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Enzyme and Microbial Technology 37 (2005) 279–294

Review

Hydroxynitrile lyases: At the interface of biology and chemistry

Monica Sharma, Nitya Nand Sharma, Tek Chand Bhalla∗

Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, Himachal Pradesh 171005, India

Received 2 February 2005; accepted 25 April 2005

Abstract

Hydroxynitrile lyases are the versatile group of enzymes, which play a significant defensive role in plant system against microbial attack. Inchemical industries, hydroxynitrile lyase is used as an important industrial biocatalyst for the synthesis of chiral cyanohydrins by exploitingthe reversible enzymatic reaction. Cyanohydrins are biologically active compounds used in synthesis of�-amino alcohols,�-hydroxy ketonesand�-hydroxy acids, which have importance as fine chemicals, pharmaceuticals and agrochemicals. NMR and inhibition studies reveledthe involvement of different amino acids at the active site and proved that the hydroxynitrile lyases generally utilize acid/base catalysismechanism. Protein engineering and site directed mutagenesis have been used to change the active site and alter the substrate specificity ofvarious hydroxynitrile lyases. Many recombinant hydroxynitrile lyases have been expressed inEscherichia coli, Saccharomyces cerevisiaea©

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syn-iningp at-

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ndPichia pastoris.2005 Elsevier Inc. All rights reserved.

eywords:Hydroxynitrile lyases; Cyanohydrins; Recombinant hydroxynitrile lyases

ontents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2792. Classification of hydroxynitrile lyases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2823. Distribution of hydroxynitrile lyases and their function in nature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2834. Hydroxynitrile lyase reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2835. Substrate specificity of hydroxynitrile lyases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2866. Biochemical and molecular properties of hydroxynitrile lyases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2867. Recombinant hydroxynitrile lyases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2878. Mechanism of reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2889. Chemo enzymatic transformation of cyanohydrins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29110. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

. Introduction

Hydroxynitrile lyases or oxynitrilases (HNLs E.C.4.2.1.0, E.C.4.2.1.11, E.C.4.2.1.37 and E.C.4.2.1.39) are the

∗ Corresponding author. Tel.: +91 177 2832154/2831948;

enzymes, which catalyze enantioselective cleavage andthesis of cyanohydrins. Cyanohydrins are alcohol contaa cyano group i.e. having a cyano and a hydroxyl groutached to the same carbon atom. In 1837 Friedrich Wohlerdetected the hydroxynitrile lyase activity for the first timealmond, which cleaves the cyanohydrins into aldehydes

ax: +91 177 2832154.E-mail address:[email protected] (T.C. Bhalla).

HCN [1]. In recent years hydroxynitrile lyases have emergedas potential biocatalysts for the synthesis of a range of chiral

141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2005.04.013

280 M. Sharma et al. / Enzyme and Microbial Technology 37 (2005) 279–294

Table 1Chemical structure and application of some cyanohydrin derivatives

S. no. Products Chemical structure Application

1 (R)-Salbutamol Bronchodilator[60]

2 (R)-Terbutaline Bronchodilator[60]

3 (S)-13-Hydroxyoctadeca (9Z, 11E)-dienoic acid Biological activity against rice blastdisease and a chiral synthon[60]

4 (S)-Amphetamines Designer drug used medically in psy-chological treatments and central ner-vous system stimulant[60]

5 l-Ephedrine Stimulant and bronchodilator[86]

6 (R)-Pantolactone Chiral building block and chiral aux-iliaries, constituents of coenzyme A,bactericide, A growth factor[70]

7 Diltiazem Cardiac drug[76]

8 Terpenoids[55] (R)-ketone cyanohydrins

9 Pheromones[55]

M. Sharma et al. / Enzyme and Microbial Technology 37 (2005) 279–294 281

Table 1 (Continued)


10 Antibiotics[55] Synthesis of semi syntheticcephalosporins antibiotics

11 Amino acids Component of biologically activecompounds[35,85]

12 Thalidomide Curb morning sickness in pregnantwomen[94]

13 Denopamine Cardiac drug[108]

14 Tembamide Hypoglycaemic activity[108,109]

15 Aegeline Adrenaline like and insecticide activ-ity [108,109]

16 Lipitor Hypercholestrolemia treatment[110]

17 Pyrethroids Insecticide[111]


Table 1 (Continued)


18 Mandelamine Urinary antiseptics[112]

19 Mandelonitrile Germicide[113]

20 Mandelic marine complexes Cosmetics[113]

21 SAMMA Mandelic acid condensation product Blocks HIV, HSV-1 and HSV-2[114]

22 2(R)-Hydroxy-4-phenyl butyric acid Angiotension converting enzymeinhibitor (Ciba Geigy) andhyperglycemia[115]

23 Norstatine Intermediate for antitumor agent, ren-nin inhibitors and HIV virus protease-inhibitor [116] Asahi chemicals)

24 Sphingosine Marker for early detection of cancerand have the HIV binding biologicalproperties[117,118]

compounds, which find applications in pharmaceutical, agro-chemical and cosmetic formulations. These enzymes catalyzethe carbon–carbon bond formation in organic synthesis[2,3].Cyanohydrins are biologically active compounds and are ex-cellent synthon as these can be readily converted into a vari-ety of valuable starting materials for synthesis of�-hydroxyketones,�-amino alcohols and�-hydroxy carboxylic acids[4–6]. Cyanohydrin derivatives are essential constituents ofcommercial products e.g. pyrethroid insecticides cyperme-thrin and fluvalinate[6]. In the present article some biochemi-cal characteristics, mechanism of action and possible applica-tions of hydroxynitrile lyases will be reviewed (seeTable 1).

2. Classification of hydroxynitrile lyases

The hydroxynitrile lyases characterized so far fallinto two major groups: HNL I and HNL II based on

the presence or absence of flavin adenine dinucleotide(FAD) [7–9] (Table 2). This cofactor is not involved innet redox reaction but their removal causes inactivation ofthe enzyme[10,11] i.e. it provides structural stability toenzyme structure or it is present as an evolutionary remnant[10,12,13]. The flavin adenine dinucleotide generally bindscovalently to a hydrophobic region near the catalytic siteof enzyme. The FAD containing (R)-(+)-mandelonitrilelyases (MDL-E.C. 4.2.1.10) have been isolated fromplants belonging to Prunoideae and Maloideae subfamiliesof the Rosaceae as major seed storage proteins whichplay an active role in cyanogenesis[14]. FAD lackinghydroxynitrile lyase found inSorghum bicolor[15,16],Manihot esculenta[17],Phlebodium aureum[18] andLinumusitatissimum[19], is less prevalent protein and has diversephysicochemical properties like substrate specificity, mass,glycosylation, isoelectric point, structure and amino acidsequence[7,20].


Table 2Characteristics of hydroxynitrile lyases I and II

Characteristics HNL I HNL II

Cofactor FAD containing FAD lackingGlycosylation N-glycosylated –Chiral aglycon Mandelonitrile Acetone cyanohydrin, (S)-mandelonitrile, (S)-4-hydroxy mandelonitrile,

(R)-2-butanone cyanohydrinsCyanogenic glycosides R-prunasin andR-amygdalin (S)-Dhurrin, (S)-sambunigrin, linamarine, lotaustralineCarbohydrate content 30% 9%Isoforms Presence of Isoenzymes –Homology Oxidoreductases (30%) �/� Hydrolases, serine carboxypeptidases, Zn2+ containing alcohol dehydrogenaseMolecular weight 50–80 kDa 28–42 kDaIsoelectric pH 4.2–4.8 3.9–4.6

3. Distribution of hydroxynitrile lyases and theirfunction in nature

The phenomenon of cyanogenesis (i.e. hydroxynitrilelyase mediated release of cyanide group in the form of HCNon microbial attack) is widely observed in higher plants. Ithas been reported in more than the 3000 species of vascu-lar plant taxa comprising 105 families of flowering plants,pteridophytes (ferns) and gymnosperms. Prominent amongthese are the plants belonging to families like Linaceae, Eu-phorbiaceae, Clusiaceae, Olacaceae, Rosaceae, Gramineae(monocotyledons) and Filitaceae (ferns)[21] Table 3. In ad-dition to plants, the cyanogenesis is also reported in taxonom-ically diverse group of organisms like bacteria (Chromobac-terium violaceum, few species of Pseudomonas)[22–24],fungi, lichen, millipedes (Apheloria corrugata), arthropodsand insects (Zygaena trifollii, a moth). Based on amino acidsequence homology and other characteristics of the HNLs,these are considered to be evolved by convergent evolution(Fig. 1).

Endogenous cyanide containing compounds occurmostly as cyanoglycosides (cyanohydrin stabilized by aglycosidically linked sugar moiety) and cyanolipids in plants[7]. Major function of these compounds in plant systemseems to be defense against herbivoral, fungal attack ormechanical injury[25]. Beside their role in defense mech-anism, cyanogenic glycosides are also utilized as nitrogensource in amino acid anabolism where HCN is refixed by�-cyanoalanine synthetase and catabolized to�-cyanoalaninewith l-cysteine. This�-cyanoalanine is further hydrolyzed tol-asparagine by�-cyanoalanine hydrolase[26–28](Fig. 2).

Sixty different types of cyanogenic glycosides has been re-ported which are derived from amino acid precursors like va-line, isoleucine, leucine, phenylalanine and tyrosine (Fig. 3).

4. Hydroxynitrile lyase reactions

Hydrocyanation of aldehydes and ketones to produce chi-ral cyanohydrin was first carried out by Rosenthaler in 1909

Table 3Sources of hydroxynitrile lyases

Species (family) Cyanogenic glycoside(s) Natural substrate Cofactor Sequence homology

Prunus serotina—PsHNL (Rosaceae)[104] Amygdalin, prunasin (R)-mandelonitrile FAD Flavoprotein[39] 30%P asinP asinPP asinMMPL stralin es

H

S)

S ses

M stralin

XS

runus lyonii—PlyHNL (Rosaceae)[14] Amygdalin, prunrunus laurocerasus—PlHNL (Rosaceae)[106] Amygdalin, prunrunus capuli—PcHNL (Rosaceae)[105] –runus amygdalus—PaHNL (Rosaceae)[54] Amygdalin, prunammea Americana—MaHNL (Clusiaceae)[105]alus communis—McHNL (Rosaceae)[54]hlebodium aureum—PhaHNL (Filitaceae)[18] (R)-Vicianininum usitatissimum—LuHNL (Linaceae)[19] Linamarin, lotau

evea brasiliensis—HbHNL (Euphorbiaceae)[56] Linamarin

orghum bicolour—SbHNL (Gramineae)[52,16] Dhurrin

orghum vulgare—SvHNL (Gramineae)[16] Dhurrin

anihot esculenta—MeHNL (Euphorbiaceae)[17,38] Linamarin, lotau

imenia Americana—XaHNL (Olacaceae)[88] Linamarinambucus nigra—SnHNL (Caprifoliaceae)[19] –

(R)-mandelonitrile FAD –(R)-mandelonitrile FAD –

(R)-mandelonitrile FAD –(R)-mandelonitrile FAD Cholesterol oxidase[26]

(R)-mandelonitrile FAD –(R)-mandelonitrile FAD –(R)-mandelonitrile FAD –Acetone cyanohydrin,

(R)-2-butanone cyanohydrinNone Alcohol de-hydrogenas

(40%)Acetone cyanohydrin None Rice protein (unknown

function)[109](S)-4-hydroxymandelonitrile None Wheat Serine

carboxypeptidases (60%[93]

(S)-4-hydroxymandelonitrile None Serine carboxy-peptida[93]

Acetone cyanohydrin,(R)-2-butanone cyanohydrin

None Rice protein (unknownfunction)[107]

(S)-mandelonitrile None –(S)-mandelonitrile None –


Fig. 1. Phylogenetic tree of hydroxynitrile lyases. Sources of HNLs: LuHNL—Linum usitatissimum, HbHNL—Hevea brasiliensis, MaHNL—Mammeaamericana, MeHNL—Manihot esculenta, XaHNL—Ximenia americana, SbHNL—Sorghum bicolor, SvHNL—Sorghum vulgare, SnHNL—Sambucusnigra, PhaHNL—Phlebodium aureum, PlyHNL—Prunus lyonii, PaHNL—Prunus amygdalus, PlHNL—Prunus laurocerasus, PcHNL—Prunus capuli,PsHNL—Prunus serotinal.

using hydroxynitrile lyases from almond[29]. It catalyzesthe asymmetric condensation of HCN with aldehydes or ke-tones to yield cyanohydrins[30–32](Fig. 4). Almond is mostcommon source of (R)-hydroxynitrile lyase. Defatted almondmeal is used as an inexpensive catalyst and does not requireimmobilization as enzyme is naturally immobilized in themeal matrix that also provide stability to it[33–35]. (S)-HNLis not easily available and thus can be cloned and over ex-pressed in microbial expression system[32,36–41].

Enantiomeric or specific synthesis of cyanohydrin isinfluenced by reaction medium, cyanide source, watercontent, buffer pH and temperature during the hydroxynitrilelyase catalyzed reaction. Increase in enantiomeric excessof the product can be obtained by carrying out reaction in

aqueous medium and at low pH, however, at low pH thestability of enzyme is affected significantly and using excessenzyme in the reaction can circumvent this problem[42,43].Alternatively, the use of HNL in biphasic system providesmany advantages like increased substrate concentration(1–1.5 M), high product yield, easy down stream process-ing as enzyme remain partitioned to one of the phases[43–45].

Most solvent used in biphasic reactions are ethyl acetate,diisopropyl and other ethers[46–55]. Enzymes in diisopropylether (DIPE)/water interface is more active than in ethylacetate. Studies have shown that while using biphasic systemenzyme activity was at interface rather than in one phase ofsolvent system[56–58]. After complete reaction in biphasic

ic glyc
Fig. 2. HCN released due to catabolism of cyanogen oside can be used as nitrogen source for amino acid synthesis.


Fig. 3. Major cyanogenic glycosides that differs in side chains.

system the aqueous phase having enzyme can be removedby simple phase separation step and can be reused, however,biphasic systems have few disadvantages like enzymeactivity is highly affected by the solvent hydrophobicity[42]. In the absence of moisture or water, catalytic activity ofenzyme reduces to zero, while in excess of water significantchemical background reaction occurs[59].

Hydrogen cyanide is the most preferred cyanide source incyanohydrin synthesis[35,46,48,50,60–62]. Besides HCN,several different cyanide sources like potassium cyanide arebeing used in biotransformation. It acts as an effective source

Fig. 4. General reaction catalyzed by hydroxynitrile lyase.

of cyanide in the low pH range of aqueous buffer[63–65].The spontaneous addition of HCN will result in reduction ofenantiomeric purity of the product because at high concen-tration HCN becomes insoluble leading to its instantaneousrelease from reaction mixture. Alternatively, the additionof hydrogen cyanide in the reaction can be replaced by itsindirect generation by addition of the acid to the aqueous so-lution of alkali cyanide in transhydrocyanation process. Thisslow diffusion of HCN gives advantage over spontaneousaddition and results in high enantiomeric purity and yield[53,54,66].

Temperature has profound effect on the course ofreaction. Enzyme stability as well as enantiomeric productyield is enhanced at low temperature−5 to 4◦C [57].However, further decrease in temperature (i.e. below−5◦C)freezes the water present in reaction mixture, which leads tocomplete loss of enzyme activity. The enantiomeric excessof product decreases significantly in aqueous/alcoholicbuffer system because of the background chemical reactions.Engineering of reaction conditions of hydroxynitrile lyasemediated organic synthesis can minimize the backgroundnon-enzymatic reaction and will further improve yield andenantiomeric purity of the product.


The enantiomeric purity of the cyanohydrin or derivativesproduced through HNL mediated reactions is routinelydetermined by using chiral HPLC[67,68], chiral GC[49,51,64,69,70–72], HPLC[54], GC[48,49,63,73–75]andNMR [33,34,73,75,76]techniques. Thin layer chromatogra-phy involving KMnO4 dip is most convenient technique forthe detection of cyanohydrins in the reaction mixture[77].NMR is also used to confirm the desired product formed byhydroxynitrile lyase.

Many workers have also carried out immobilizationof hydroxynitrile lyases on different matrices like ECTE-OLA cellulose (ion exchanger)[74,78,79], DEAE cellulose,sepharose 4B, pore glass beads[74], silica gel[80], micro-crystalline cellulose (Avicel)[49,74], eupergil C, nitrocellu-lose [50], celite, liquid crystals and PVAL hydrogels[81].Recently CLEA (cross linked enzyme aggregate) technologyhas emerged for immobilization of hydroxynitrile lyases[82].

5. Substrate specificity of hydroxynitrile lyases

Hydroxynitrile lyase differs in their stereospecificity andsubstrate affinity. PaHNL has broad substrate specificity andapplicability [83]. Kyler explored the active site dimen-sion of this enzyme with different substrates[84]. Brussee[ tea hicha iliza-t sa en-g ulkya ce ofa thism

s anc ge ofs nyls

and ketones. XaHNL has limited occurrence in nature andit has fairly good affinity for aromatic aldehydes[88]. Newsources of hydroxynitrile lyases like PhaHNL catalyse aro-matic and heterocyclic carbonyls rather than aliphatic sub-strates and find use in selected applications[17].

6. Biochemical and molecular properties ofhydroxynitrile lyases

Hydroxynitrile lyases from about 15 sources have beenreported so far and few of them are fully characterized whilesearch for novel hydroxynitrile lyases is going on. TheseHNLs have different conformations, molecular weights, pHoptima, pI values, FAD requirements and kinetic properties(Table 4).

Structures of catalytic site of hydroxynitrile lyases havebeen explored and studied by chemical mutagenesis and X-ray crystallography. HbHNL was the first enzyme whose3D crystal structure was elucidated followed by other HNLs[36,89]. The X-ray diffraction studies have been carried outwith crystal of PaHNL and MeHNL[88]. Enzymes are ofwidely different phylogenetic origin like subtilisin, cysteineproteases, eucaryotic serine proteases and�/� hydrolases andshare the common structural motif of eight� sheets connectedby � helices beside similar protein folds[90,91]. HbHNLb ieda t bya

rox-y rs ofG mily( ep ues,w 9%).O cor-r onei aturep

TB

H s

MHPSSSXPPPPPMML

R

33–35,62,85]and Kanerva[52,86] studied the substraffinity using crude almond extract and almond bran, wre cheaper sources of enzyme and do not require immob

ion. (S)-hydroxynitrile lyase enzyme ofHevea brasiliensindManihot esculentahave been altered through proteinineering or site directed mutagenesis by substituting bmino acids (e.g. tryptophan) which constrict the entranctive sites with less bulky amino acids like alanine andade the enzyme to accept large size of substrates[87].LuHNL is a less availableR-selective enzyme and ha

arrow substrate range[41,83] while HbHNL and MeHNLatalyse the synthesis of cyanohydrins from a wide ranubstrate like aliphatic, aromatic, heteroaromatic carbo

able 4iochemical properties of hydroxynitrile lyases

NLs Mol. wt. (kDa) KM

eHNL 30 110 mM 101R mMbHNL 30 3.4 mMhaHNL 20 0.85 mMbHNL 38, 18 0.8�MvHNL 38, 18 –nHNL – –aHNL 36.5 0.53 mMlyHNL 59 93�MaHNL 75 0.59 mMsHNL 60 0.17�MlHNL 60 –cHNL – –aHNL – –cHNL 75–84 –uHNL 42 2.5 , 1.9R

—recombinant enzyme.

elongs to�/� hydrolase super family having deeply burctive site within protein and linked to outer environmennarrow channel flanked by apolar residues.The amino acid sequence of FAD containing hyd

nitrile lyases showed that they are related to membelucose–methanol–choline (GMC) oxidoreductase fa

30% sequence identity)[59]. Hydroxynitrile lyases are throteins of size ranging from 250 to 664 amino acid residhich share a number of highly conserved regions (>8ne of these regions, located in the N-terminal section,

esponds to the FAD ADP-binding domain, while others located in the center of the enzyme protein as a signattern[92].

VMax pH Isoelectric point

124 138R 3.5–5.4 4.4, 4.1, 4.60.058 mM/min 5.5–6.0 –– 6.5

– 5.5 –– 5.0–5.5 –– 5.0–5.5 –1031 IU 5.5 –

450× 103 5.5 4.7537800 n/mM 5.5–6.0 –

– 6.0–7.0 4.58–4.63– 5.5–6.0 4.2–4.4– 5.5 –– 5.5 –– 5.5 –

71R 5.5 4.70–4.85


Topology of HbHNL differs slightly from�/� hydrolasefamily. It lacks first two� sheets and has a continued capregion where lies catalytic active site[90]. A study on molec-ular cloning of SbHNL gene has shown that it lacks intronsand has a stretch of 23 A (adenine) bases which suggests thatit might have evolved by a reverse transcription event duringevolution from serine carboxypeptidase. It has 11 stranded� sheets surrounded by more than a dozen� helices[93].

PaHNL has 4N-glycosylation sites at Asn118, Asn135,Asn352, Asn392 and Asn-X-Ser/Thr on the consensus se-quence[59].N-glycosylation constituents are Man3GluNAc2(Mannose-N-acetyl glucosamine). PaHNL has two domainsone for FAD binding and other for substrate binding. TheFAD cofactor is completely isolated from solvent and isdeeply buried within the enzyme molecule. The FAD bindingdomain mainly has� and� sheets and surrounded by six�helices (H1, H2, H3, H4, H5, H6 and H9) and two 310 typehelices (a protein secondary structure containing three aminoacid residues per turn and hydrogen bonds in 10 atom rings),� meander (single sheet formed by� hairpin repeats) and�� motif in central parallel sheet and the later is responsiblefor FAD binding. The substrate-binding site is characterizedby 6-stranded� sheets (C0–C5), surrounded by helices H4,H7 and H8 and one 310 type helix. PaHNL has 24% homol-ogy to glucose oxidase (GOX) ofPenicillium amagasakienseand cholestrol oxidase (CHOX) ofStreptomycessp.[59].

LuHNL has an ADP binding�� domain and cat-alytic domain containing two Zn2+, which are not directlyinvolved in catalysis [19]. Some hydroxynitrile lyasesrequire post-translational modifications for activity e.g.(R)-hydroxynitrile lyase and (S)-hydroxynitrile lyase ofS.bicolorare highly glycosylated and require post-translationalproteolytic processing for their activity[36]. Hydroxynitrilelyase ofPrunus serotinais present in isoforms and thismultiplicity is either due to allelic differences at the struc-tural loci or due to gene duplication and post-translationalmodifications ultimately resulting in isoenzyme forms[7].This heterogeneity in genes provides organisms specificity,regulation and differential expression.

7. Recombinant hydroxynitrile lyases

The hydroxynitrile lyases of some plants have beencloned in microbial systems since the latter can be easilytransformed and cultured in a short time for large-scaleproduction. HbHNL gene has been over expressed inEscherichia coliusing strong inducibletacpromoter and thegene product formed inclusion bodies, which were insolubleand inactive[36,37,43]. The most successful heterologousexpression of HbHNL was achieved inSaccharomyces cere-v

Fig. 5. Reaction mechanism of hydroxyn

isiae and methanol induciblePichia pastorisexpression

itrile lyase ofHevea brasiliensis[95].


Fig. 6. Reaction mechanism of hydroxynitrile lyase ofPrunus amygdalus[99].

system in soluble and highly active form comprising about60% of total protein of the cell[36,50]. Likewise MeHNLwas over expressed inE. coli and 40 l scale fermentationgave 40000 IU of hydroxynitrile lyase for (S)-cyanohydrinsynthesis[38,39]. LuHNL has been cloned and expressed inE. colias well as inP. pastoris[19,41].

A recombinantP. pastoriscontaining HbHNL gene wascultured using high cell density culture technique in a 30 l lab-oratory fermenter, which yielded 23 g/l HbHNL and was pu-rified by one-step ion exchange chromatography[36]. RocheDiagnostics, Penzberg, Germany has produced HNLs up to50,000 l fermenter scale with approximately similar yield aslaboratory scale and used for the synthesis of (S)-cyanohydrinof 3-phenoxybenzaldehyde, which is an important intermedi-ate for synthetic pyrethroid. SbHNL recombinant expressionin prokaryotic system failed since it required complexposttranslational modification of the native enzyme[15].

8. Mechanism of reaction

Inhibition of hydroxynitrile lyases by histidine and serinemodifying reagents suggest the involvement of different

amino acids in active site. Inhibition studies with nativestructure of HbHNL exhibited the involvement of three watermolecules at the reaction site. The fully conserved watermolecule is hydrogen bonded to histidine 235-carbonyloxygen and side chain of glutaric acid 79 and histidine 103[94]. Catalytic reactions in HbHNL proceed by nucleophiledisplacement. HbHNL uses the catalytic triad of serine-80,histidine 235 and aspartate 207 residues. The centralhistidine acts as an active species and increases the basicityof serine 80 resulting in the abstraction of a proton fromthe hydroxyl group of cyanohydrin. Proposed catalyticmechanism showed that lysine 236 plays a critical role incatalytic action at low pH values by stabilizing the negativecharge of cyanide ion, which is present in direct contact withcyanide moiety of the substrate[89,95–97](Fig. 5).

NMR spectroscopy has shown that there is a short dis-tance between aspartate-207 and histidine 235, which formstrong hydrogen bond with competitive inhibitors that causesignificant downfield shift. Though the structure of HbHNLand MeHNL are super imposable, in MeHNL lysine 236 doesnot participate in catalysis[98].

FAD dependent PaHNL relies on general acid /base catal-ysis by imidazole of a conserved catalytic histidine 496[37].

ydroxy
Fig. 7. Reaction mechanism of h nitrile lyase ofSorghum bicolor[100].


Fig. 8. Reaction mechanism of hydroxynitrile lyase ofManihot esculenta[101].

Histidine 496 acts as a base and deprotonates the hydroxylgroup of the substrate[6]. Negativity of cyanide ion is neu-tilized by pronounced positive electrostatic potential at theactive site by two positively charged residues arginine 300and lysine 361. PaHNL binds FAD cofactor in its oxidizedform. The reversal of electrostatic potential upon FAD re-moval or modification makes the complex unstable at activesite leading to inactivation of enzyme[99] (Fig. 6).

During catalysis in SbHNL hydrogen bonds are formedbetween substrate hydroxyl group, serine 158 and oxygenatom of tryptophan 270-carboxyl group. A water molecule issuspended between other carboxylate group of tryptophan-

270 and nitrile group of the substrate. Here the carboxylatemoiety of tryptophan acts as catalytic base causing abstrac-tion of proton from cyanohydrin hydroxyl group, while wa-ter at active site transfers this proton to cyanide resulting incarbon–carbon bond cleavage[100] (Fig. 7).

In MeHNL serine was reported to play a pivotal role incatalytic nucleophile addition reactions i.e. different residuesinvolved in the catalysis are joined by hydrogen bonding e.g.serine-80[101,102]forms hydrogen bond to imidazole nitro-gen of histidine-236, which is further stabilized by hydrogenbonding with aspartate. Serine-80 functions as a strong baseresulting in the formation of negatively charged oxyanionhole. This is further stabilized by an oxygen hole formedby the amide nitrogen of serine-80 and glycine-78 backbone,which is present as a consensus motif. Due to this stabilizationthere is an increase in positive charge on alpha carbon of hy-droxynitrile resulting in cyanide group release[16,101,102](Fig. 8).

In hydroxynitrile lyases ofP. serotina,P. lyonii,X. ameri-cana,L.usitatissimumandS.vulgarecysteine was found to beinvolved at the active site[89]. During catalysis the substrateand product access the active site in a sequential manner e.g.PaHNL follows uni-bi mechanism in which aldehydes bindsfirst to active site followed by HCN. Inhibition studies showedthat cyanide ion and its analogues act as inhibitors of enzymealdehydes complex, and this suggests that during cyanogen-e thanf ea es at
Fig. 9. Cyanide group transformations.
sis HCN binds to enzyme aldehydes complex ratherree cyanide ion[71]. Generally hydroxynitrile lyases utilizcid/base catalysis mechanism. The amino acid residu


Fig. 10. Transformation of hydroxyl group.

Fig. 11. Transformation of O-protected cyanohydrins.


active site of these enzymes differ significantly but share thecommon motif for cyanogenesis.

9. Chemo enzymatic transformation of cyanohydrins

Cyanohydrins can be synthesized using hydroxynitrilelyases, which offer high enantioselectivity, broad substratevariability and high yield in the synthesis of cyanohydrins ortheir derivatives. Chiral cyanohydrins are promising versa-tile building blocks since they can be used to create tailoredmolecules and have large number of applications in chemicalindustries. These cyanohydrins either serve as intermediatesubstrates or precursors that can further be converted intovaluable products. Cyanohydrins so produced can be trans-formed into variety of products using chemical reactions like(a) reaction of cyano group[51,55,103,104](solvolysis, grig-nard reaction and hydride addition) (Fig. 9) (b) reaction ofhydroxyl group[33] (c) reaction ofR substituents and (d)conversion of OH group to a good leaving group to allow nu-cleophilic displacement with inversion of configuration[93].Cyanohydrins and a range of their structural variants havewide applications in pharmaceutical industry[35,60,68,86].Alpha-sulfonyloxynitriles undergo SN2 reactions to give va-riety of products (Fig. 10).

The protective groups provide cyanohydrins stabilitya e in-c ions[ , es-t F)e drinsa ma-t s ofs

1

nics Mosto ys-t inl ondi-t haves sedf e ap andd e hy-d elec-t lingc tratea en-z buta fortst r thes

Acknowledgements

We thank the Council of Scientific and Industrial Research(CSIR), New Delhi, India, for providing financial assistancein the form of Junior Research Fellowship to Ms. MonicaSharma and Mr. Nitya Nand Sharma.

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