Regulation of directed mutations of Asp-68 and Tyr-70 in H-protein on the overall enzyme activity of the glycine cleavage system
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.
Glycine cleavage system
Reductive glycine pathway