Hahn F M, Eubanks L M, Testa C A, Blagg B S, Baker J A, Poulter C D
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
J Bacteriol. 2001 Jan;183(1):1-11. doi: 10.1128/JB.183.1.1-11.2001.
In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-xylulose 5-phosphate synthase A and B, containing a His(6) tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-D-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-xylulose 5-phosphate synthase A and B: K(m)(pyruvate) = 0.61 and 3.0 mM, K(m)(D-glyceraldehyde 3-phosphate) = 150 and 120 microM, and V(max) = 1.9 and 1.4 micromol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-D-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.
在真细菌、绿藻和植物叶绿体中，类异戊二烯生物合成的关键中间体异戊烯基二磷酸是通过甲基赤藓糖醇磷酸途径合成的。基本类异戊二烯单元的五个碳原子是通过丙酮酸和3-磷酸-D-甘油醛连接而成的。该反应由硫胺素二磷酸依赖性酶1-脱氧-D-木酮糖5-磷酸合酶催化。在荚膜红细菌中，两个开放阅读框（ORF）携带编码1-脱氧-D-木酮糖5-磷酸合酶的基因。ORF 2816位于光合作用相关基因簇中，与该细菌光合作用机制合成所需的大多数基因在一起，而ORF 2895位于基因组的其他位置。由ORF 2816和ORF 2895编码的蛋白质，即1-脱氧-D-木酮糖5-磷酸合酶A和B，含有一个His(6)标签，在大肠杆菌中合成，并分两步纯化至纯度大于95%。1-脱氧-D-木酮糖5-磷酸合酶A似乎是一种由68 kDa亚基组成的同型二聚体。开发了一种新的测定方法，并测定了1-脱氧-D-木酮糖5-磷酸合酶A和B的以下稳态动力学常数：K(m)(丙酮酸)=0.61和3.0 mM，K(m)(3-磷酸-D-甘油醛)=150和120 microM，在200 mM柠檬酸钠（pH 7.4）中V(max)=1.9和1.4微摩尔/分钟/毫克。编码1-脱氧-D-木酮糖5-磷酸合酶B的ORF补充了大肠杆菌FH11菌株中被破坏的必需dxs基因。
