Shulami Smadar, Yaniv Orit, Rabkin Emilia, Shoham Yuval, Baasov Timor
Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
Extremophiles. 2003 Dec;7(6):471-81. doi: 10.1007/s00792-003-0346-3. Epub 2003 Aug 29.
3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase, catalyzes the aldol-type condensation between phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate (A5P) to produce the unusual 8-carbon sugar KDO8P, and inorganic phosphate. A 15.5-kb segment containing the kdsA gene from the hyperthermophilic bacterium Aquifex pyrophilus was cloned from a genomic library and sequenced. The native kdsA gene lacks a typical ribosome binding site, but contains a conserved U,A-rich sequence upstream to the start codon. The purified kdsA gene product catalyzes the formation of KDO8P from its natural substrates, PEP and A5P, as determined by (1)H NMR analysis. KDO8P synthase showed maximum activity at 80 degrees C and pH 5.5-6.0 at 10-min reaction assay. At temperatures of 70, 80, and 90 degrees C, the enzyme exhibited half-lives of 8.0, 2.25, and 0.5 h, respectively. The kinetic constants at 60 degrees C were K(m)(A5P)=70 microM, K(m)(PEP)=290 microM, and k(cat)=4 s(-1). The isolated enzyme contained 0.19 and 0.26 mol iron and zinc, respectively, per mole of enzyme subunit. Treatment with metal chelators eliminated enzyme activity, and by the addition of several divalent metal ions, the activity was restored and even exceeded the original activity. These results indicate that A. pyrophilus KDO8P synthase is a metal-dependent enzyme. A C11A mutant of KDO8P synthase from A. pyrophulis retained less than 1% of the wild-type activity and was shown to be incapable of metal binding.
3-脱氧-D-甘露-2-辛酮酸-8-磷酸（KDO8P）合酶催化磷酸烯醇丙酮酸（PEP）与D-阿拉伯糖-5-磷酸（A5P）之间的醛醇型缩合反应，生成异常的8碳糖KDO8P和无机磷酸盐。从嗜热细菌嗜热栖热菌的基因组文库中克隆并测序了一个包含kdsA基因的15.5 kb片段。天然的kdsA基因缺乏典型的核糖体结合位点，但在起始密码子上游含有一个保守的富含U、A的序列。通过（1）H NMR分析确定，纯化的kdsA基因产物催化从其天然底物PEP和A5P形成KDO8P。在10分钟反应测定中，KDO8P合酶在80℃和pH 5.5 - 6.0时表现出最大活性。在70℃、80℃和90℃的温度下，该酶的半衰期分别为8.0小时、2.25小时和0.5小时。在60℃时的动力学常数为K(m)(A5P)=70 microM，K(m)(PEP)=290 microM，k(cat)=4 s(-1)。每摩尔酶亚基，分离得到的酶分别含有0.19摩尔铁和0.26摩尔锌。用金属螯合剂处理会消除酶活性，而通过添加几种二价金属离子，活性得以恢复，甚至超过原始活性。这些结果表明嗜热栖热菌KDO8P合酶是一种金属依赖性酶。来自嗜热栖热菌的KDO8P合酶的C11A突变体保留的野生型活性不到1%，并且被证明无法结合金属。
