Butty Frank D, Aucoin Monique, Morrison Leslie, Ho Nathan, Shaw Gary, Creuzenet Carole
Department of Microbiology and Immunology, Infectious Diseases Research Group, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Biochemistry. 2009 Aug 18;48(32):7764-75. doi: 10.1021/bi901065t.
6-Deoxyheptose is found within the surface polysaccharides of several bacterial pathogens. In Yersinia pseudotuberculosis, it is important for the barrier function of the O-antigen in vitro and for bacterial dissemination in vivo. The putative C6 dehydratase DmhA and C4 reductase DmhB, that were identified as responsible for 6-deoxyheptose synthesis based on genetics data, represent potential therapeutical targets. Their detailed biochemical characterization is presented herein. The substrate, GDP-D-glycero-D-manno-heptose, was synthesized enzymatically from sedoheptulose 7-phosphate using overexpressed and purified GmhA/B/C/D enzymes from Aneurinibacillus thermoaerophilus. Overexpressed and purified DmhA used this substrate with high efficiency, as indicated by its K(m) of 0.23 mM and k(cat) of 1.1 s(-1). The mass spectrometry (MS) analysis of the reaction product was consistent with a C6 dehydration reaction. DmhB could readily reduce this compound in the presence of NAD(P)H to produce GDP-6-deoxy-D-manno-heptose, as indicated by MS and NMR analyses. DmhA also used GDP-mannose as a substrate with a K(m) of 0.32 mM and a k(cat) of 0.25 min(-1). This kinetic analysis indicates that although the K(m) values for GDP-mannose and GDP-manno-heptose were similar, the genuine substrate for DmhA is GDP-manno-heptose. DmhB was also able to reduce the GDP-4-keto-6-deoxymannose produced by DmhA, although with poor efficiency and exclusively in the presence of NADPH. This study is the first complete biochemical characterization of the 6-deoxyheptose biosynthesis pathway. Also, it allows the screening for inhibitors, the elucidation of substrate specificity determinants, and the synthesis of carbohydrate antigens of therapeutic relevance.
6-脱氧庚糖存在于多种细菌病原体的表面多糖中。在假结核耶尔森氏菌中，它对体外O抗原的屏障功能以及体内细菌传播至关重要。基于遗传学数据被确定为负责6-脱氧庚糖合成的假定C6脱水酶DmhA和C4还原酶DmhB是潜在的治疗靶点。本文介绍了它们详细的生化特性。底物GDP-D-甘油-D-甘露庚糖是使用嗜热栖热放线菌中过表达和纯化的GmhA/B/C/D酶从景天庚酮糖7-磷酸酶促合成的。过表达和纯化的DmhA高效利用该底物，其K(m)为0.23 mM，k(cat)为1.1 s(-1)。反应产物的质谱（MS）分析与C6脱水反应一致。MS和NMR分析表明，DmhB在NAD(P)H存在下可轻松还原该化合物以产生GDP-6-脱氧-D-甘露庚糖。DmhA也使用GDP-甘露糖作为底物，K(m)为0.32 mM，k(cat)为0.25 min(-1)。该动力学分析表明，尽管GDP-甘露糖和GDP-甘露庚糖的K(m)值相似，但DmhA的真正底物是GDP-甘露庚糖。DmhB也能够还原DmhA产生的GDP-4-酮-6-脱氧甘露糖，尽管效率很低且仅在NADPH存在下。这项研究是6-脱氧庚糖生物合成途径的首次完整生化特性描述。此外，它还允许筛选抑制剂、阐明底物特异性决定因素以及合成具有治疗相关性的碳水化合物抗原。
