PDBsum entry 1sci

Lyase

PDB id: 1sci

Contents

Protein chain

256 a.a. *

Ligands

SO4 ×4

Waters ×261

* Residue conservation analysis

PDB id:

1sci

Name:

Lyase

Title:

K236l mutant of hydroxynitrile lyase from hevea brasiliensis

Structure:

(S)-acetone-cyanohydrin lyase. Chain: a. Synonym: hydroxynitrile lyase. Engineered: yes. Mutation: yes

Source:

Hevea brasiliensis. Organism_taxid: 3981. Tissue: leaf. Gene: hnl. Expressed in: pichia pastoris. Expression_system_taxid: 4922.

Resolution:

2.18Å R-factor: 0.171 R-free: 0.212

Authors:

K.Gruber,G.Gartler,B.Krammer,H.Schwab,C.Kratky

Key ref:

K.Gruber et al. (2004). Reaction mechanism of hydroxynitrile lyases of the alpha/beta-hydrolase superfamily: the three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236. J Biol Chem, 279, 20501-20510. PubMed id: 14998991 DOI: 10.1074/jbc.M401575200

Date:

12-Feb-04 Release date: 29-Jun-04

Protein chain

P52704  (HNL_HEVBR) -  (S)-hydroxynitrile lyase from Hevea brasiliensis

Seq: 257 a.a.

Struc: 256 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.4.1.2.47 - (S)-hydroxynitrile lyase.
• Reaction:
  1. a monosubstituted aliphatic (S)-hydroxynitrile = an aldehyde + hydrogen cyanide
  2. a disubstituted aliphatic (S)-hydroxynitrile = a ketone + hydrogen cyanide
  3. an aromatic (S)-hydroxynitrile = an aromatic aldehyde + hydrogen cyanide

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M401575200

J Biol Chem 279:20501-20510 (2004)

PubMed id: 14998991

Reaction mechanism of hydroxynitrile lyases of the alpha/beta-hydrolase superfamily: the three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236.

K.Gruber, G.Gartler, B.Krammer, H.Schwab, C.Kratky.

ABSTRACT

The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the alpha/beta-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for the Hevea enzyme but denied for the Manihot enzyme. We applied a freeze-quench method to prepare crystals of the complex of HbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys(236) the catalytic base His(235) would be protonated at neutral pH. All of this suggests that Lys(236) is instrumental for catalysis in several ways, i.e. by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN(-), and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of alpha/beta-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.

Selected figure(s)

Figure 2.
FIG. 2. The proposed mechanism of the reaction catalyzed by HbHNL formulated for the cyanohydrin cleavage direction (21).

Figure 3.
FIG. 3. The mechanism proposed for the reaction catalyzed by MeHNL (24).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 20501-20510) copyright 2004.

Figures were selected by an automated process.