Creuzenet C, Schur M J, Li J, Wakarchuk W W, Lam J S
University of Guelph, Department of Microbiology, Guelph, Ontario N1G 2W1, Canada.
J Biol Chem. 2000 Nov 10;275(45):34873-80. doi: 10.1074/jbc.M006369200.
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.
FlaA1是幽门螺杆菌中一种功能未知的可溶性小蛋白。它在许多医学相关细菌的毒力方面具有同源物，这些同源物至关重要。FlaA1作为带组氨酸标签的蛋白被过表达，并通过镍螯合和阳离子交换色谱法纯化至同质。分光光度法测定、毛细管电泳和质谱分析表明，FlaA1是一种新型的对UDP-GlcNAc具有特异性的双功能C(6)脱水酶/C(4)还原酶。它将UDP-GlcNAc转化为UDP-4-酮-6-甲基-GlcNAc中间体，该中间体被立体特异性还原为UDP-QuiNAc。在平衡时可获得高达80%的底物转化率。UDP-GlcNAc的K(m)和V(max)分别为159微摩尔和65皮摩尔/分钟。获得FlaA1的全部活性不需要外源辅因子。额外的NADH仅低效地用于还原步骤。FlaA1的生化特性对于阐明医学相关细菌中导致2,6-脱氧糖形成的生物合成途径很重要。它明确地确定了该途径的第一步，并提供了制备底物UDP-QuiNAc的方法，而UDP-QuiNAc是研究下游酶所必需的。
