The crystal structure and catalytic mechanism of hydroxynitrile lyase from passion fruit, Passiflora edulis
The crystal structure and catalytic mechanism of hydroxynitrile lyase from passion fruit, Passiflora edulis
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DOI：
10.1111/febs.14339
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发表时间：
2018-01-01
期刊：
影响因子：
5.4
通讯作者：
Asano, Yasuhisa
中科院分区：
文献类型：
--
作者：
Motojima, Fumihiro;Nuylert, Aem;Asano, Yasuhisa
羟基腈裂解酶（HNL）是用于合成手性氰醇的酶。来自西番莲的HNL（PeHNL）是R-选择性的，并且是迄今为止已知的最小的HNL。PeHNL及其C-末端肽缺失衍生物的晶体结构通过分子置换法使用来自白杨的热稳定蛋白SP1的模板结构分别在2.8和1.8埃分辨率下测定。PeHNL属于二聚体α + β桶超家族，由二聚体中间的中心β桶组成。还确定了与（R）-扁桃腈（（R）-MAN）络合的PeHNL的结构。（R）-MAN的羟基与活性位点中的His 8和Tyr 30形成氢键，而腈基朝向Glu 54的羧基，不像其他HNL，它通常与碱性残基相互作用。突变分析结果表明，His 8-Asn 101催化二联体是酶促反应的关键。在PeHNL-（R）-MAN复合物中，His-Nd 1和Asn 101-O delta 1之间的氢键长度很短（类似于2.6埃），这将增加His 8的碱性以从（R）-MAN的羟基中提取质子。从腈基释放的氰离子从质子化的His 8中提取质子以生成氰化氢。因此，PeHNL活性位点中的His 8在反应中既充当一般酸又充当一般碱。
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.