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。
Abstract
The reductive glycine pathway is considered to be the most promising one carbon (C1) synthesis pathway, 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 overall 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%, respectively. 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 residues of H-protein that hinders or promotes the release of lipoamide.
关键词
C1合成 /
还原甘氨酸途径 /
甘氨酸裂解酶系 /
H-蛋白
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Key words
C1 synthesis /
reductive glycine pathway /
glycine cleavage system /
H-protein
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