Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity
Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity
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DOI：
10.1371/journal.pone.0235341
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发表时间：
2020-06-30
期刊：
影响因子：
3.7
通讯作者：
Kazlauskas, Romas J.
中科院分区：
文献类型：
--
作者：
Jones, Bryan J.;Evans, Robert L., III;Kazlauskas, Romas J.
属于α/β-水解酶折叠超家族的羟基腈裂解酶（HNL）在大约1亿年前从酯酶进化而来。在α/β-水解酶折叠超家族中的祖先羟基腈裂解酶的重建产生了具有催化活性的羟基腈裂解酶HNL 1。HNL 1的几个性质不同于现代橡胶树HNL（HbHNL）。HNL 1与HbHNL相比更有利于更大的底物，与扁桃腈裂解相比，HNL 1对酯水解（乙酸对硝基苯酯）的催化混杂性是其两倍，并且比HbHNL高35 ℃的不可逆热失活抗性。我们假设HNL 1的X射线晶体结构可能揭示了这些特性差异的分子基础。解析到1.96埃分辨率的X射线晶体结构显示出预期的α/β-水解酶折叠，但与HbHNL相比，活性位点大60%。这种较大的活性位点与其从酯酶的进化相呼应，因为来自烟草的相关酯酶SABP 2也具有比HbHNL大38%的活性位点。HNL 1中较大的活性位点可能是其接受较大羟基腈底物的能力。定点突变HbHNL扩大活性位点增加其混杂酯酶活性50倍，与HNL 1中较大的活性位点是其混杂酯酶活性的主要原因一致。尿素诱导的HNL 1解折叠表明，它的解折叠不如HbHNL完全（HNL 1的m值= 0.63，HbHNL的m值= 0.93 kcal/mol中心点M），这可能是HNL 1在加热时更好地抵抗不可逆失活的能力的原因。HNL 1的结构显示氢键网络的变化，这可能会稳定折叠结构的区域。
Hydroxynitrile lyases (HNL's) belonging to the alpha/beta-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the alpha/beta-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared toHbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 degrees C higher than forHbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-angstrom resolution shows the expected alpha/beta-hydrolase fold, but a 60% larger active site as compared toHbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site thanHbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis ofHbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely thanHbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol center dot M forHbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.