Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
Mol Microbiol. 2011 Jan;79(1):76-93. doi: 10.1111/j.1365-2958.2010.07432.x. Epub 2010 Nov 2.
Rubrobacter xylanophilus is the only actinobacterium known to accumulate the organic solute mannosylglycerate (MG); moreover, the accumulation of MG is constitutive. The key enzyme for MG synthesis, catalysing the conversion of GDP-mannose (GDP-Man) and D-3-phosphoglycerate (3-PGA) into the phosphorylated intermediate mannosyl-3-phosphoglycerate and GDP, was purified from R. xylanophilus cell extracts and the corresponding gene was expressed in E. coli. Despite the related solute glucosylglycerate (GG) having never been detected in R. xylanophilus, the cell extracts and the pure recombinant mannosyl-3-phosphoglycerate synthase (MpgS) could also synthesize glucosyl-3-phosphoglycerate (GPG), the precursor of GG, in agreement with the higher homology of the novel MpgS towards GPG-synthesizing mycobacterial glucosyl-3-phosphoglycerate synthases (GpgS) than towards MpgSs from hyper/thermophiles, known to accumulate MG under salt or thermal stress. To understand the specificity and substrate ambiguity of this novel enzyme, we determined the crystal structure of the unliganded MpgS and of its complexes with the nucleotide and sugar donors, at 2.2, 2.8 and 2.5 Å resolution respectively. The first three-dimensional structures of a protein from this extremely gamma-radiation-resistant thermophile here reported show that MpgS (GT81 family) contains a GT-A like fold and clearly explain its nucleotide and sugar-donor specificity. In the GDP-Man complex, a flexible loop ((254) RQNRHQ(259) ), located close to the active site moves towards the incoming sugar moiety, providing the ligands for both magnesium ion co-ordination and sugar binding. A triple mutant of R. xylanophilus MpgS, mimicking the (206) PLAGE(210) loop stabilizing hydrogen bond network observed for mycobacterial GpgSs, reduces significantly the affinity to GDP-Man, implicating this loop in the sugar-donor discrimination.
红球菌属木聚糖嗜热菌是唯一已知能够积累有机溶质甘露糖基甘油（MG）的放线菌；此外，MG 的积累是组成型的。催化 GDP-甘露糖（GDP-Man）和 D-3-磷酸甘油（3-PGA）转化为磷酸化中间体甘露糖-3-磷酸甘油和 GDP 的 MG 合成关键酶，从红球菌属木聚糖嗜热菌细胞提取物中纯化，并在大肠杆菌中表达相应基因。尽管相关溶质葡萄糖基甘油（GG）从未在红球菌属木聚糖嗜热菌中检测到，但细胞提取物和纯重组甘露糖-3-磷酸甘油合酶（MpgS）也可以合成 GG 的前体葡萄糖基-3-磷酸甘油（GPG），这与新型 MpgS 对合成 GG 的分枝杆菌葡萄糖基-3-磷酸甘油合酶（GpgS）的同源性高于对已知在盐或热应激下积累 MG 的嗜热/超嗜热微生物的 MpgS 更高一致。为了了解这种新型酶的特异性和底物模糊性，我们测定了未配位的 MpgS 及其与核苷酸和糖供体复合物的晶体结构，分辨率分别为 2.2、2.8 和 2.5 Å。从这个极其耐伽马辐射的耐热菌中首次报道的三种蛋白质三维结构表明，MpgS（GT81 家族）含有 GT-A 样折叠，并清楚地解释了其核苷酸和糖供体特异性。在 GDP-Man 复合物中，靠近活性位点的一个柔性环（（254）RQNRHQ（259））向进入的糖部分移动，为镁离子配位和糖结合提供配体。红球菌属木聚糖嗜热菌 MpgS 的三重突变体，模拟了分枝杆菌 GpgSs 观察到的（206）PLAGE（210）环稳定氢键网络，显著降低了与 GDP-Man 的亲和力，表明该环参与了糖供体的识别。
