Background: GlfT2 is a bifunctional galactofuranosyltransferase essential for mycobacterial cell wall biosynthesis. Results: Crystal structures of free and UDP-bound GlfT2, along with mutagenesis and kinetic studies, reveal novel details underlying substrate binding and catalysis.
Conclusion:The homotetrameric architecture, distinctive nucleotide-binding site, and unprecedented structural features underlying the bifunctional polymerase activity of GlfT2 are revealed. Significance: Novel insights into polymerizing glycosyltransferases and the design of anti-mycobacterial therapeutics are obtained.
Background: Synthesis of allolactose (lac operon inducer) from lactose requires a site for clasping glucose as an acceptor on -galactosidase. Results: The structure of the glucose site was defined, and its evolutionary conservation was determined. Conclusion: The glucose binding site defines an "allolactose synthesis motif" that is co-selected with lac repressors. Significance: Novel insights into evolutionary adaptations for regulation by allolactose are presented.
Many enzymes require a specific monovalent cation (M(+)), that is either Na(+) or K(+), for optimal activity. While high selectivity M(+) sites in transport proteins have been extensively studied, enzyme M(+) binding sites generally have lower selectivity and are less characterized. Here we study the M(+) binding site of the model enzyme E. coli β-galactosidase, which is about 10 fold selective for Na(+) over K(+). Combining data from X-ray crystallography and computational models, we find the electrostatic environment predominates in defining the Na(+) selectivity. In this lower selectivity site rather subtle influences on the electrostatic environment become significant, including the induced polarization effects of the M(+) on the coordinating ligands and the effect of second coordination shell residues on the charge distribution of the primary ligands. This work expands the knowledge of ion selectivity in proteins to denote novel mechanisms important for the selectivity of M(+) sites in enzymes.
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