DB code: S00358

RLCP classification	5.191.546000.23 : Elimination
CATH domain	3.40.50.1820 : Rossmann fold	Catalytic domain
E.C.	4.1.2.47
CSA	1dwo 1qj4
M-CSA	1dwo 1qj4
MACiE

CATH domain	Related DB codes (homologues)
3.40.50.1820 : Rossmann fold	S00544 S00344 S00517 S00525 S00526 S00720 S00723 S00724 S00725 S00919 S00057 S00374 S00345 S00347 S00348 S00346 S00350 S00352 S00353 S00355 S00356 D00189 D00210 D00539 T00253

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	Pfam
P52704	Hydroxynitrilase	EC 4.1.2.37 EC 4.1.2.37) ((S)-acetone-cyanohydrin lyase S)-acetone-cyanohydrin lyase) ((S)-hydroxynitrile lyase Oxynitrilase	[Graphical View]
P52705	Hydroxynitrilase	EC 4.1.2.37 EC 4.1.2.37) ((S)-acetone-cyanohydrin lyase S)-acetone-cyanohydrin lyase) ((S)-hydroxynitrile lyase Oxynitrilase	PF00561 (Abhydrolase_1) [Graphical View]

KEGG enzyme name
(S)-hydroxynitrile lyase (S)-cyanohydrin producing hydroxynitrile lyase (S)-oxynitrilase (S)-HbHNL (S)-MeHNL hydroxynitrile lyase oxynitrilase HbHNL MeHNL (S)-selective hydroxynitrile lyase (S)-cyanohydrin carbonyl-lyase (cyanide forming)

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P52704	HNL_HEVBR	An aliphatic (S)-hydroxynitrile = cyanide + an aliphatic aldehyde or ketone. An aromatic (S)-hydroxynitrile = cyanide + an aromatic aldehyde.	Homodimer.
P52705	HNL_MANES	An aliphatic (S)-hydroxynitrile = cyanide + an aliphatic aldehyde or ketone. An aromatic (S)-hydroxynitrile = cyanide + an aromatic aldehyde.	Homotrimer.

KEGG Pathways
Map code	Pathways	E.C.
MAP00460	Cyanoamino acid metabolism

Compound table
	Substrates	Products			Intermediates
KEGG-id	C18797	C00177	C00071	C01450
E.C.
Compound	(S)-Hydroxynitrile	cyanide	Aldehyde	Ketone
Type	carbohydrate	organic ion	carbohydrate	carbohydrate
ChEBI		17514 17514
PubChem		5975 5975

1dwoA	Unbound	Unbound	Unbound	Bound:ACN
1dwoB	Unbound	Unbound	Unbound	Unbound
1dwpA	Unbound	Unbound	Unbound	Analogue:ACT
1dwpB	Unbound	Unbound	Unbound	Analogue:ACT
1dwqA	Unbound	Unbound	Unbound	Bound:ATO
1dwqB	Unbound	Unbound	Unbound	Unbound
1e89A	Unbound	Unbound	Unbound	Unbound
1e89B	Unbound	Unbound	Unbound	Unbound
1e8dA	Bound:CNH	Unbound	Unbound	Unbound
1e8dB	Bound:CNH	Unbound	Unbound	Unbound
1eb8A	Unbound	Unbound	Unbound	Unbound
1eb8B	Unbound	Unbound	Unbound	Unbound
1eb9A	Unbound	Unbound	Bound:HBA	Unbound
1eb9B	Unbound	Unbound	Bound:HBA	Unbound
1qj4A	Unbound	Unbound	Unbound	Unbound
1yasA	Unbound	Unbound	Unbound	Analogue:HIS
2yasA	Unbound	Analogue:SCN	Unbound	Unbound
3yasA	Unbound	Unbound	Unbound	Bound:ACN
4yasA	Unbound	Unbound	Unbound	Unbound
5yasA	Unbound	Unbound	Unbound	Analogue:FAC
6yasA	Unbound	Unbound	Unbound	Unbound
7yasA	Unbound	Unbound	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
Swiss-prot;P52704, P52705 & literature [8], [11] & [12]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1dwoA	THR 11;SER 80;ASP 208;HIS 236
1dwoB	THR 11;SER 80;ASP 208;HIS 236
1dwpA	THR 11;SER 80;ASP 208;HIS 236
1dwpB	THR 11;SER 80;ASP 208;HIS 236
1dwqA	THR 11;SER 80;ASP 208;HIS 236		CSA 81(acetonylation)
1dwqB	THR 11;SER 80;ASP 208;HIS 236		CSA 81(acetonylation)
1e89A	THR 11; ;ASP 208;HIS 236				mutant S80A
1e89B	THR 11; ;ASP 208;HIS 236				mutant S80A
1e8dA	THR 11; ;ASP 208;HIS 236				mutant S80A
1e8dB	THR 11; ;ASP 208;HIS 236				mutant S80A
1eb8A	THR 11;SER 80;ASP 208;HIS 236
1eb8B	THR 11;SER 80;ASP 208;HIS 236
1eb9A	THR 11;SER 80;ASP 208;HIS 236
1eb9B	THR 11;SER 80;ASP 208;HIS 236
1qj4A	THR 11;SER 80;ASP 207;HIS 235;LYS 236
1yasA	THR 11;SER 80;ASP 207;HIS 235;LYS 236
2yasA	THR 11;SER 80;ASP 207;HIS 235;LYS 236
3yasA	THR 11;SER 80;ASP 207;HIS 235;LYS 236
4yasA	THR 11;SER 80;ASP 207;HIS 235;LYS 236
5yasA	THR 11;SER 80;ASP 207;HIS 235;LYS 236
6yasA	THR 11;SER 80;ASP 207;HIS 235;LYS 236
7yasA	THR 11;SER 80;ASP 207;HIS 235;LYS 236

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[1]	Fig.2	3
[2]	Fig.8, p.813-819	3
[3]	Fig.4, p.25833	3
[5]	Fig. 6, p.447-448	3
[6]	Scheme 3, p.186, p.193	5
[7]	Fig.2, p.997-998	4
[8]	Fig.6, p.1997	2
[11]	Fig.5, p.199	3
[12]	Fig.2, p.1019-1020	3
[13]	Fig.5, p.295-298	1

References
[1]
Resource
Comments
Medline ID
PubMed ID	8958122
Journal	Ann N Y Acad Sci
Year	1996
Volume	799
Pages	707-12
Authors	Hasslacher M, Schall M, Hayn M, Griengl H, Kohlwein SD, Schwab H
Title	(S)-hydroxynitrile lyase from Hevea brasiliensis.
Related PDB
Related UniProtKB
[2]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (1.9 ANGSTROMS).
Medline ID	96434327
PubMed ID	8805565
Journal	Structure
Year	1996
Volume	4
Pages	811-22
Authors	Wagner UG, Hasslacher M, Griengl H, Schwab H, Kratky C
Title	Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis.
Related PDB	1yas
Related UniProtKB	P52704
[3]
Resource
Comments
Medline ID
PubMed ID	8824213
Journal	J Biol Chem
Year	1996
Volume	271
Pages	25830-4
Authors	Wajant H, Pfizenmaier K
Title	Identification of potential active-site residues in the hydroxynitrile lyase from Manihot esculenta by site-directed mutagenesis.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID	9325140
Journal	Protein Expr Purif
Year	1997
Volume	11
Pages	61-71
Authors	Hasslacher M, Schall M, Hayn M, Bona R, Rumbold K, Luckl J, Griengl H, Kohlwein SD, Schwab H
Title	High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID	9094745
Journal	Proteins
Year	1997
Volume	27
Pages	438-49
Authors	Hasslacher M, Kratky C, Griengl H, Schwab H, Kohlwein SD
Title	Hydroxynitrile lyase from Hevea brasiliensis: molecular characterization and mechanism of enzyme catalysis.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID	10407140
Journal	Biochim Biophys Acta
Year	1999
Volume	1432
Pages	185-93
Authors	Hanefeld U, Straathof AJ, Heijnen JJ
Title	Study of the (S)-hydroxynitrile lyase from Hevea brasiliensis: mechanistic implications.
Related PDB
Related UniProtKB
[7]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (1.1 ANGSTROMS)
Medline ID	99423043
PubMed ID	10494852
Journal	Biol Chem
Year	1999
Volume	380
Pages	993-1000
Authors	Gruber K, Gugganig M, Wagner UG, Kratky C
Title	Atomic resolution crystal structure of hydroxynitrile lyase from Hevea brasiliensis.
Related PDB	1qj4
Related UniProtKB	P52704
[8]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (1.72 ANGSTROMS).
Medline ID	20014021
PubMed ID	10548044
Journal	Protein Sci
Year	1999
Volume	8
Pages	1990-2000
Authors	Zuegg J, Gruber K, Gugganig M, Wagner UG, Kratky C
Title	Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.
Related PDB	2yas 3yas 4yas 5yas 6yas 7yas
Related UniProtKB	P52704
[9]
Resource
Comments
Medline ID
PubMed ID	11102786
Journal	Curr Opin Biotechnol
Year	2000
Volume	11
Pages	532-9
Authors	Effenberger F, Forster S, Wajant H
Title	Hydroxynitrile lyases in stereoselective catalysis.
Related PDB
Related UniProtKB
[10]
Resource
Comments
Medline ID
PubMed ID	10679367
Journal	Curr Opin Chem Biol
Year	2000
Volume	4
Pages	103-9
Authors	Johnson DV, Zabelinskaja-Mackova AA, Griengl H
Title	Oxynitrilases for asymmetric C-C bond formation.
Related PDB
Related UniProtKB
[11]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	11173464
Journal	Acta Crystallogr D Biol Crystallogr
Year	2001
Volume	57
Pages	194-200
Authors	Lauble H, Forster S, Miehlich B, Wajant H, Effenberger F
Title	Structure of hydroxynitrile lyase from Manihot esculenta in complex with substrates acetone and chloroacetone: implications for the mechanism of cyanogenesis.
Related PDB	1dwo 1dwp 1dwq
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID	11316882
Journal	Protein Sci
Year	2001
Volume	10
Pages	1015-22
Authors	Lauble H, Miehlich B, Forster S, Wajant H, Effenberger F
Title	Mechanistic aspects of cyanogenesis from active-site mutant Ser80Ala of hydroxynitrile lyase from Manihot esculenta in complex with acetone cyanohydrin.
Related PDB	1e89 1e8d
Related UniProtKB
[13]
Resource
Comments
Medline ID
PubMed ID	11790839
Journal	Protein Sci
Year	2002
Volume	11
Pages	292-300
Authors	Dreveny I, Kratky C, Gruber K
Title	The active site of hydroxynitrile lyase from Prunus amygdalus: modeling studies provide new insights into the mechanism of cyanogenesis.
Related PDB
Related UniProtKB
[14]
Resource
Comments
Medline ID
PubMed ID	11742123
Journal	Protein Sci
Year	2002
Volume	11
Pages	65-71
Authors	Lauble H, Miehlich B, Forster S, Kobler C, Wajant H, Effenberger F
Title	Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta.
Related PDB	1eb8 1eb9
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID	12889812
Journal	Biotechnol Lett
Year	2003
Volume	25
Pages	1041-7
Authors	Yan G, Cheng S, Zhao G, Wu S, Liu Y, Sun W
Title	A single residual replacement improves the folding and stability of recombinant cassava hydroxynitrile lyase in E. coil.
Related PDB
Related UniProtKB

Comments
This enzyme belongs to the alpha/beta hydrolase superfamily, which has a catalytic triad (Ser/Asp/His). The earlier literature ([1], [2] & [5]) proposed a catalytic mechanism, in which Ser80 acts as a nucleophilic residue to form a tetrahedral intermediate with substrate, as that in serine proteases. However, more recent papers ([3], [6], [7], [8], [11] & [12]) proposed an alternative mechanism, in which catalytic residues act as acid/base or stabilizers. This enzyme catalyzes a reversible reaction. In the forward direction, cyanohydrin will be degradaded into acetone and cyanide, accompanied by elimination and double-bond formation, whilst cyanohydrin will be synthesized from acetone and cyanide, by addition to the double-bond, in the reverse direction. The forward reaction (elimination and double-bond formation reaction) proceeds as follows (see ([3], [6], [7], [8], [11] & [12]): (1) Ser80, modulated by His235, acts as a general base, to abstract a proton from cyanohydrin OH-group, leading to the elimination of the cyanide (or nitrile). During this reaction, Thr11 stabilizes the oxygen atom of the OH-group, whilst His235 and Lys236 (PDB; 1qj4; enzyme from Havea brasiliensis) stabilize the eliminated group, cyanide (or nitrile). (2) His235 acts as a general acid, to donate a proton to the eliminated group, the cyanide ion. In the enzyme from Manihot esculenta, however, the corresponding lysine residue, Lys237, does not seem to be involved in the stabilization, according to the literature [11] & [12]. The reverse reaction (addition to double-bond) adopts an analogous mechanism, using the same catalytic residues.

Comments

This enzyme belongs to the alpha/beta hydrolase superfamily, which has a catalytic triad (Ser/Asp/His).
The earlier literature ([1], [2] & [5]) proposed a catalytic mechanism, in which Ser80 acts as a nucleophilic residue to form a tetrahedral intermediate with substrate, as that in serine proteases.
However, more recent papers ([3], [6], [7], [8], [11] & [12]) proposed an alternative mechanism, in which catalytic residues act as acid/base or stabilizers.
This enzyme catalyzes a reversible reaction. In the forward direction, cyanohydrin will be degradaded into acetone and cyanide, accompanied by elimination and double-bond formation, whilst cyanohydrin will be synthesized from acetone and cyanide, by addition to the double-bond, in the reverse direction.
The forward reaction (elimination and double-bond formation reaction) proceeds as follows (see ([3], [6], [7], [8], [11] & [12]):
(1) Ser80, modulated by His235, acts as a general base, to abstract a proton from cyanohydrin OH-group, leading to the elimination of the cyanide (or nitrile). During this reaction, Thr11 stabilizes the oxygen atom of the OH-group, whilst His235 and Lys236 (PDB; 1qj4; enzyme from Havea brasiliensis) stabilize the eliminated group, cyanide (or nitrile).
(2) His235 acts as a general acid, to donate a proton to the eliminated group, the cyanide ion.
In the enzyme from Manihot esculenta, however, the corresponding lysine residue, Lys237, does not seem to be involved in the stabilization, according to the literature [11] & [12].
The reverse reaction (addition to double-bond) adopts an analogous mechanism, using the same catalytic residues.

Created	Updated
2004-04-04	2012-10-03