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  • FlaA1, a new bifunctional UDP-GlcNAc C6 Dehydratase/ C4 reductase from Helicobacter pylori.

FlaA1, a new bifunctional UDP-GlcNAc C6 Dehydratase/ C4 reductase from Helicobacter pylori.

The Journal of biological chemistry (2000-08-24)
C Creuzenet, M J Schur, J Li, W W Wakarchuk, J S Lam
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
4-(Dimethylamino)benzaldehyde, ACS reagent, 99%
Sigma-Aldrich
4-(Dimethylamino)benzaldehyde, 98%
Supelco
4-(Dimethylamino)benzaldehyde solution, 10 g/L in isopropanol, for TLC derivatization
Sigma-Aldrich
4-(Dimethylamino)benzaldehyde, puriss. p.a., reag. Ph. Eur., ≥99% (perchloric acid titration)
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4-(Dimethylamino)benzaldehyde, suitable for histochemical demonstration of nitro blue tetrazolium reduction in neutrophils
Millipore
Kovac′s reagent for indoles, suitable for microbiology
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Kovac′s reagent for indoles, suitable for microbiology