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  • Title: HDX-MS Reveals Substrate-Dependent, Localized EX1 Conformational Dynamics in the Retaining GT-B Glycosyltransferase, MshA.
    Author: Karki R, Hennek JT, Chen W, Frantom PA.
    Journal: Biochemistry; 2023 Sep 05; 62(17):2645-2657. PubMed ID: 37589157.
    Abstract:
    Glycosyltransferases (GTs) are well-characterized with respect to static 3D structures and molecular dynamics simulations, but there is a lack of reports on in-solution dynamics on time scales relevant to turnover. Here, backbone amide hydrogen/deuterium exchange followed by mass spectrometry (HDX-MS) was used to investigate the in-solution dynamics of the model retaining GT MshA from Corynebacterium glutamicum (CgMshA). CgMshA has a GT-B fold and catalyzes the transfer of N-acetyl-glucosamine (GlcNAc) from UDP-GlcNAc to l-myo-inositol-1-phosphate in the first step in mycothiol biosynthesis. HDX-MS results identify several key regions of conformational changes in response to UDP-GlcNAc binding, including residues 159-198 in the N-terminal domain and residues 323-354 in the C-terminal domain. These regions also exhibited substrate-dependent EX1 exchange kinetics consistent with conformational tension on the milliseconds to seconds time scale. A potential source of this conformational change is the flexible β4/α5 loop in the C-terminal domain, which sits at the interface of the two domains and likely interacts with the GlcNAc ring of UDP-GlcNAc. In contrast to UDP-GlcNAc, the UDP-CgMshA product complex exhibited severe decreases in deuterium incorporation, suggesting a less dynamic conformation. The HDX-MS results are complemented by solvent viscosity effects of 1.8-2.3 on the CgMshA kcat value, which are consistent with product release as a rate-determining step and possibly a direct role for protein dynamics in catalysis. The identification of in-solution dynamics that are sensitive to substrate binding allows for the proposal of a more detailed mechanism in CgMshA including conformation tension between the donor sugar and the flexible C-terminal domain β4/α5 loop providing sufficient conformational sampling for substrate-assisted catalysis to occur.
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