Nowicki Matthew W, Kuaprasert Buabarn, McNae Iain W, Morgan Hugh P, Harding Marjorie M, Michels Paul A M, Fothergill-Gilmore Linda A, Walkinshaw Malcolm D
Structural Biochemistry Group, Institute of Structural and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh, UK.
J Mol Biol. 2009 Dec 4;394(3):535-43. doi: 10.1016/j.jmb.2009.09.041. Epub 2009 Sep 23.
The structures of Leishmania mexicana cofactor-independent phosphoglycerate mutase (Lm iPGAM) crystallised with the substrate 3-phosphoglycerate at high and low cobalt concentrations have been solved at 2.00- and 1.90-A resolutions. Both structures are very similar and the active site contains both 3-phosphoglycerate and 2-phosphoglycerate at equal occupancies (50%). Lm iPGAM co-crystallised with the product 2-phosphoglycerate yields the same structure. Two Co(2+) are coordinated within the active site with different geometries and affinities. The cobalt at the M1 site has a distorted octahedral geometry and is present at 100% occupancy. The M2-site Co(2+) binds with distorted tetrahedral geometry, with only partial occupancy, and coordinates with Ser75, the residue involved in phosphotransfer. When the M2 site is occupied, the side chain of Ser75 adopts a position that is unfavourable for catalysis, indicating that this site may not be occupied under physiological conditions and that catalysis may occur via a one-metal mechanism. The geometry of the M2 site suggests that it is possible for Ser75 to be activated for phosphotransfer by H-bonding to nearby residues rather than by metal coordination. The 16 active-site residues of Lm iPGAM are conserved in the Mn-dependent iPGAM from Bacillus stearothermophilus (33% overall sequence identity). However, Lm iPGAM has an inserted tyrosine (Tyr210) that causes the M2 site to diminish in size, consistent with its reduced metal affinity. Tyr210 is present in trypanosomatid and plant iPGAMs, but not in the enzymes from other organisms, indicating that there are two subclasses of iPGAMs.
墨西哥利什曼原虫非辅因子依赖性磷酸甘油酸变位酶（Lm iPGAM）在高钴和低钴浓度下与底物3-磷酸甘油酸结晶，其结构已分别在2.00埃和1.90埃分辨率下解析。两种结构非常相似，活性位点中3-磷酸甘油酸和2-磷酸甘油酸的占有率相等（均为50%）。Lm iPGAM与产物2-磷酸甘油酸共结晶得到相同的结构。活性位点内有两个Co(2+)，其配位几何结构和亲和力不同。M1位点的钴具有扭曲的八面体几何结构，占有率为100%。M2位点的Co(2+)以扭曲的四面体几何结构结合，占有率仅为部分，且与参与磷酸转移的Ser75配位。当M2位点被占据时，Ser75的侧链采取不利于催化的位置，这表明该位点在生理条件下可能未被占据，催化可能通过单金属机制发生。M2位点的几何结构表明，Ser75有可能通过与附近残基形成氢键而非通过金属配位来被激活进行磷酸转移。Lm iPGAM的16个活性位点残基在嗜热脂肪芽孢杆菌的锰依赖性iPGAM中保守（总体序列同一性为33%）。然而，Lm iPGAM有一个插入的酪氨酸（Tyr210），导致M2位点尺寸减小，与其降低的金属亲和力一致。Tyr210存在于锥虫和植物的iPGAM中，但不存在于其他生物体来源的酶中，这表明存在两类iPGAM。
