Department of Biochemistry, Molecular Biology and Biophysics and The Biotechnology Institute, University of Minnesota, Saint Paul, Minnesota, United States of America.
PLoS One. 2020 Jun 30;15(6):e0235341. doi: 10.1371/journal.pone.0235341. eCollection 2020.
Hydroxynitrile lyases (HNL's) belonging to the α/β-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/β-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 to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. 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-Å resolution shows the expected α/β-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL 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 than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), 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.
羟腈裂解酶（HNL's）属于α/β-水解酶折叠超家族，大约在 1 亿年前从酯酶进化而来。在α/β-水解酶折叠超家族中对祖先羟腈裂解酶的重建产生了具有催化活性的羟腈裂解酶 HNL1。与现代橡胶树羟腈裂解酶（HbHNL）相比，HNL1 具有几个不同的性质。与 HbHNL 相比，HNL1 更倾向于较大的底物，对酯水解（对硝基苯乙酸酯）的催化杂化程度是 mandelonitrile 裂解的两倍，并且对不可逆热失活的抗性比 HbHNL 高 35°C。我们假设 HNL1 的 X 射线晶体结构可能揭示这些性质差异的分子基础。X 射线晶体结构解析至 1.96-Å 分辨率，显示出预期的α/β-水解酶折叠，但与 HbHNL 相比，活性位点大 60%。这个更大的活性位点反映了它从酯酶进化而来，因为相关的烟草酯酶 SABP2 的活性位点也比 HbHNL 大 38%。HNL1 中较大的活性位点可能是其能够接受较大羟腈底物的原因。将 HbHNL 进行定点突变以扩大活性位点，使其杂化酯酶活性增加了 50 倍，这与 HNL1 中较大的活性位点是其杂化酯酶活性的主要原因相一致。HNL1 的脲诱导去折叠表明，它的去折叠程度不如 HbHNL 完全（m 值为 0.63 对于 HNL1 与 0.93 kcal/mol·M 对于 HbHNL），这可能解释了 HNL1 在加热时更好地抵抗不可逆失活的能力。HNL1 的结构显示氢键网络的变化，这可能稳定折叠结构的区域。
