Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
Mol Microbiol. 2012 Aug;85(3):513-34. doi: 10.1111/j.1365-2958.2012.08124.x. Epub 2012 Jul 12.
The enzymes phosphomannomutase (PMM), phospho-N-acetylglucosamine mutase (PAGM) and phosphoglucomutase (PGM) reversibly catalyse the transfer of phosphate between the C6 and C1 hydroxyl groups of mannose, N-acetylglucosamine and glucose respectively. Although genes for a candidate PMM and a PAGM enzymes have been found in the Trypanosoma brucei genome, there is, surprisingly, no candidate gene for PGM. The TbPMM and TbPAGM genes were cloned and expressed in Escherichia coli and the TbPMM enzyme was crystallized and its structure solved at 1.85 Å resolution. Antibodies to the recombinant proteins localized endogenous TbPMM to glycosomes in the bloodstream form of the parasite, while TbPAGM localized to both the cytosol and glycosomes. Both recombinant enzymes were able to interconvert glucose-phosphates, as well as acting on their own definitive substrates. Analysis of sugar nucleotide levels in parasites with TbPMM or TbPAGM knocked down by RNA interference (RNAi) suggests that, in vivo, PGM activity is catalysed by both enzymes. This is the first example in any organism of PGM activity being completely replaced in this way and it explains why, uniquely, T. brucei has been able to lose its PGM gene. The RNAi data for TbPMM also showed that this is an essential gene for parasite growth.
磷酸甘露糖变位酶（PMM）、磷酸 N-乙酰葡萄糖胺变位酶（PAGM）和磷酸葡萄糖变位酶（PGM）这三种酶可将磷酸可逆地从甘露糖、N-乙酰葡萄糖胺和葡萄糖的 C6 和 C1 羟基之间转移。虽然在布氏锥虫基因组中发现了 PMM 和 PAGM 酶的候选基因，但令人惊讶的是，PGM 却没有候选基因。TbPMM 和 TbPAGM 基因在大肠杆菌中被克隆和表达，TbPMM 酶被结晶并以 1.85 Å 的分辨率解决了其结构。针对重组蛋白的抗体将内源性 TbPMM 定位于寄生虫血循环形式的糖体内，而 TbPAGM 则定位于细胞质和糖体内。两种重组酶都能够相互转化葡萄糖磷酸，并且可以作用于自己的明确底物。通过 RNA 干扰（RNAi）敲低 TbPMM 或 TbPAGM 的寄生虫中糖核苷酸水平的分析表明，在体内，PGM 活性由这两种酶共同催化。这是在任何生物体中首次完全以这种方式替代 PGM 活性的例子，这也解释了为什么独特的布氏锥虫能够失去其 PGM 基因。针对 TbPMM 的 RNAi 数据还表明，这是寄生虫生长所必需的基因。
