Phillips C, Dohnalek J, Gover S, Barrett M P, Adams M J
Department of Biochemistry, Oxford University, Rex Richards Building, Oxford, OX1 3QU, UK.
J Mol Biol. 1998 Sep 25;282(3):667-81. doi: 10.1006/jmbi.1998.2059.
The three-dimensional structure of 6-phosphogluconate dehydrogenase (6PGDH) from the parasitic protozoan Trypanosoma brucei has been solved at 2.8 A resolution. This pentose phosphate pathway enzyme is NADP-dependent; NADPH generated in the reaction protects against oxidative stress. The enzyme crystallises in the space-group P3121 with a dimer in the asymmetric unit and cell dimensions a=b=135.13 A, c=116.74 A, alpha=beta=90 degrees, gamma=120 degrees. The structure has refined to R=18.6% (Rfree=27.3%) with good geometry. The amino acid sequence of T. brucei 6PGDH is only 35% identical to that of the sheep liver enzyme and significant activity differences have been observed. The active dimer assembles with the C-terminal tail of one subunit threaded through the other, forming part of the substrate binding site. The tail of T. brucei 6PGDH is shorter than that of the sheep enzyme and its terminal residues associate tightly with the second monomer. The three-dimensional structure shows this generates additional interactions between the subunits close to the active site; the coenzyme binding domain is thereby associated more tightly with the helical domain. Three residues, conserved in all other known sequences, are important in creating a salt bridge between monomers close to the substrate binding site. The differences could explain the 200-fold enhanced affinity observed for the substrate analogue 6-phospho-2-deoxy-D-gluconate and suggest targets for anti-parasite drug design. The coenzyme binding domain of 6PGDH has a beta-alpha-beta fold; while in most species the "fingerprint" sequence is GxAxxG, in the T. brucei enzyme it is GxGxxG. Additional interactions between the enzyme and the coenzyme bis-phosphate are likely in the parasite 6PGDH, accounting for greater inhibition (40-fold) of 2'5'-ADP. While the core of the T. brucei dimer was restrained during refinement, several conformational differences have been found between the monomers; those at the coenzyme binding site suggest the molecule could be asymmetric during the enzyme reaction.
寄生原生动物布氏锥虫6-磷酸葡萄糖酸脱氢酶（6PGDH）的三维结构已在2.8埃分辨率下解析出来。这种戊糖磷酸途径酶依赖于NADP；反应中生成的NADPH可抵御氧化应激。该酶以P3121空间群结晶，不对称单元中有一个二聚体，晶胞参数a = b = 135.13埃，c = 116.74埃，α = β = 90°，γ = 120°。结构精修后的R值为18.6%（自由R值为27.3%），几何结构良好。布氏锥虫6PGDH的氨基酸序列与绵羊肝脏酶的氨基酸序列仅有35%的同一性，且已观察到显著的活性差异。活性二聚体由一个亚基的C末端尾巴穿过另一个亚基组装而成，形成底物结合位点的一部分。布氏锥虫6PGDH的尾巴比绵羊酶的尾巴短，其末端残基与第二个单体紧密结合。三维结构表明，这在靠近活性位点的亚基之间产生了额外的相互作用；辅酶结合结构域因此与螺旋结构域结合得更紧密。在所有其他已知序列中保守的三个残基，对于在靠近底物结合位点的单体之间形成盐桥很重要。这些差异可以解释观察到的对底物类似物6-磷酸-2-脱氧-D-葡萄糖酸亲和力增强200倍的现象，并为抗寄生虫药物设计提供靶点。6PGDH的辅酶结合结构域具有β-α-β折叠；虽然在大多数物种中“指纹”序列是GxAxxG，但在布氏锥虫酶中是GxGxxG。寄生虫6PGDH中酶与辅酶双磷酸之间可能存在额外的相互作用，这解释了2'5'-ADP的抑制作用更强（40倍）的原因。虽然在精修过程中布氏锥虫二聚体的核心受到了限制，但在单体之间发现了几个构象差异；辅酶结合位点处的差异表明该分子在酶反应过程中可能是不对称的。
