Discovery and Structural Analysis to Improve the Enantioselectivity of Hydroxynitrile Lyase from Parafontaria laminata Millipedes for (R)-2-Chloromandelonitrile Synthesis

Abstract

Hydroxynitrile lyase (HNL) catalyzes the reversible synthesis and degradation of cyanohydrins, which are important synthetic intermediates for fine chemical and pharmaceutical industries. Here, we report the discovery of HNL from Parafontaria laminata (PlamHNL) millipedes, purification of the HNL to homogeneity, expression of the gene for the enzyme in heterologous expression hosts, and increase in the reaction rate and enantioselectivity in the synthesis of 2-chloromandelonitrile by protein engineering. The recombinant PlamHNL expressed in Pichia pastoris is glycosylated and has a higher thermostability and pH stability than the nonglycosylated HNL expressed in Escherichia coli. PlamHNL showed a unique wide substrate specificity among other millipede HNLs acting on various cyanohydrins, including 2-chloromandelonitrile, a key intermediate for the antithrombotic agent clopidogrel. We solved the X-ray crystal structure of the PlamHNL and found that the catalytic residues were almost identical to those of HNL from Chamberlinius hualienensis, although the forming binding cavity was different. In order to improve the catalytic activity and stereoselectivity, a computational structure-guided directed evolution approach was performed by an enzyme-substrate docking simulation at all of the residues that were exposed on the surface of the active site. The PlamHNL-N85Y mutant showed higher conversion (91% conversion with 98.2% ee of the product) than the wild type (76% conversion with 90% ee of the product) at pH 3.5 and 25 °C for 30 min of incubation. This study shows the diversity of millipede HNLs and reveals the molecular basis for improvement of the activity and stereoselectivity of the wild-type HNL to increase the reaction rate and enantioselectivity in the synthesis of 2-chloromandelonitrile.

Figure 1

(A–C) Effect of pH on (A) initial velocity, (B) enantiomeric excess, and (C) enzyme stability. (D–F) Effect of temperature on (D) initial velocity, (E) enantiomeric excess, and (F) enzyme stability. The reaction was performed in citrate buffer (400 mM, pH 4.0), benzaldehyde (50 mM), and KCN (100 mM). Solid and gray bars indicate PlamHNL-P. The dashed line and open bar indicate PlamHNL-E.

Figure 2

Crystal structure of PlamHNL. (A) Overall structure of the PlamHNL dimer connecting with two intermolecular disulfide bonds between C101 of one molecule and C179 of another. There were three intramolecular disulfide bonds between C23–C129, C55–C173, and C125–C139. The gray grid shows the space available for substrate binding. (B) Superimposed structure of PlamHNL (magenta) and ChuaHNL (green). (C) Superimposed side chain and surface of the active pocket of PlamHNL (magenta) and ChuaHNL (green). (D, E) close-up views of the side chain-forming cavity and entrance channel of PlamHNL and ChuaHNL, respectively.

Figure 3

(A) Amino acid sequence and secondary-based alignment of HNLs from millipedes. The secondary structure elements are shown as follows: α-helices, medium squiggles with α symbols; 3₁₀-helices, squiggles with η symbols; β-sheets, arrows with β symbols; and strict β-turns, TT letters. The pairs of cysteine residues forming disulfide bonds are shown as green digits at the bottom of the alignment. The red background shows strictly conserved residues. Similar groups are highlighted with a yellow background. Residues conserved between groups are boxed. The proposed catalytic residues are surrounded by blue boxes. Numbers refer to the PlamHNL sequence. (B, C) Close-up views of the active site structure of PlamHNL complexed with (B) benzaldehyde (yellow) and (C) benzaldehyde and thiocyanate (magenta). The catalytic residues (R58, Y60, D76, Y124, and K138) are shown in blue. The hydrogen bonds are shown as black dotted lines, and their distances are labeled. Water molecules are depicted as red spheres (wat1 and wat2).

Figure 4

(A, B) Docking model of the (A) wild type and (B) PlamHNL-N85Y mutant complexed with (R)-2-chloromandelonitrile (magenta). The crystal structures of wild type and mutant are colored in blue. The catalytic residues (R58, Y60, D76, Y124, and K138) are shown in yellow. The hydrogen bonds are shown as black dotted lines, and their distances are labeled. The CH−π is shown as light green dotted lines. N85 and N85Y are colored in green.

Figure 5

Enantioselectivity for (R)-2-chloromandelonitrile synthesis of purified enzymes from PlamHNL wild type (black bar or circle open) and mutant N85Y (cross bar or triangle up solid). (A) Effect of enzyme quantity. (B) Effect of low pH conditions. (C, D) Time course of (C) total (R + S)-2-chloromandelonitrile production and (D) their enantiomeric excess in the reaction of citrate buffer (300 mM, pH 3.5) containing 2-chlorobenzaldehyde (50 mM), KCN (100 mM), and enzyme (4 U) at 25 °C.