Publications

• A two-base mechanism for Escherichia coli ADP-L-glycero-D-manno-heptose 6-epimerase.

  Morrison J.P., Tanner M.E.

  ADP-l-glycero-d-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the inversion of configuration at C-6' ' of the heptose moiety of ADP-d-glycero-d-manno-heptose and ADP-l-glycero-d-manno-heptose. The epimerase HldD operates in the biosynthetic pathway of l-glycero-d-manno-heptose, ... >> More

  ADP-l-glycero-d-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the inversion of configuration at C-6' ' of the heptose moiety of ADP-d-glycero-d-manno-heptose and ADP-l-glycero-d-manno-heptose. The epimerase HldD operates in the biosynthetic pathway of l-glycero-d-manno-heptose, which is a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. Previous studies support a mechanism in which HldD uses its tightly bound NADP+ cofactor to oxidize directly at C-6' ', generating a ketone intermediate. A reduction of the ketone from the opposite face then occurs, generating the epimeric product. How the epimerase is able access both faces of the ketone intermediate with correct alignment of the three required components, NADPH, the ketone carbonyl, and a catalytic acid/base residue, is addressed here. It is proposed that the epimerase active site contains two catalytic pockets, each of which bears a catalytic acid/base residue that facilitates reduction of the C-6' ' ketone but leads to a distinct epimeric product. The ketone carbonyl may access either pocket via rotation about the C-5' '-C-6' ' bond of the sugar nucleotide and in doing so presents opposing faces to the bound cofactor. Evidence in support of the two-base mechanism is found in studies of two single mutants of the Escherichia coli K-12 epimerase, Y140F and K178M, both of which have severely compromised epimerase activities that are more than 3 orders of magnitude lower than that of the wild type. The catalytic competency of these two mutants in promoting redox chemistry is demonstrated with an alternate catalytic activity that requires only one catalytic base: dismutation of a C-6' ' aldehyde substrate analogue (ADP-beta-d-manno-hexodialdose) to an acid and an alcohol (ADP-beta-d-mannuronic acid and ADP-beta-d-mannose). This study identifies the two catalytic bases as tyrosine 140 and lysine 178. A one-step enzymatic conversion of mannose into ADP-beta-mannose is also described and used to make C-6' '-substituted derivatives of this sugar nucleotide. << Less

  Biochemistry 46:3916-3924(2007) [PubMed] [EuropePMC]

• Characterization of the ADP-beta-D-manno-heptose biosynthetic enzymes from two pathogenic Vibrio strains.

  Shi Z., Tang Y., Wang Z., Wang M., Zhong Z., Jia J., Chen Y.

  ADP-activated β-D-manno-heptoses (ADP-β-D-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-D-glycero-β-D-manno-heptose (ADP-D,D-manno-heptose) and its C-6'' epimer, ADP-L-glycero-β-D-manno-heptose (ADP-L,D-manno-he ... >> More

  ADP-activated β-D-manno-heptoses (ADP-β-D-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-D-glycero-β-D-manno-heptose (ADP-D,D-manno-heptose) and its C-6'' epimer, ADP-L-glycero-β-D-manno-heptose (ADP-L,D-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-D,D-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-β-D-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-D,D-manno-heptose and the epimerase that converts it to ADP-L,D-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that β-D-mannose 1-phosphate, but not α-D-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldE_VC (from V. cholerae) and HldE_VP (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldE_VC and HldE_VP together with the E. coli-derived HldE_EC were thus carried out using β-D-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-β-D-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains. KEY POINTS: • Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-β-D-manno-heptoses. • HldEs from two Vibrio strains and E. coli could activate β-D-mannose 1-phosphate to ADP-β-D-mannose. • Comparable nucleotidyltransfer efficiencies were observed in the kinetic studies of HldEs. << Less

  Appl Microbiol Biotechnol 108:267-267(2024) [PubMed] [EuropePMC]