polygalacturonases: active site analyses and mechanism of action

Indian Journal of Biotechnology Vol 5, April 2006, pp 148-162

Polygalacturonases: Active site analyses and mechanism of action P Palanivelu*

Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India

Received 1 October 2004; revised 5 May 2005; accepted 29 June 2005

Polygalacturonases from various sources have been analyzed by ClustalW and T-COFFEE, for identification of conserved and functional motifs in them. All the 104 polygalacturonases analyzed by the above programs, revealed four highly conserved motifs, viz., NTD, G/QDD, G/SHG and RIK among them. Distance conservation between the motifs was also observed. Based on the available evidences from chemical modification studies on active site amino acids, site-directed mutagenesis, protein sequence analysis and X-ray crystallographic data, a mechanism of action is proposed for this group of enzymes.

Keywords: polygalacturonases, protein sequence analysis, active site amino acids, distance conservation between motifs, mechanism of action

IPC Code: Int. Cl.8 C12N9/38, 9/40

Introduction Pectin, like cellulose, is one of the most important structural cell-wall polysaccharides of all higher plants. It constitutes 1/3 of the macromolecules of primary cell wall and is an abundant constituent of middle lamella, where it acts as a cementing material to bind adjacent cells. Pectin, is a relatively diverse group of acidic heteropolysaccharides, which vary both in their composition and molecular weight. The pectin chains are essentially made up of 1-4 α-D-galacturonic acid residues that are partly esterified (up to 75%) with methanol. Rhamnose residues are found interspersed in the main chain at about 25 to 30 residues, which makes the so-called “hairy regions” on the pectin chains. The rhamnose residues are linked via β,1-2 and α,1-4 bonds to the D-galacturonate residues. Some of the galacturonate residues are also acetylated (~ 5%) at C-2 and C-3 positions. The main chain usually contains side chains of variable lengths, often consisting of galactans, xylans, arabinans, arbinogalactans, rhamnogalactu-ronans, etc., and branch-off from the main chain at rhamnose residues. Enzymatic de-esterification of pectin yields a polymer known as pectic acid or polygalacturonic acid (PGA). Polygalacturonases (PGases) are the major group of enzymes, which depolymerize pectin/pectic acid by

cleaving the glycosidic bonds by hydrolytic reaction. These include both endoPGases (EC 3.2.1.15) and exoPGases. The exoPGases are of two types, viz., those which catalyze the hydrolytic cleavage of one galacturonic acid residue at a time from the non-reducing end (EC 3.2.1.67) and those which catalyze the hydrolytic cleavage of two galacturonic acid residues at a time from the non-reducing end (EC 3.2.1.82). The other group of enzymes which depolymerize pectin are pectin/pectate lyases, which cleave glycosidic bonds via β-elimination to yield oligomers that are 4, 5 unsaturated at the non-reducing end. In addition to the above enzymes, a novel enzyme, rhamnogalacturonase splits galacturonic-rhamnopyranosyl linkages at the so-called hairy regions of pectin1. All PGases belong to the glycoside hydrolase family 28. Pectinases are extensively used in food industries for extraction and clarification of fruit juices. Microbial pectinolytic enzymes, mainly produced by fungi, are utilized in many large-scale industrial processes. The addition of commercial preparations of pectinolytic enzymes greatly enhances yield of juice. Enzymatic degradation of pectin by fungal pectinase preparations is routinely used to maximize juice yields from fruits like grapes, apples, raspberries, strawberries, blackberries, tomatoes, peaches, pears, avocados and mangoes. Up to 40% more juices are extracted after treatment with the pectinases.

_______________ *Tel: 91-452-2458208; Fax: 91-452-2459105 E-mail: [email protected]

PALANIVELU: ACTIVE SITE & MECHANISM OF ACTION OF POLYGALACTURONASES

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Pectinolytic enzyme preparations exhibiting high levels of polygalacturonase activity are also used in the maceration of vegetables and fruits. Enzymatic maceration is used in the production of fruit nectars, "pulpy" drinks and in the preparation of baby foods. Pectinolytic enzymes are also used to aid the extraction of essential oils, coffee and pigments. Pectinolytic enzymes find application in the field of veterinary medicine as a remedy to treat cattle suffering from indigestion. Materials and Methods The protein sequence data for PGases were obtained from the CaZy home page (www.afmb.cnrs-mrs.fr/cazy) where all these enzymes are grouped under family 28. Only those PGases, which are characterized as exo- and endoPGases, were used for this analysis. The sequences were converted to FastA format and then analyzed by ClustalW or T-COFFEE programs available at ExPASy site (www.expasy.org). To further analyze the involvement of His in catalysis, a homogenous preparation of a PGase from the thermophilic fungus, Thermomyces lanuginosus was treated with the active site directed reagent diethyl pyrocarbonate (DEPC), which inactivates active site His by alkylation. The PGase was assayed as described earlier2. Results and Discussion Highly Conserved Amino Acid Motifs in Polygalacturonases Both endo- and exoPGases have been studied in great detail from a large number of sources. Genes, which encode these enzymes have also been cloned and sequenced from bacteria, yeasts, molds and also from variety of plants. In order to find out whether the active site His is completely conserved (100%) in all PGases, (both in exo- and endoPGases) multiple sequence analysis of all the PGases that are characterized as exo or endo from glycoside hydrolase Family 28 was analyzed to identify highly conserved motifs among them by ClustalW or T-COFFEE progams.

Figs. 1A-1F, 2 and 3 show the results of protein sequence analysis of all the characterized endo- and exoPGases, respectively, from various sources. It is clear from the analysis, that four regions are strictly/highly conserved (indicated by blocks) in all PGases. Small variations observed in few of the sequences are summarized in Table 1. It is interesting to note that in all the 104 enzymes (87 enzymes

belonging to endoPGases, (EC 3.2.1.15), 10 enzymes belonging to EC 3.2.1.67 and 7 enzymes from EC 3.2.1.82), only four motifs are almost completely conserved, irrespective of whether the enzyme is endo- or exo acting and also irrespective of the source from which it was isolated. The following conserved amino acid residues characterize these four motifs:

1. NTD (e.g., 184-186 in P26213) 2. G/QDD (e.g., 206-208 in P26213) 3. G/SHG (e.g., 228-230 in P26213) 4. RIK (e.g., 262-264 in P26213)

Among these four regions, the two catalytic regions (G/QDD and G/SHG) are strictly conserved and the other two substrate-binding regions are highly conserved in all PGases. Table 2 shows that not only the sequences are highly conserved but also the distances between these conserved motifs are almost precisely maintained (only representative sequences are shown under each group), suggesting their functional importance in substrate binding and catalysis which is further corroborated by site-directed mutagenesis of the residues in the conserved motifs and also by X-ray crystallographic studies. Active Site Amino Acids in Glycosidases All glycosidases irrespective of whether they belong to inverting or retention type, use two highly conserved carboxylic amino acids to bring about hydrolysis of the susceptible glycosidic bond in sugars. By protein sequence analysis, site-directed mutagenesis and active site labeling studies, these two carboxylic amino acids are identified as an Asp and a Glu, which act like a pair of scissors. The distance between the two amino acids are ~5. 5 Å for retaining glycosidases and ~9.5 Å for inverting glycosidases at their catalytic sites. Generally, in all glycosidases the Glu acts as a proton donor and the Asp acts as a nucleophile. The nucleophile either stabilizes the transition state intermediate or form an “Enzyme–Substrate” complex as in levansucrases3, cyclodextrin glycosyltransferases4, α-amylases5, and xylanases6,7, after the susceptible glycosidic bond is cleaved by the proton donated by the Glu. Recently, it has been shown even in lysozyme that the nucleophile Asp52 actually forms a covalent intermediate and not involved in stabilizing the oxocarbenium ion intermediate through electrostatic interactions as once proposed8. Thus, not only α-glycosidases but β- glycosidases also operate via covalent catalysis.

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Fig. 1A⎯ClustalW (1.82) multiple sequence alignment of EndoPGases (EC 3.2.1.15). Conserved motifs are indicated by blocks.


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Fig. 1B⎯ClustalW (1.82) multiple sequence alignment of EndoPGases (EC 3.2.1.15). Conserved motifs are indicated by blocks.

Fig. 1C⎯ClustalW (1.82) multiple sequence alignment of EndoPGases (EC 3.2.1.15). Conserved motifs are indicated by blocks.


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Fig. 1D⎯ClustalW (1.82) multiple sequence alignment of EndoPGases (EC 3.2.1.15). Conserved motifs are indicated by blocks.


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Fig. 1E⎯ClustalW (1.82) multiple sequence alignment of EndoPGases (EC 3.2.1.15). Conserved motifs are indicated by blocks.

Fig. 1F⎯ClustalW (1.82) multiple sequence alignment of EndoPGases (EC 3.2.1.15). Conserved motifs are indicated by blocks.


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Palanivelu9 has analyzed about 50 different glycosidases belonging to different groups of enzymes such as invertases, levanases and levansucrases for their active site regions by protein sequence analysis and found that in all these enzymes Glu acts as proton donor and Asp serves as nucleophile. Asp is not Proton Donor in Majority of Glycosidase Families There are 92 glycoside hydrolase families in the CaZy site. Only in 6 of these families Asp is suggested/implicated as a proton donor, which includes the glycoside hydrolase family 28 also. In all other 86 glycoside hydrolase families, Glu is implicated/proved as proton donor. However, in glycoside hydrolase family 28, that covers all PGases, two Asp residues are implicated, where one acts as proton donor and the other serves as a nucleophile.

Asp is usually implicated as a proton donor only in some proteases known as aspartic proteases such as pepsin, chymosin, renin, cathepsins-D and E, which works optimally at highly acidic pH values. Apparently, it suggests only under such highly acidic condition the catalytic aspartic acid residue is protonated, i.e., could carry a proton and thus, could act as a proton donor. But some recent papers on PGases have implicated an Asp as a proton donor even though both Asp and His are found in the catalytic site10-13. Therefore, in order to find out whether aspartic proteases and PGases show any similarity in their active site, a multiple sequence analysis of well-known aspartic proteases (as mentioned above) was performed (Fig. 4). As it is clear from Fig. 4, there is no similarity in the active site between the two groups. The following examples will substantiate that Glu (and not Asp) is the proton

Fig. 2⎯Clustal format for T-COFFEE multiple sequence alignment of ExoPGases (EC 3.2.1.67). Conserved motifs are indicated by blocks.

Fig. 3⎯Clustal format for T-COFFEE multiple sequence alignment of ExoPGases (EC 3.2.1.82). Conserved motifs are indicated by blocks.


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donor in many glycosidases. Lysozymes contain both Asp and Glu at their catalytic site14. It is the Glu, which donates the proton resulting hydrolysis of the susceptible glycosidic bond, and not the Asp. The Asp acts as a nucleophile and forms an enzyme-substrate complex8. X-ray crystallographic analysis has shown that both the catalytic residues are placed juxtaposition on the same plane on the susceptible bond (forming subsite E)15. Synthetic enzymes (synzymes), synthesized using Merriefield’s solid phase peptide synthesis, such as (Phe-Glu)n copolymer exhibited high lysozyme activity, and (Phe-Glu-X)n polymer ( X could be any hydrophobic amino acid like Ala) showed glucosidase activity. In both the synzymes, Glu acts as the proton donor. Other glycosidases such as β-N-acetyl-fucosaminidase H16, peptide-N-glucosidase F117, β-1,4 glucanases7, xylanases6, cellulases18, cyclodextrin glycosyltransferases19 and β-amylases20 employ Glu as the proton donor.

Furthermore, the structures of several α-amylases have been determined recently. Interestingly, all these structures again show a cluster of three carboxylic acids in the active site viz., Asp197, Glu233 and Asp300

(numbering as per human pancreatic amylase). Rydberg et al21 have shown recently by site-directed mutagenesis that among the three carboxylic acids Asp197 is the catalytic nucleophile whereas Glu233

is the proton donor. Moreover, Watanabe et al22 have also identified a Glu204

and an Asp200 as the essential residues in the

chitinase from Bacillus circulans. It is interesting to note that Glu→Asp mutant enzyme was essentially inactive corroborating that Asp did not act as a proton donor. This point was further confirmed by X-ray crystallographic studies of a chitinase from Hordeum vulgare, where Glu67 is shown to be the proton donor23. Furthermore, a highly conserved Glu286

is shown to be involved in proton translocation in cytochromes too24. A recent study also reveals that Glu is the essential proton transfer gate during the catalytic

Table 1⎯Variation observed (in few of the sequences) among the highly conserved regions Motif Type PGase Type NTD DD GHG RIK Endo-15 (Fig 1A) NTDG/A G/QDDC* GHGL/ISI/VGS RI*K (N in P26509) (V in O59925) (Fig. 1B) NTDG/A G/QDDC GHGI/LSIGSL/VG RI*K (V in Q9178) (Fig. 1C) NT*DG/A G/QDDC G*HG-SIGS RI*K (S in O13466) (S in O00104) (V in O22311 & O59934) (Fig. 1D) NTDG/A G/QDDC* GHG—I/L*SV/IGS* R*IK (D in P94293 & (T in O97400 & (H in O97400) H in P77818) G in P94293) (Fig. 1E) NT*DG/A** G/QDDC GHGI/LSI/VGSL/I/V RI/VK (*S in Q9HF67) (**V in Q9HFP1) (Fig. 1F) NT*D G/QDDC GHGI/LSI/VGSI/VG RI*K (S in Q9Y7V9) (V in 2 sequences) Exo-67 NT*D G*DDC G/SHGI/LSI/VGSLG RIK (S in 074255) (T in O74255) Exo-82* NNG*DG GDD GHG GLRA*KS (A in Q53241) (L in Q53241 & G in Q60045) (*In Exo-82 type a highly conserved GGGA is observed 5 amino acids downstream to RA*K motif). Note: Numbers in brackets indicate the accession number. * Indicates the amino acid replaced.


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cycle of the [NiFe] hydrogenase25. These evidences prove that Glu is the preferred proton donor not only in glycosidases but also in cytochromes and hydrogenases as well. Histidine Acts as Proton Donor in Many Enzymes As there is no evidence for a completely or a highly conserved Glu in PGases, it is likely the His which was found in the catalytic site by X-ray crystallographic analysis in these enzymes could act as a proton donor in these enzymes as elaborated elsewhere. In fact, there

are reports where His acts as proton donor in glycosidic bond cleavage reactions. The following results and observations favour the placement of highly conserved His as the proton donor in the catalytic site of PGases. All PGases so far characterized exhibited moderately acidic pH optima (around 5 to 6). Therefore, Schroter et al suggested a protonated His in the active site26. Rao et al27 identified a Trp and a His in a PGase from Aspergillus ustus at the active site by active-site directed chemical modifications. They proposed a

Table 2⎯Distance conservation analysis between motifs

Highly and completely conserved regions among exo - and endo polygalacturonases from various sources were analyzed for distance conservation between motifs. Only some of the representative sequences are taken for illustration. The conservation of distance between the conserved motifs has not been reported earlier. Such precise distance conservation among the highly conserved motifs further corroborates their importance in substrate binding and catalysis. (Sequences are from SWISSPROT/ TrEMBL database) Bacteria 1) 201NTD ……….. 202GDD …………..250GHGMSIGS ………… 880RIK-E. carotovora P 26509 → 21aa ← → 28 aa ← → 30 aa ←

2) 243NTD ……….. 265GDD …………..291THGISIGS ………… 331RIK - X. campestris Q8P582 → 22aa ← → 26 aa ← → 40 aa ←

3) 293NTD ……….. 315GDD …………..342GHGLSIGS ………… 377RIK-P. syringae Q87Y49 → 22aa ← → 27 aa ← → 35 aa ←

Yeasts 1) 177NTD ……….. 199QDD ……….. 221GHGISVGS ….……….255RIK - S. cerevisiae (endo) P 47180 → 22 aa ← → 22 aa ← → 34 aa ← 2) 178NTD ………. 200QDD …………. 222GHGLSIGS ……….. 256RIK-K. marxianus (endo) Q6TA03 → 22 aa ← → 22 aa ← → 34 aa ←

Higher fungi 1) 178NTD ……… 200QDD ………… 222GHGLSIGS ………… 256RIK-A. niger (endo II) P 26214 → 22 aa ← → 22 aa ← → 34 aa ← 2) 221NTD ……… 243GDD …………. 266SHGISVGS ………… 302RIK-A. tubingensis (exo) Q 0093 → 22aa ← → 23 aa ← → 36 aa ← 3) 227NTD ………249GDD ………… 272SHGISVGS ………… 308RIK-C. carbonum (exo) Q 00359 → 22 aa ← → 23 aa ← → 36 aa ←

Plants 1) 218NTD ………240GDD ………… 263GHGLSVGS ………… 298RIK-A. thaliana (flower) P 49062 → 22 aa ← → 23 aa ← → 35 aa ← 2) 190NTD ………212GDD ………… 235GHGISVGS ………… 270RIK-N. tabacum (pollen) Q 05967 → 22 aa ← → 23 aa ← → 35 aa ← 3) 156NTD ………178GDD ………… 201GHGISVGS ………… 236RIK-O. organensis (pollen) P 24548 → 22 aa ← → 23 aa ← → 35 aa ← 4) 247NTD ………269GDD ………… 292GHGISIGS ……………327RIK-L. esculentum (fruit) P 05117 → 22 aa ← → 23 aa ← → 35 aa ←


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mechanism involving a single proton transfer from His. But their study did not disclose which one, out of the 7 His residues, is actually involved in catalysis and there is no mention of any nucleophile in their proposed mechanism. However, by site-directed mutagenesis, Caprari et al28 have identified that His234 is the critical amino acid in the PGase of the plant pathogenic fungus, Fusarium moniliforme. In fact, this critical His is from the highly conserved region GH234GXSIGS. A mutant enzyme carrying Lys in place of His (H234→K) had completely lost the activity suggesting the importance of His234 in catalysis. A recent study by site-directed mutagenesis of an endoPGase II from A. niger11 and Fusarium moniliforme13 revealed that the His located in the conserved region as mentioned above is important in bringing about catalysis. In the endoPGase II from A. niger12 four replacements of the critical His223 were made as shown; H223 →A, H223 →C, His223 →S and H223

→Q. The KM for the first mutant enzyme was

similar to that of native enzyme but the activity was only 0.5% suggesting that, as Ala is not a proton donor even though the mutant enzyme could bind the substrate almost with same affinity. The Cys, Ser and Gln replacements showed 5-, 10- and 7 fold less affinity for the substrate and exhibited about 1, 0.1 and 0.02% activity, respectively. Thus, these data further confirm the importance of His in catalysis. Furthermore, site-directed mutagenesis studies with the Fusarium moniliforme enzyme has also thrown more light on the critical Asp212 and His188 residues (numbering from the F. moniliforme enzyme), which involve in catalysis. For example, the following mutant enzymes, D191→A; D212 →E; D213 →E; D212→N; D213→N and R267→A showed no activity. The mutant enzymes K269→E and H188→P exhibited only 0.026% and 0.35% activities, respectively. If the D212 is the proton donor as suggested in the recent reports10-13, the mutant enzyme D212→E would have shown some activity because E can also act as an effective proton donor. The complete absence of activity in the above mutant enzyme again suggests that

Fig. 4⎯ClustalW (1.82) multiple sequence alignment of aspartic proteases. Note: The blocks indicate the active site Asp (proton donor) regions in these enzymes


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the D212 and D213 may not act as a proton donor but could act as a nucleophile as suggested in this communication. Furthermore, the H188→P mutant enzyme drastically affected the interaction with PGIP-2, a competetive inhibitor of PGases, suggesting that the highly conserved His188 is infact in the active site. So far, only few PGases (form E. caratovora10, A. niger11 and F. moniliforme13) have been studied in detail by crystallographic methods. All three enzymes exhibit almost similar structural motifs: i.e., fold into a right-handed parallel β-helix with 10 complete turns and a small α- helical region at the N-terminus. In addition to this, these enzymes contain two loop regions, which form a “tunnel like” substrate-binding site. It is interesting to note that all the four conserved regions are located in this cleft region. An Asp in G/QDD region and a His from GHGMSIGS region are placed juxtaposition within the expected 5.5 Å which could optimally serve as a nucleophile and a proton donor, respectively. Therefore, based on the above evidences, in this communication, using His as the proton donor, a mechanism of action is proposed for PGases. The following results and observations further support the proposed mechanism here. His is implicated as proton donor in other hydrolases like exo-(1,3)β-D glucanase29, mung bean glucosidase I30, aminopeptidase31,32 and S1 nuclease33. Moreover, His is implicated in lyases viz., in hydroxynitrile lyase34 and also in pectin and pectate lyases35 as the acid/base catalyst. The most well studied example, where His acts as aproton donor, is the pancreatic ribonucleases. The ribonucleases use two His and a Lys at their active site, viz., His12 , His119 and Lys43 (numberings form bovine pancreatic RNase A). In RNases, the two histidines act differently, i.e., one acts as a proton donor (His119) and other acts as the proton acceptor. Therefore, the absence of a completely conserved Glu in PGases but a completely conserved His in all PGases, suggests His could be favoured as a proton donor in PGases, which is further corroborated by biochemical, site-directed mutagenesis and X-ray crystallographic studies also. Acidic Polysaccharide Hydrolases and Active Sites Amino Acids It is interesting to note that both RNases and PGases have many active site similarities. For example, both the enzymes are inactivated by active site-directed reagents such as DEPC and iodoacetamide. Our studies with the active site directed reagents on a homogenous preparation of a PGase from the thermophilic fungus, Thermomyces lanuginosus, also support His in the

active site. We found that p-chloromercurybenzoate (PCMB) did not inhibit the PGase even at 4 mM (but an invertase from the same source was almost completely inactivated). Iodoacetamide at 1 and 4 mM inactivated the PGase from the thermophilic fungus by 74% and 94%, respectively36. DEPC also inactivated the fungal enzyme completely at 0.1%, the same concentration, which was used to inactivate RNases, suggesting the involvement of a His in catalysis. Thus, the above data from our laboratory on active site-directed analysis also corroborate the involvement of a His in catalysis. Similar observations were reported for an endoPGase from A. ustus where DEPC inhibited the enzyme and PCMB did not inhibit the enzyme27. Again, iodoacetamide inactivation of RNases has been studied in detail. It specifically reacts to the active site histidine residues of RNases viz., His12 and His119 (numbering from bovine RNase). Interestingly, the major product of the reaction is with the His119, which is again the proton donor in RNases. Furthermore, it has also been proved by site-directed mutagenesis of the catalytic residues of bovine pancreatic deoxyribonuclease I that H134 and H252 provide general acid and general base catalysis37. Further analysis of substrate binding regions of the two different enzymes also showed a striking similarity. i.e., similar to NTD regions in PGases, NTF regions are found in all pancreatic RNases. About a dozen pancreatic RNases from different sources were analyzed by ClustalW and found in all RNases, the NTF region is completely conserved (Fig. 5). The Thr45 (numbering form bovine RNase) of RNases is shown to be involved in making multiple hydrogen bonding with uracil in the substrate. Similarly, Thr of the NTD region is placed in PGases which could form multiple hydrgen bonding with the substrate. The Arg and Lys of RIK region, which is found in the substrate binding cleft also could make mutiple hydrogen bonding with the substrate. Thus, the first and last completely conserved triplets could act like two hands in holding the substrate, producing necessary strain and distortion on the susceptible glycosidic bond and the middle two highly conserved regions involve in the hydrolysis of the glycosidic bond, as elaborated in Fig. 6. Moreover, it has also been shown by site-directed mutagenesis that heparinases from Flavobacterium heparinum, which degrade heparin (also known as heparinic acid) made of repeating units (D- glucosamine and L- iduronic acid or D-glucuroinc acid) also uses His (H203 in heparinase I38 and H451in heparinase II39) as the catalytic amino acid. Again in


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hyaluronate lyase from Streptococcus which degrades hyaluronic acid (heteropolysaccharide made up of repeating units of D-glucuronic acid and N-acetylglucosamine, β,1→3 linked) also uses His in the active site. It is the H479 which participates in the initial abstraction of hydrogen ion from the glucuronic acid

residues of hyaluronan40. In chondroitinsases also, which degrade the acidic polysaccharides chondroitins, (similar to hyaluronic acid where N- acetylglusosamine is replaced by N-acetylgalactosamine) it has been shown by site-directed mutagenesis that the H225, is in the active site41. Besides, site-directed mutagenesis of

Fig. 5⎯Multiple sequence alignment of ribonucleases (pancreatic), showing catalytic regions and substrate binding regions, which are similar to Pgases. Note: Blocks indicate the substrate binding and catalytic amino acid (His) regions.


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Fig.6⎯Proposed mechanism of action for PGases (The numbering is from the endoPGase II of A. niger).


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an alginate lyase which degrades alginic acid (polymannuronic acid) also proved that the H204, is critical for lyase activity42. Thus, it appears that in almost all these enzymes which hydrolyze acidic polysaccharide polymers His is used as proton donor. Recent crytallographic data on two more PGases viz., one from Stereum purpureum43 and the other from A. niger44 (endopolygalacturonase I) have also shown that the His195, Arg226, and Lys228 in S. purpureum and His229, Arg262 and Lys264 in A. niger actually forms the +1 substarte binding site and makes hydrogen bond with the carboxylate of the substrate. Based on the available evidences from chemical modification studies on active site amino acids, site-directed mutagenesis, protein sequence analysis and X-ray crystallographic data, the following mechanism of action is proposed for this group of enzymes (Fig. 6):

1. Positioning of active site amino acids on the susceptible glycosidic bond. The 178NTD and 256RIK motifs interact with the substrate on both the sides of the susceptible glycosidic bond through multiple hydrogen bonds and produce necessary strain and distortion on the susceptible glycosidic bond. The catalytic amino acids His223 and Asp201 are positioned on the susceptible glycosidic bond.

2. Proton transfer from the catalytic His223 to the susceptible glycosidic bond.

3. Glycosidic bond is cleaved with release of the first product and with simultaneous formation of covalent bond between the substrate and the catalytic site nucleophile Asp201.

4. The other active site Asp residue (Asp202) positions a water molecule for a nucleophilic attack.

Nucleophilic attack by the water molecule, resulting in the release of second product and restoration of active site of the enzyme. Acknowledgement The author wishes to thank Prof S Krishnaswamy, Centre of excellence in Bioinformatics, School of Biotechnology, and Dr R Usha, School of Biotechnology for useful suggestions on the manuscript and Bioinformatics Centre, Madurai Kamaraj University, for the Internet facilities. References 1 Koford L V, Kauppinen S, Christgau S, Andersen L N,

Heldt-Hansen H P et al, Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus, J Biol Chem, 269 (1994) 29182-29189.

2 Sathish Kumar S & Palanivelu P, Production and properties of pectinolytic enzymes from the thermophilic fungus, Thermomyces lanuginosus, World J Microbiol Biotechnol, 14 (1998) 781-782.

3 Chambert R & Treboul G G, Levansucrase of Bacillus subtilis: Characterization of a stabilized fructosyl-enzyme complex and identification of an aspartyl residue as the binding site of the fructosyl groups, Eur J Biochem, 71 (1976) 493-508.

4 Vitdehang J C M, Mosi R, Kalk K H, van der Vien B A, Dijkhuizen L et al, X-ray structures along with the reaction pathway of cyclodextrin glycosyltranferase elucidate catalysis in the α-amylase family, Nat Struct Biol, 6 (1991), 432- 436.

5 Tao B Y, Reilly P J & Robyt J F, Detection of covalent intermediate in the mechanism of porcine pancreatic α-amylase by using 13C nuclear magnetic resonance, Biochim Biophys Acta, 995 (1987) 214-220.

6 Jeffries T W, Biochemistry and genetics of microbial xylanases, Curr Opin Biotechnol, 7 (1996) 337-342.

7 White A, Tull D, Johns K, Withers S G & Rose D R, Crystallographic observation of a covalent catalytic intermediate in β-glycosidase, Nat Struct Biol, 3 (1996) 149-153.

8 Vocadlo D J, Davies G J, Laine R & Withers S G, Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate, Nature (Lond), 412 (2001) 835-838.

9 Palanivelu P, Fructofuronosyl hydrolases and transferases: Active sites and mechanism of action, Indian J Microbiol, 38 (1998) 177-185.

10 Pickergill R, Smith D, Worboys K & Jenkins J, Crystal structure of polygalacturonase from Erwinia carotovora subsp. carotovora, J Biol Chem, 273 (1998) 24660-24664.

11 van Santen Y, Benen J A E, Schroter K H, Kalk K H, Armand S et al, 1.68-Ǻ Crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis, J Biol Chem, 274 (1999) 30474-33480.

12 Armand S, Wagemaker M J M, Torres P S, Kester H C M, van Santen, Y et al, The active topology of Aspergillus niger endopolygalacturonase II as site-directed mutagenesis, J Biol Chem, 275 (2000) 691-696.

13 Federici L, Caprari C, Mattei B, Savino C, Di Matteo A et al, Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein), Proc Natl Acad Sci USA, 98 (2001) 13425-13430.

14 Malcolm B A, Rosenberg S, Corey M J, Allen J S, de Baetselier A & Kirsch J A, Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme, Proc Natl Acad Sci USA, 86 (1989) 133-137.

15 Blake C C, Koenig D F, Mair G A, North A C T, Philips D C & Sarma V R, Structure of hen egg white lysozyme, Nature (Lond), 206 (1965) 757-761.

16 Rao V, Cui T, Guan & van Roey P, Mutations of endo-β-N-acetyl glucosaminidase-H active site residues Asp130 and Glu 132: Activities and conformations, Protein Sci, 8 (1999) 2338-2346.

17 Van Roey P, Rao V, Plummer T H & Tarentino A, Crystal structure of endo-β-N-acetylglucosaminidase-F1, an α/β barrel enzyme adapted for a complex substrate, Biochemistry, 33 (1994) 13989-13996.


162

18 Ducrose V, Czjzek M, Belaich A, Gaudin C, Fierobe H P et al, Crystal structure of the catalytic domain of a bacterial cellulase belonging to family 5, Structure, 3 (1995) 939-949.

19 Strokopytov B, Penninga D, Rozeboom H J, Kalk K H, Dijkhuizen L & Dijkstra B W, X-ray structure of cyclodextrin glycosyltransferase complexed with acarbose: Implications for the catalytic mechanism of glycosidases, Biochemistry, 34 (1995) 2234-2240.

20 Mikami B, Degano M, Hehre E J & Sacchettini J C, Crystal structure of soybean β-amylase reacted with β-maltose and maltal: Active site components and their apparent roles in catalysis, Biochemistry, 33 (1994) 7779-77787.

21 Rydberg E H, Sidhu G, Vo H C, Hewitt J, Cote H C F et al, Cloning, mutagenesis and structural analysis of human pancreatic α-amylase in Pichia pastoris, Protein Sci, 8 (1999) 635- 643.

22 Watanabe T, Kobori K, Miyashita K, Fujii T, Sakai H et al, Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity, J Biol Chem, 268 (1993) 18567-18572.

23 Hart P J, Pfluger H D, Monzingo A F, Hollis T & Robertus J D, The redefined crystal structure of an endochitinase from Hordeum vulgare L. seeds at 1.8 Ǻ resolution, J Biol Chem, 248 (1995) 402-403.

24 Verkhovskaya M L, Horsman A G, Puustenan A, Riguad J L, Morgan J E et al, Glutamic acid 286 in subunit I of cytochrome bo3 is involved in proton translocation, Proc Natl Acad Sci USA, 94 (1997) 10128-10131.

25 Dementin S, Burlat B, De Lacey D L, Pardo A, Adryanczyk P C et al, Glutamate is the essential proton transfer gate during the catalytic cycle of the [NiFe] hydrogenase, J Biol Chem, 279 (2004) 10508-10513.

26 Schroter K H, Arkema A, Kester H C M, Visser J & Dijkstra B W, Crystallographic and preliminary charecterization of endopolygalacturonase II form Aspergillus niger, J Mol Biol, 243 (1999) 351-352.

27 Rao N M, Kembhavi A A & Pant A, Implication of tryptophan and histidine in the active site of endopolygalacturonase from Aspergillus ustus: Elucidation of reaction mechanism, Biochim Biophys Acta, 1296 (1996) 167-173.

28 Caprari C, Mattei B, Basile M L, Salvi G, Crescenzi V et al, Mutagenesis of endopolygalacturonase from Fusarium moniliforme: Histidine residue 234 is critical for enzymatic and macerating activities and not for binding to polygalacturonase-inhibiting protein (PGIP), Mol Plant Microbe Interact, 9 (1996) 617-624.

29 Jeffcoat R & Kirkwood S, Implication of histidine at the active site of exo-β-(1,3)-D-glucanase from basidomycete sp QM 806, J Biol Chem, 262 (1987) 1088-1091.

30 Zeng Y C & Elbein A D, Purification to homogeneity and properties of plant glucosidase I, Arch Biochem Biophys, 355 (1998) 26-34.

31 Helene A, Beaumont A & Roques B P, Functional residues at the active site of aminopeptidase N, Eur J Biochem, 196 (1991) 385-393.

32 Iturrioz X, Vazeux G, Celerier J, Corval P & Llorens-Cortes C, Histidine 450 plays a critical role in catalysis and, with Ca2+, contributes to the substrate specificity of aminopeptidase A, Biochemistry, 39 (2000) 3061-3068.

33 Gite S, Reddy G & Shankar V, Active-site characterization of SI nuclease: Involvement of histidine in catalysis, Biochem J, 288 (1992) 571-576.

34 Zuegg J, Gruber K, Gugganig M, Wagner U G & Kratky C, Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis, Protein Sci, 8 (1999) 1990-2000.

35 Vitali J, Schick B, Kester H C M, Visser J & Jurnak F, The three dimensional structure of Aspergillus niger pectin lyase B at 1.7 Ǻ resolution, Plant Physiol, 116 (1998) 69-80.

36 Sathish Kumar S & Palanivelu P, Purification and characterization of a polygalacturonase from the thermophilic fungus, Thermomyces lanuginosus, World J Microbiol Biotechnol, 15 (1999) 643-646.

37 Jones S J, Worrall A F & Connolly B A, Site-directed mutagenesis of the catalytic residues of bovine pancreatic deoxyribonuclease-I, J Mol Biol, 264 (1996) 1153-1163.

38 Godavarti R, Cooney C L, Langer R & Sasisekaran R, Heparinase-I from Flavobacterium heparinum. Identification of a critical histidine residue essential for catalysis as probed by chemical modification and site-directed mutagenesis, Biochemistry, 35 (1996) 6846-6852.

39 Shriver Z, Hu Y & Sasisekaran R, Heparinase-II from Flavobacterium heparinum: Role of histidine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis, J Biol Chem, 273 (1998) 10160-10167.

40 Lin B, Averett W F & Pritchard D G, Identification of a histidine residue essential for enzymatic activity of group B streptococcal hyaluronate lyase, Biochem Biophys Res Commun, 231 (1997) 379-382.

41 Huang W, Boju L, Tkalec L, Su H, Yang H O et al, Active site of chondroitin AC lyase revealed by the structure of enzyme oligosaccharide complexes and mutagenesis, Biochemistry, 40 (2001) 2359-2372.

42 Preston L A, Wong T Y, Bender C L & Schiller N L, Characterization of alginate lyase from Pseudomonas syrigae pv syringae, J Bacteriol, 182 (2000) 6268-6271.

43 Shimizu T, Nakatsu T, Miyairi K, Okuno T & Kato H, Active site architecture of endopolygalacturonase I from Stereum purpureum revealed by crystal structures in native and ligand-bound forms at atomic resolution, Biochemistry, 41 (2002) 6651-6659.

44 van Pouderoyen G, Snijder H J, Benen J A E & Dijkstra B W, Structural insights into the processivity of endopolygalacturonase I from Aspergillus niger, FEBS Lett, 55 (2003) 462-466.

polygalacturonases: active site analyses and mechanism of action

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