Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis

Wagner, U.G.; Hasslacher, Meinhard; Griengl, Herfried; Schwab, Helmut; Kratky, Christoph

doi:10.1016/s0969-2126(96)00088-3

Cited by 103 publications

(87 citation statements)

References 39 publications

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“…However, high amounts of 2-mercapoethanol, which would lead to the dissociation of such a dimer into both subunits [23], did not affect the activity of PNAE. The dimerization obviously does not depend on cysteine residues, but most probably takes place via a contact area of apolar residues [12], as the sequence comparison between the HbHNL and PNAE gives a pronounced homology of 75% (Fig. 3) at these apolar sections.…”

Section: Discussionmentioning

confidence: 96%

“…Mechanistic aspects of the reaction catalyzed were investigated by site-directed mutagenesis, replacing the amino acids of the putative catalytic triad and individual cysteine residues by alanine in order to characterize the mutant enzymes and to localize essential cysteines. Modelling experiments of PNAE based on the X-ray data of HNL from H. brasiliensis [11,12] are described, allowing for the first time a deeper insight into a particular step of Rauvolfia alkaloid biosynthesis. …”

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confidence: 99%

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Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Mattern-Dogru¹,

Ma²,

Hartmann³

et al. 2002

European Journal of Biochemistry

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In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epivellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the a/b hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By sitedirected mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the a/b hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the a/b hydrolase enzyme superfamily.Keywords: polyneuridine aldehyde esterase; active-site residues; site-directed mutagenesis; modelling; a/b hydrolase enzyme superfamily.Polyneuridine aldehyde esterase (PNAE, EC 3.1.1) is a central enzyme in a 10-step biosynthetic pathway expressed in the medicinal plant Rauvolfia serpentina Benth. ex Kurz. The pathway delivers the antiarrhythmic monoterpenoid indole alkaloid ajmaline [1,2]. From the enzymatic point of view, this is presently one of the most detailed investigated examples of a pathway leading to a natural product. Reactions of this complex biosynthetic sequence are catalyzed by membrane-bound and soluble enzymes such as cytochrome P450-dependent hydroxylases, a synthase, several reductases, a methyltransferase and a set of hydrolases including a b-glucosidase, an acetylesterase and the herein described methylesterase PNAE [3]. These enzymes exhibit a high degree of substrate specificity, PNAE being the most specific. PNAE is located in the middle of the biosynthetic chain starting from tryptamine and secologanin and catalyses the conversion of polyneuridine aldehyde into the next stable pathway intermediate, epivellosimine (Fig. 1). The enzyme has been detected in and partially characterized from cell suspension cultures of R. serpentina [4,5]. PNAE has also been highly enriched from cultured plant cells and the cDNA was recently functionally expressed in Escherichia coli [6]. Sequence alignment studies showed highest homologies to an esterase involved in pathogen defence in rice [7] that hydrolyses naphthol esters, and to hydroxynitrile lyases (HNLs) from Hevea brasiliensis [8] and Manihot esculenta [9]. The alignment suggested that PNAE is a new member of the a/b hydrolase superfamily that contains the putative catalytic triad serine, aspartic acid and histidine [10].For the present communication, a more detailed characterization of PNAE was performed by testing the influence of various inhibitors, HNL substrates and by structure elucid...

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Section: Discussionmentioning

confidence: 96%

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confidence: 99%

Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Mattern-Dogru¹,

Ma²,

Hartmann³

et al. 2002

European Journal of Biochemistry

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“…The~S!-HNLs from Manihot esculenta~Euphoribiacae; Wajant & Pfizenmaier, 1996! and Hevea brasiliensis~Euphoribi-acae;Hasslacher et al, 1996aHasslacher et al, , 1996bHasslacher et al, , 1997aWagner et al, 1996! belong to the a0b hydrolase superfamily. It appears that nature has invented oxynitrilases at least twice, once derived from FADdependent oxidoreductases, and once from a0b hydrolases~Ollis et al., 1992;Cygler et al, 1993!. The~S!-HNL from H. brasiliensis~Hb-HNL!…”

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confidence: 99%

Three‐dimensional structures of enzyme‐substrate complexes of the hydroxynitrile lyase from hevea brasiliensis

et al. 1999

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The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL! and several substrate and0or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Og. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.Keywords: biocatalysis; cyanohydrin formation; enzyme mechanism; hydroxynitrile lyase; oxynitrilase; protein crystallography Hydroxynitrile lyases~EC 4.2.1.39! catalyze the cleavage of cyanohydrins to yield hydrocyanic acid plus the corresponding aldehyde or ketone~Fig. 1!. The release of HCN serves as a defense against herbivores and microbial attack for a variety of plants Conn, 1981;Hickel et al., 1996;Wajant & Effenberger, 1996! and is initiated by a b-glycosidases-mediated degradation of cyanoglycosides, forming a sugar and the a-hydroxynitrile~cyanohydrin!. In aqueous solution, cyanohydrin cleavage occurs spontaneously above pH 5, while the enzyme-catalyzed reaction also occurs at lower pH values~down to pH ; 3; Hickel et al., 1996!.

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“…Keywords: Substrate specificity; active-site tunnel mutant; crystal structure Hydroxynitrile lyases (HNLs) constitute a diverse family of enzymes that catalyze the stereospecific cleavage of a wide range of cyanohydrins into aldehydes or ketones and hydrogen cyanide (cyanogenesis) , which have recently become the subject of intensive structural studies (Wagner et al 1996;Zuegg et al 1999; Lauble et al 2001a,b). Apart from the interesting aspects of their catalytic function, HNLs are of practical importance as biocatalysts for the reverse reaction of cyanogenesis, that is, the stereoselective addition of HCN to carbonyl compounds (Scheme 1).…”

Section: Introductionmentioning

confidence: 99%

“…The crystal structure of the MeHNL-W128A substratefree form at 2.1 Å resolution indicates that the W128A substitution has significantly enlarged the active-site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL-W128A-4-hydroxybenzaldehyde complex structure at 2.1 Å resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.Keywords: Substrate specificity; active-site tunnel mutant; crystal structure Hydroxynitrile lyases (HNLs) constitute a diverse family of enzymes that catalyze the stereospecific cleavage of a wide range of cyanohydrins into aldehydes or ketones and hydrogen cyanide (cyanogenesis) , which have recently become the subject of intensive structural studies (Wagner et al 1996;Zuegg et al 1999; Lauble et al 2001a,b). Apart from the interesting aspects of their catalytic function, HNLs are of practical importance as biocatalysts for the reverse reaction of cyanogenesis, that is, the stereoselective addition of HCN to carbonyl compounds (Scheme 1).…”

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confidence: 99%

Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta

Lauble¹

2002

Protein Science

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Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme. This residue was therefore substituted in the mutant MeHNL-W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild-type MeHNL and MeHNL-W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively). However, the specific activities of MeHNL-W128A for the unnatural substrates mandelonitrile and 4-hydroxymandelonitrile are increased 9-fold and ∼450-fold, respectively, compared with the wild-type MeHNL. The crystal structure of the MeHNL-W128A substratefree form at 2.1 Å resolution indicates that the W128A substitution has significantly enlarged the active-site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL-W128A-4-hydroxybenzaldehyde complex structure at 2.1 Å resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.Keywords: Substrate specificity; active-site tunnel mutant; crystal structure Hydroxynitrile lyases (HNLs) constitute a diverse family of enzymes that catalyze the stereospecific cleavage of a wide range of cyanohydrins into aldehydes or ketones and hydrogen cyanide (cyanogenesis) , which have recently become the subject of intensive structural studies (Wagner et al. 1996;Zuegg et al. 1999; Lauble et al. 2001a,b). Apart from the interesting aspects of their catalytic function, HNLs are of practical importance as biocatalysts for the reverse reaction of cyanogenesis, that is, the stereoselective addition of HCN to carbonyl compounds (Scheme 1).Although the HNL from Manihot esculenta (MeHNL) accepts a broad range of carbonyl compounds as substrates , MeHNL-catalyzed cyanohydrin formation is limited by bulky substituents with respect to conversion and ee-value, as shown, for example, for 3-phenoxybenzaldehyde, an important starting material for pyrethroids Bühler 2000).On the basis of the X-ray structure of MeHNL (Lauble et al. 1999), first attempts have been undertaken to correlate the substrate specificity with structural features of the threedimensional structure of the enzyme. The X-ray structure of

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Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis

Cited by 103 publications

References 39 publications

Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Three‐dimensional structures of enzyme‐substrate complexes of the hydroxynitrile lyase from hevea brasiliensis

Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta

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