Abstract
Hydroxynitrile lyases are versatile enzymes that enantiospecifically cope with cyanohydrins, important intermediates in the production of various agrochemicals or pharmaceuticals. We determined four atomic resolution crystal structures of hydroxynitrile lyase from Hevea brasiliensis: one native and three complexes with acetone, isopropyl alcohol, and thiocyanate. We observed distinct distance changes among the active site residues related to proton shifts upon substrate binding. The combined use of crystallography and ab initio quantum chemical calculations allowed the determination of the protonation states in the enzyme active site. We show that His(235) of the catalytic triad must be protonated in order for catalysis to proceed, and we could reproduce the cyanohydrin synthesis in ab initio calculations. We also found evidence for the considerable pK(a) shifts that had been hypothesized earlier. We envision that this knowledge can be used to enhance the catalytic properties and the stability of the enzyme for industrial production of enantiomerically pure cyanohydrins.
Highlights
Hydroxynitrile lyases (HNLs),2 EC 4.2.1.39, catalyze the cleavage of cyanohydrins into hydrocyanic acid (HCN) and the corresponding aldehyde or ketone (Fig. 1)
We show that His235 of the catalytic triad must be protonated in order for catalysis to proceed, and we could reproduce the cyanohydrin synthesis in ab initio calculations
In addition to the ligands in the active site in the structures ACET, isopropyl alcohol (ISOP), and SCN, several sulfate ions from the crystallization buffer were found in all structures on the protein surface and in intermolecular contact regions
Summary
Sample Preparation and Crystallization—Hb-HNL was obtained from the company DSM and purified as described elsewhere [17]. In order to minimize the number of atoms, the amino acids involved in substrate contact were modeled as follows: His235 as imidazole, Asp207 as acetic acid, Ser as methanol, and Lys236 as aminoethane The coordinates for these residues and the ligands acetone and thiocyanate/isothiocyanic acid were taken from the refined crystal structures. Structure short name Space group/cell (average) EMBL/DORIS beam line Resolution range (Å) Unique reflections Completeness (%) Redundancy Rsym (%) I/(I)a Wilson B (Å2) R factor for the refined models (%) (all data) No of atoms (total) No of solvent atoms No of heteroatoms Overall B protein (Å2) DPI b Estimated minimum/maximum coordinate error (Å) r.m.s. deviation 1–2 distances r.m.s. deviation 1–3 distances Ligand occupancy a Outermost shell. Various protonation states were tried for all models (Fig. 2)
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