Hydroxynitrile lyases (HNLs) are enzymes used in the synthesis of chiral cyanohydrins. The HNL from Passiflora edulis (PeHNL) is R‐selective and is the smallest HNL known to date. The crystal structures of PeHNL and its C‐terminal peptide depleted derivative were determined by molecular replacement method using the template structure of a heat stable protein, SP1, from Populus tremula at 2.8 and 1.8 Å resolution, respectively. PeHNL belongs to dimeric α+β barrel superfamily consisting of a central β‐barrel in the middle of a dimer. The structure of PeHNL complexed with (R)‐mandelonitrile ((R)‐MAN) was also determined. The hydroxyl group of (R)‐MAN forms hydrogen bonds with His8 and Tyr30 in the active site, whereas the nitrile group is oriented toward the carboxyl group of Glu54, unlike other HNLs, where it interacts with basic residues typically. The results of mutational analysis indicate that the catalytic dyad of His8‐Asn101 is critical for the enzymatic reaction. The length of the hydrogen bond between His‐Nδ1 and Asn101‐Oδ1 is short in the PeHNL‐(R)‐MAN complex (~ 2.6 Å), which would increase the basicity of His8 to abstract a proton from the hydroxyl group of (R)‐MAN. The cyanide ion released from the nitrile group abstracts a proton from the protonated His8 to generate a hydrogen cyanide. Thus, the His8 in the active site of PeHNL acts both as a general acid and a general base in the reaction. Enzymes http://www.chem.qmul.ac.uk/iubmb/enzyme/EC4/1/2/10.html Database Structural data are available in PDB database under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5XZQ, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5XZT, and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5Y02.

A hydroxynitrile lyase from the passion fruit Passiflora edulis (PeHNL) was isolated from the leaves and showed high stability in biphasic co-organic solvent systems for cyanohydrin synthesis. Cyanohydrins are important building blocks for the production of fine chemicals and pharmaceuticals. Thus, to enhance production yields of PeHNL for industrial applications, we cloned and expressed recombinant PeHNL in Escherichia coli BL21(DE3) and Pichia pastoris GS115 cells without a signal peptide sequence. The aim of this study is to determine the effect of N-glycosylation on enzyme stability and catalytic properties in microbial expression systems. PeHNL from leaves (PeHNL-N) and that expressed in P. pastoris (PeHNL-P) were glycosylated, whereas that expressed in E. coli (PeHNL-E) was not. The enzymes PeHNL-N and PeHNL-P showed much better thermostability, pH stability, and organic solvent tolerance than the deglycosylated enzyme PeHNL-E and the deglycosylated mutant N105Q from P. pastoris (PeHNL-P-N105Q). The glycosylated PeHNL-P also efficiently performed transcyanation of (R)-mandelonitrile with a 98 % enantiomeric excess in a biphasic system with diisopropyl ether. These data demonstrate the efficacy of these methods for improving enzyme expression and stability for industrial application through N-glycosylation.

Hydroxynitrile lyases (HNLs), which are key enzymes in cyanogenesis, catalyze the cleavage of cyanohydrins into carbonyl compounds and hydrogen cyanide. Since HNLs also catalyze the reverse reaction, they are used industrially for the asymmetric synthesis of cyanohydrins, which are valuable building blocks of pharmaceuticals and fine chemicals. HNLs have been isolated from cyanogenic plants and bacteria. Recently, an HNL from the cyanogenic millipede Chamberlinius hualienensis was shown to have the highest specific activity for (R)-mandelonitrile synthesis, along with high stability and enantioselectivity. However, no HNLs have been isolated from other cyanogenic millipedes. We identified and characterized HNLs from 10 cyanogenic millipedes in the Paradoxosomatidae and Xystodesmidae. Sequence analyses showed that HNLs are conserved among cyanogenic millipedes and likely evolved from one ancestral gene. The HNL from Parafontaria tonominea was expressed in Escherichia coli SHuffle T7 and showed high specific activity for (R)-mandelonitrile synthesis and stability at a range of pHs and temperatures. The stability of millipede HNLs is likely due to disulfide bond(s). The E. coli cells expressing HNL produced (R)-mandelonitrile with 97.6% enantiomeric excess without organic solvents. These results demonstrate that cyanogenic millipedes are a valuable source of HNLs with high specific activity and stability.

We determined the X‐ray crystallographic structure of a unique hydroxynitrile lyase from the cyanogenic millipede, Chamberlinius hualienensis, and elucidated the reaction mechanism. We revealed that it belongs to the lipocalins, a family of proteins active in the transport small hydrophobic molecules. It is one of a few lipocalins with enzyme activities. This addition expands the evolutionary relationship of the enzyme to a new family of proteins.

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