash301 2007
[9] S F Sousa P A Fernandes and M J Ramos ldquoProtein-ligand docking current status and future challengesrdquo ProteinsStructure Function and Genetics vol 65 no 1 pp 15ndash26 2006
[10] R D Taylor P J Jewsbury and JW Essex ldquoA review of protein-small molecule docking methodsrdquo Journal of Computer-AidedMolecular Design vol 16 no 3 pp 151ndash166 2002
[11] M A Thompson ldquoMolecular docking using ArgusLab anefficient shape-based search algorithm and AScore scoring
functionrdquo in Proceedings of the ACS Meeting Philadelphia PaUSA March-April 2004 172 CINF 42
[12] R Wang X Fang Y Lu and S Wang ldquoThe PDBbind databasecollection of binding affinities for protein-ligand complexeswith known three-dimensional structuresrdquo Journal ofMedicinalChemistry vol 47 no 12 pp 2977ndash2980 2004
[13] R Reitzer K Gruber G Jogl et al ldquoGlutamate mutase fromClostridium cochlearium the structure of a coenzyme B
12-
dependent enzyme provides new mechanistic insightsrdquo Struc-ture vol 7 no 8 pp 891ndash902 1999
[14] N Shibata J Masuda T Tobimatsu et al ldquoA new modeof B12
binding and the direct participation of a potassiumion in enzyme catalysis X-ray structure of diol dehydrataserdquoStructure vol 7 no 8 pp 997ndash1008 1999
[15] M Koutmos S Datta K A Pattridge J L Smith and RG Matthews ldquoInsights into the reactivation of cobalamin-dependent methionine synthaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no44 pp 18527ndash18532 2009
[16] S Joy P S Nair R Hariharan and M R Pillai ldquoDetailedcomparison of the protein-ligand docking efficiencies ofGOLDa commercial package and arguslab a licensable freewarerdquo InSilico Biology vol 6 no 6 pp 601ndash605 2006
[17] M Thompson ArgusLab 401 Planaria software LLC SeattleWash USA 2004
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
![Page 4: Research Article Molecular Docking Study on the …downloads.hindawi.com/archive/2013/312183.pdfResearch Article Molecular Docking Study on the Interaction of Riboflavin (Vitamin B](https://reader033.vdocuments.site/reader033/viewer/2022042116/5e93aafdc60b2918c71e72c8/html5/thumbnails/4.jpg)
4 Journal of Computational Medicine
Table 2 List of hydrogen bonds between riboflavin and coenzyme B12 containing enzymes
S no Enzyme name No of H-bonds Amino acid residue atom Ligand atom H-bond distance (A)
1 Glutamate mutase (1ccw) 2 Gly120 (NH) (O2) 252Thr121 (NH) (N1) 272Cys302 (SH) (N5) 250
2 Diol dehydratase (1egm) 3 Ser301 (O) (N1) 296Arg699 (N) (O2) 282
3 Methionine synthase (3iv9) 2 His759 (NH) (O4) 268His1145 (NH) (O2) 272
300Val 336Gln 374Phe
302Cys301Ser
222Thr282 373Met
699Arg296
250
226Tyr
654Leu 656Asn
659Leu
662Gln
Figure 3 Docking of riboflavin into enzyme diol dehydratase[14] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink color in thick stick
were formed between the nitrogen (N1) of riboflavin andoxygen atom of Ser301 at a distance of 296 A and betweennitrogen of Arg699 and oxygen atom (O2) of riboflavin ata distance of 282 A The E-subunit comprises of active siteof the enzyme which has been found to pack against theupper face of B
12cofactor and may facilitate the transfer of
51015840-adenosyl radical from cofactor to substrate Therefore itmay be assumed that the riboflavin may interfere with thecatalytic process of the enzyme by binding at the interfaceof cofactor B
12and the enzymersquos active site and thus inhibit
the radical shuttling mechanism The AScore value obtainedfor this complex was minus698 kcalmol which indicates thatriboflavin significantly binds with the enzyme diol dehy-dratase
33 Methionine Synthase Figure 3 shows the binding ofriboflavin near the cofactor making contact with amino acidresidue His 759 The oxygen atom (O4) of the carbonylgroup of riboflavin was hydrogen bonded to N-atom of theHis 759 at a distance of 268 A Another hydrogen bonding
1148Lys
1145His
1176Ser
1039Asp
268
759His
272
753Thr
836Thr
834Gly
831Leu
Figure 4 Docking of riboflavin into enzyme methionine synthase[15] Riboflavin and protein binding sites are shown in elementcolors in wireframe and thick stick models respectively whereascyanocobalamin is represented in pink color in thick stick
was seen between the N-atom of His1145 and the carbonyloxygen (O2) of riboflavin at a distance of 272 AThe dockingAScore of the ligand-protein complex was minus607 kcalmolThe crystal structure of methionine synthase revealed thatthe enzyme is present in ldquobase-offrdquo or ldquoHis-onrdquo form withHis759 as the lower axial ligand His759 has been found toact as transient intermediate in the reductive reactivation andconformational transition of cobalt following methylationin methionine synthase-dependent enzyme catalysis [15]Therefore we may assume that riboflavin may inhibit theimportant catalytic step of the enzyme that is reactivationand methylation probably by making contact with the sameamino acid residue (His759) that is involved in the reactionThe binding energy also shows a significant ligand-receptorcomplex with this enzyme
4 Conclusion
By analyzing the docking results we hypothesized thatriboflavin might have inhibitory activity against cobalamincoenzymesThe enzyme glutamate mutase has been found tobe the most susceptible protein target for the studied ligandand the riboflavin showed the best binding affinity with thisenzyme having least binding energy of minus713 kcalmol
Journal of Computational Medicine 5
Glutamate mutase and diol dehydratase can be consid-ered as important targets for the development of antibioticsOther useful therapeutic drugs can also be synthesized toenhance the function of enzyme methionine synthase as itconverts homocystein into methionine and requires methyl-cobalamin coenzyme for its function Inactivation of thiscoenzyme leads to the elevated levels of homocystein in bloodand urine andmay result in clotting and in long-term damageto the arteries (stroke and heart attack) Therefore theinteraction of vitamin B
2and B
12and their role in important
metabolic reactions in the body should be considered beforepreparing multivitamin complexes
The fact that riboflavin participates in energy-basedmetabolic reactions may play an important role in enzymecatalysis which depends on a delicate energy balance fordifferent reaction pathways The local amino acids andsubstrates in the active sites of the enzymes may function inthis respect
Further experimental approaches can be adopted toprobe the effect of structural alterations of flavin groupof compounds in the catalytic properties of coenzymeB12-dependent enzymes
References
[1] K Gruber B Puffer and B Krautler ldquoVitamin B12-derivatives-
enzyme cofactors and ligands of proteins and nucleic acidsrdquoChemical Society Reviews vol 40 no 8 pp 4346ndash4363 2011
[2] L Randaccio S Geremia N Demitri and JWuerges ldquoVitaminB12 unique metalorganic compounds and the most complex
vitaminsrdquoMolecules vol 15 no 5 pp 3228ndash3259 2010[3] R Banerjee and SW Ragsdale ldquoThemany faces of vitamin B
12
catalysis by cobalamin-dependent enzymesrdquo Annual Review ofBiochemistry vol 72 pp 209ndash247 2003
[4] R G Matthews ldquoCobalamin and corrinoid dependentenzymesrdquo in Metal Ions in Life Sciences A Sigel H Sigeland R K O Sigel Eds vol 6 pp 53ndash114 Royal Society ofChemistry Cambridge UK 2009
[5] P J Kasper and U Ryde ldquoHow the Co-C bond is cleavedin coenzyme B
12enzymes a theoretical studyrdquo Journal of the
American Chemical Society vol 127 no 25 pp 9117ndash9128 2005[6] L Randaccio S Geremia and J Wuerges ldquoCrystallography of
vitamin B12proteinsrdquo Journal of Organometallic Chemistry vol
692 no 6 pp 1198ndash1215 2007[7] B Krautler ldquoOrganometallic chemistry of B
12coenzymesrdquo in
Metal Ions in Life Sciences A Sigel H Sigel and R K O SigelEds vol 6 pp 1ndash51 Royal Society of Chemistry CambridgeUK 2009
[8] A Kahraman R JMorris R A Laskowski and JMThorntonldquoShape variation in protein binding pockets and their ligandsrdquoJournal of Molecular Biology vol 368 no 1 pp 283ndash301 2007
[9] S F Sousa P A Fernandes and M J Ramos ldquoProtein-ligand docking current status and future challengesrdquo ProteinsStructure Function and Genetics vol 65 no 1 pp 15ndash26 2006
[10] R D Taylor P J Jewsbury and JW Essex ldquoA review of protein-small molecule docking methodsrdquo Journal of Computer-AidedMolecular Design vol 16 no 3 pp 151ndash166 2002
[11] M A Thompson ldquoMolecular docking using ArgusLab anefficient shape-based search algorithm and AScore scoring
functionrdquo in Proceedings of the ACS Meeting Philadelphia PaUSA March-April 2004 172 CINF 42
[12] R Wang X Fang Y Lu and S Wang ldquoThe PDBbind databasecollection of binding affinities for protein-ligand complexeswith known three-dimensional structuresrdquo Journal ofMedicinalChemistry vol 47 no 12 pp 2977ndash2980 2004
[13] R Reitzer K Gruber G Jogl et al ldquoGlutamate mutase fromClostridium cochlearium the structure of a coenzyme B
12-
dependent enzyme provides new mechanistic insightsrdquo Struc-ture vol 7 no 8 pp 891ndash902 1999
[14] N Shibata J Masuda T Tobimatsu et al ldquoA new modeof B12
binding and the direct participation of a potassiumion in enzyme catalysis X-ray structure of diol dehydrataserdquoStructure vol 7 no 8 pp 997ndash1008 1999
[15] M Koutmos S Datta K A Pattridge J L Smith and RG Matthews ldquoInsights into the reactivation of cobalamin-dependent methionine synthaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no44 pp 18527ndash18532 2009
[16] S Joy P S Nair R Hariharan and M R Pillai ldquoDetailedcomparison of the protein-ligand docking efficiencies ofGOLDa commercial package and arguslab a licensable freewarerdquo InSilico Biology vol 6 no 6 pp 601ndash605 2006
[17] M Thompson ArgusLab 401 Planaria software LLC SeattleWash USA 2004
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
![Page 5: Research Article Molecular Docking Study on the …downloads.hindawi.com/archive/2013/312183.pdfResearch Article Molecular Docking Study on the Interaction of Riboflavin (Vitamin B](https://reader033.vdocuments.site/reader033/viewer/2022042116/5e93aafdc60b2918c71e72c8/html5/thumbnails/5.jpg)
Journal of Computational Medicine 5
Glutamate mutase and diol dehydratase can be consid-ered as important targets for the development of antibioticsOther useful therapeutic drugs can also be synthesized toenhance the function of enzyme methionine synthase as itconverts homocystein into methionine and requires methyl-cobalamin coenzyme for its function Inactivation of thiscoenzyme leads to the elevated levels of homocystein in bloodand urine andmay result in clotting and in long-term damageto the arteries (stroke and heart attack) Therefore theinteraction of vitamin B
2and B
12and their role in important
metabolic reactions in the body should be considered beforepreparing multivitamin complexes
The fact that riboflavin participates in energy-basedmetabolic reactions may play an important role in enzymecatalysis which depends on a delicate energy balance fordifferent reaction pathways The local amino acids andsubstrates in the active sites of the enzymes may function inthis respect
Further experimental approaches can be adopted toprobe the effect of structural alterations of flavin groupof compounds in the catalytic properties of coenzymeB12-dependent enzymes
References
[1] K Gruber B Puffer and B Krautler ldquoVitamin B12-derivatives-
enzyme cofactors and ligands of proteins and nucleic acidsrdquoChemical Society Reviews vol 40 no 8 pp 4346ndash4363 2011
[2] L Randaccio S Geremia N Demitri and JWuerges ldquoVitaminB12 unique metalorganic compounds and the most complex
vitaminsrdquoMolecules vol 15 no 5 pp 3228ndash3259 2010[3] R Banerjee and SW Ragsdale ldquoThemany faces of vitamin B
12
catalysis by cobalamin-dependent enzymesrdquo Annual Review ofBiochemistry vol 72 pp 209ndash247 2003
[4] R G Matthews ldquoCobalamin and corrinoid dependentenzymesrdquo in Metal Ions in Life Sciences A Sigel H Sigeland R K O Sigel Eds vol 6 pp 53ndash114 Royal Society ofChemistry Cambridge UK 2009
[5] P J Kasper and U Ryde ldquoHow the Co-C bond is cleavedin coenzyme B
12enzymes a theoretical studyrdquo Journal of the
American Chemical Society vol 127 no 25 pp 9117ndash9128 2005[6] L Randaccio S Geremia and J Wuerges ldquoCrystallography of
vitamin B12proteinsrdquo Journal of Organometallic Chemistry vol
692 no 6 pp 1198ndash1215 2007[7] B Krautler ldquoOrganometallic chemistry of B
12coenzymesrdquo in
Metal Ions in Life Sciences A Sigel H Sigel and R K O SigelEds vol 6 pp 1ndash51 Royal Society of Chemistry CambridgeUK 2009
[8] A Kahraman R JMorris R A Laskowski and JMThorntonldquoShape variation in protein binding pockets and their ligandsrdquoJournal of Molecular Biology vol 368 no 1 pp 283ndash301 2007
[9] S F Sousa P A Fernandes and M J Ramos ldquoProtein-ligand docking current status and future challengesrdquo ProteinsStructure Function and Genetics vol 65 no 1 pp 15ndash26 2006
[10] R D Taylor P J Jewsbury and JW Essex ldquoA review of protein-small molecule docking methodsrdquo Journal of Computer-AidedMolecular Design vol 16 no 3 pp 151ndash166 2002
[11] M A Thompson ldquoMolecular docking using ArgusLab anefficient shape-based search algorithm and AScore scoring
functionrdquo in Proceedings of the ACS Meeting Philadelphia PaUSA March-April 2004 172 CINF 42
[12] R Wang X Fang Y Lu and S Wang ldquoThe PDBbind databasecollection of binding affinities for protein-ligand complexeswith known three-dimensional structuresrdquo Journal ofMedicinalChemistry vol 47 no 12 pp 2977ndash2980 2004
[13] R Reitzer K Gruber G Jogl et al ldquoGlutamate mutase fromClostridium cochlearium the structure of a coenzyme B
12-
dependent enzyme provides new mechanistic insightsrdquo Struc-ture vol 7 no 8 pp 891ndash902 1999
[14] N Shibata J Masuda T Tobimatsu et al ldquoA new modeof B12
binding and the direct participation of a potassiumion in enzyme catalysis X-ray structure of diol dehydrataserdquoStructure vol 7 no 8 pp 997ndash1008 1999
[15] M Koutmos S Datta K A Pattridge J L Smith and RG Matthews ldquoInsights into the reactivation of cobalamin-dependent methionine synthaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no44 pp 18527ndash18532 2009
[16] S Joy P S Nair R Hariharan and M R Pillai ldquoDetailedcomparison of the protein-ligand docking efficiencies ofGOLDa commercial package and arguslab a licensable freewarerdquo InSilico Biology vol 6 no 6 pp 601ndash605 2006
[17] M Thompson ArgusLab 401 Planaria software LLC SeattleWash USA 2004
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
![Page 6: Research Article Molecular Docking Study on the …downloads.hindawi.com/archive/2013/312183.pdfResearch Article Molecular Docking Study on the Interaction of Riboflavin (Vitamin B](https://reader033.vdocuments.site/reader033/viewer/2022042116/5e93aafdc60b2918c71e72c8/html5/thumbnails/6.jpg)
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom