FlaA1, a new bifunctional UDP-GlcNAc C6 Dehydratase/ C4 reductase from Helicobacter pylori.
FlaA1 is a small soluble protein of unknown function in Helicobacter pylori. It has homologues that are essential for the virulence of numerous medically relevant bacteria. FlaA1 was overexpressed as a histidine-tagged protein and purified to homogeneity by nickel chelation and cation exchange chromatography. Spectrophotometric assays, capillary electrophoresis, and mass spectrometry analyses showed that FlaA1 is a novel bifunctional C(6) dehydratase/C(4) reductase specific for UDP-GlcNAc. It converts UDP-GlcNAc into a UDP-4-keto-6-methyl-GlcNAc intermediate, which is stereospecifically reduced into UDP-QuiNAc. Substrate conversions as high as 80% were obtained at equilibrium. The K(m) and V(max) for UDP-GlcNAc were 159 microm and 65 pmol/min, respectively. No exogenous cofactor was required to obtain full activity of FlaA1. Additional NADH was only used with poor efficiency for the reduction step. The biochemical characterization of FlaA1 is important for the elucidation of biosynthetic pathways that lead to the formation of 2,6-deoxysugars in medically relevant bacteria. It establishes unambiguously the first step of the pathway and provides the means of preparing the substrate UDP-QuiNAc, which is necessary for the study of downstream enzymes.
Creuzenet
C
C
University of Guelph, Department of Microbiology, Guelph, Ontario N1G 2W1, Canada.
Schur
M J
MJ
Li
J
J
Wakarchuk
W W
WW
Lam
J S
JS
D001426:Bacterial Proteins; D001547:Benzaldehydes / Q000494:pharmacology; D015153:Blotting, Western; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D019075:Electrophoresis, Capillary; D005816:Genetic Complementation Test; D005947:Glucose / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology*; D006639:Histidine / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D016296:Mutagenesis; D009532:Nickel / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors
D001426:Bacterial Proteins; D001547:Benzaldehydes; C417241:FlaA1 protein, Helicobacter pylori; D007202:Indicators and Reagents; D006639:Histidine; D009532:Nickel; D010088:Oxidoreductases; D002237:Carbohydrate Dehydrogenases; C046670:UDP-N-acetylglucosamine dehydrogenase; D006836:Hydro-Lyases; D005947:Glucose; C510241:p-dimethylaminobenzaldehyde