The crystal structure and catalytic mechanism of hydroxynitrile lyase from passion fruit, Passiflora edulis

被引:14
|
作者
Motojima, Fumihiro [1 ,2 ,3 ]
Nuylert, Aem [1 ,2 ]
Asano, Yasuhisa [1 ,2 ,3 ]
机构
[1] Toyama Prefectural Univ, Biotechnol Res Ctr, 5180 Kurokawa, Imizu, Toyama 9390398, Japan
[2] Toyama Prefectural Univ, Dept Biotechnol, 5180 Kurokawa, Imizu, Toyama 9390398, Japan
[3] JST, ERATO, Asano Act Enzyme Mol Project, 5180 Kurokawa, Imizu, Toyama 9390398, Japan
基金
日本科学技术振兴机构;
关键词
catalytic mechanism; crystal structure; dimeric alpha plus beta barrel; hydroxynitrile lyase; Passiflora edulis; ACTIVE-SITE; HEVEA-BRASILIENSIS; (S)-HYDROXYNITRILE LYASE; MOLECULAR-CLONING; BIOCATALYTIC PROPERTIES; 3-DIMENSIONAL STRUCTURE; SUBSTRATE-BINDING; ESCHERICHIA-COLI; IDENTIFICATION; EXPRESSION;
D O I
10.1111/febs.14339
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 angstrom resolution, respectively. PeHNL belongs to dimeric alpha+beta barrel superfamily consisting of a central beta-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-Nd1 and Asn101-O delta 1 is short in the PeHNL-(R)-MAN complex (similar to 2.6 angstrom), 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.
引用
收藏
页码:313 / 324
页数:12
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