Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, Christchurch, New Zealand.
Biochemistry. 2011 Nov 1;50(43):9318-27. doi: 10.1021/bi201231e. Epub 2011 Oct 10.
The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the reaction between phosphoenolpyruvate and arabinose 5-phosphate (A5P) in the first committed step in the biosynthetic pathway for the formation of 3-deoxy-D-manno-octulosonate, an important component in the cell wall of Gram-negative bacteria. KDO8P synthase is evolutionarily related to the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase, which uses erythrose 4-phosphate in place of A5P. The A5P binding site in KDO8P synthase is formed by three long loops that extend from the core catalytic (β/α)(8) barrel, β2α2, β7α7, and β8α8. The extended β7α7 loop is always present in KDO8P synthase yet is not observed for DAH7P synthase. Modeling of this loop indicated interactions between this loop and the extended β2α2 loop; both loops provide key hydrogen-bonding contacts with A5P. The two absolutely conserved residues on the β7α7 loop (Gln and Ser) were mutated to Ala in both the metal-dependent KDO8P synthase from Acidithiobacillus ferrooxidans and the metal-independent KDO8P synthase from Neisseria meningitidis. In addition, mutants were constructed for both enzymes with the extended β7α7 loop excised to match the DAH7P synthase architecture. Removal of the loop extension severely hindered efficient catalysis, dramatically increasing the K(m)(A5P) and reducing the k(cat) for both enzymes. Excision of the complete loop was far more detrimental to catalysis than the double mutations of the two conserved Gln and Ser residues. Therefore, the presence of the entire extended β7α7 loop is important for efficient catalysis by KDO8P synthase, with the loop acting to promote efficient and productive binding of A5P.
3-脱氧-D-甘露辛酮糖 8-磷酸（KDO8P）合酶在 3-脱氧-D-甘露辛酮糖生物合成途径的第一步中催化磷酸烯醇丙酮酸与阿拉伯糖 5-磷酸（A5P）之间的反应，3-脱氧-D-甘露辛酮糖是革兰氏阴性细菌细胞壁的重要组成部分。KDO8P 合酶与莽草酸途径的第一个酶，3-脱氧-D-阿拉伯庚酮糖 7-磷酸（DAH7P）合酶在进化上有关，后者用赤藓糖 4-磷酸代替 A5P。KDO8P 合酶的 A5P 结合位点由三个从核心催化（β/α）（8）桶延伸的长环形成，β2α2、β7α7 和 β8α8。延伸的β7α7 环始终存在于 KDO8P 合酶中，但在 DAH7P 合酶中未观察到。该环的建模表明该环与延伸的β2α2 环之间存在相互作用；两个环都与 A5P 提供关键氢键接触。β7α7 环上的两个绝对保守残基（谷氨酰胺和丝氨酸）在来自 Acidithiobacillus ferrooxidans 的金属依赖型 KDO8P 合酶和来自 Neisseria meningitidis 的金属非依赖型 KDO8P 合酶中均突变为丙氨酸。此外，两种酶的突变体均构建有切除延伸的β7α7 环以匹配 DAH7P 合酶结构。除去环延伸严重阻碍了有效催化，极大地增加了两种酶的 K(m)(A5P)并降低了 k(cat)。切除整个环比对两个保守谷氨酰胺和丝氨酸残基的双突变更不利于催化。因此，完整延伸的β7α7 环的存在对 KDO8P 合酶的有效催化很重要，该环的作用是促进 A5P 的有效和高产结合。
