摘要
还原甘氨酸途径被认为是最有前景的C1 (one carbon)合成途径,其核心酶系是甘氨酸裂解酶系。在前期研究中,我们在甘氨酸裂解酶系H-蛋白"解开自保护"过程的研究中初步锁定了H-蛋白空腔内的潜在关键氨基酸残基为Ser-67、Asp-68和Tyr-70,并且证明Ser-67位点对甘氨酸酶系的整体酶活有重要影响。本文对H-蛋白的Asp-68和Tyr-70位点进行了侧链带正电突变(H-D68K、H-D68H、H-D68R和H-Y70K、H-Y70H、H-Y70R突变体),以及侧链非极性突变(H-D68G、H-D68V、H-D68M、H-D68L和H-Y70G、H-Y70V、H-Y70M、H-Y70L突变体),并测定了各突变体在甘氨酸裂解方向上的酶活。结果发现,Asp-68位带正电突变倾向降低甘氨酸酶系的整体酶活,Asp-68位非极性突变、Tyr-70位带正电突变及非极性突变在总体上倾向于维持或提升整体酶活。其中,相对野生型H-蛋白,H-D68R突变体的酶活下降了90.2%,H-Y70R、H-D68G和H-Y70L突变体的酶活分别提高了75.6%、53.6%和146%。硫辛酰胺与H-蛋白空腔内的氨基酸相互作用的分析结果表明,甘氨酸裂解酶系整体酶活的变化是由于H-蛋白的68和70位残基的突变阻碍或促进硫辛酰胺的释放。
The reductive glycine pathway is considered to be the most promising one carbon(C1)synthesis path-way,and its core enzyme is the glycine cleavage system(GCS).In a previous study,we preliminarily identified the potential key amino acid residues in the H-protein cavity as Ser-67,Asp-68 and Tyr-70 in a study of the“unlocking self-protection”process of H-protein in the glycine cleavage system,and showed that the Ser-67 site had an important impact on the overall enzyme activity of the glycine cleavage system.In this paper,side-chain positively charged mutations(H-D68K,H-D68H,H-D68R and H-Y70K,H-Y70H,H-Y70R mutants)and side-chain nonpolar mutations(H-D68G,H-D68V,H-D68M,H-D68L and H-Y70G,H-Y70V,H-Y70M,H-Y70L mutants)were performed on the Asp-68 and Tyr-70 sites of H-protein,and the enzyme activities of each mutant in the glycine cleavage direction were determined.The results showed that positively charged mutations at Asp-68 tended to decrease the overall enzyme activity of the glycine cleavage system,while nonpolar mutations at Asp-68,positively charged mutations and nonpolar mutations at Tyr-70 tended to maintain or increase the over-all enzyme activity.Compared with the wild-type H-protein,the enzyme activity of the H-D68R mutant decreased by 90.2%,and those of H-Y70R,H-D68G and H-Y70L mutant increased by 75.6%,53.6%and 146%,re-spectively.An analysis of the interactions between lipoamide and residues in the cavity of H-protein showed that the change in the overall enzyme activity of the glycine cleavage system was due to the mutation at 68 and 70 resi-dues of H-protein that hinders or promotes the release of lipoamide.
作者
张涵
李宇辰
聂晶磊
任杰
曾安平
ZHANG Han;LI YuChen;NIE JingLei;REN Jie;ZENG AnPing(Beijing Advanced Innovation Center for Soft Matter Science and Engineering,Beijing University of Chemical Technology,Beijing 100029,China;Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100081,China;Hamburg University of Technology,Hamburg 21073,Germany)
出处
《北京化工大学学报(自然科学版)》
CAS
CSCD
北大核心
2021年第6期48-56,共9页
Journal of Beijing University of Chemical Technology(Natural Science Edition)