Morandi P, Valzasina B, Colombo C, Curti B, Vanoni M A
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
Biochemistry. 2000 Feb 1;39(4):727-35. doi: 10.1021/bi9920329.
To contribute to the understanding of glutamate synthase and of beta subunit-like proteins, which have been detected by sequence analyses, we identified the NADPH-binding site out of the two potential ADP-binding regions found in the beta subunit. The substitution of an alanyl residue for G298 of the beta subunit of Azospirillum brasilense glutamate synthase (the second glycine in the GXGXXA fingerprint of the postulated NADPH-binding site) yielded a protein species in which the flavin environment and properties are unaltered. On the contrary, the binding of the pyridine nucleotide substrate is significantly perturbed demonstrating that the C-terminal potential ADP-binding fold of the beta subunit is indeed the NADPH-binding site of the enzyme. The major effect of the G298A substitution in the GltS beta subunit consists of an approximately 10-fold decrease of the affinity of the enzyme for pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH. By combining kinetic measurements and absorbance-monitored equilibrium titrations of the G298A-beta subunit mutant, we conclude that also the positioning of its nicotinamide portion into the active site is altered thus preventing the formation of a stable charge-transfer complex between reduced FAD and NADP(+). During the course of this work, the Azospirillum DNA regions flanking the gltD and gltB genes, the genes encoding the GltS beta and alpha subunits, respectively, were sequenced and analyzed. Although the Azospirillum GltS is similar to the enzyme of other bacteria, it appears that the corresponding genes differ with respect to their arrangement in the chromosome and to the composition of the glt operon: no genes corresponding to E. coli and Klebsiella aerogenes gltF or to Bacillus subtilis gltC, encoding regulatory proteins, are found in the DNA regions adjacent to that containing gltD and gltB genes in Azospirillum. Further studies are needed to determine if these findings also imply differences in the regulation of the glt genes expression in Azospirillum (a nitrogen-fixing bacterium) with respect to enteric bacteria.
为了促进对通过序列分析检测到的谷氨酸合酶和β亚基样蛋白的理解，我们在β亚基中发现的两个潜在ADP结合区域中确定了NADPH结合位点。用丙氨酰残基取代巴西固氮螺菌谷氨酸合酶β亚基的G298（假定的NADPH结合位点的GXGXXA指纹中的第二个甘氨酸）产生了一种蛋白质，其中黄素环境和性质未改变。相反，吡啶核苷酸底物的结合受到显著干扰，表明β亚基的C端潜在ADP结合折叠确实是该酶的NADPH结合位点。GltSβ亚基中G298A取代的主要影响是该酶对吡啶核苷酸的亲和力降低了约10倍，而对NADPH还原酶的速率几乎没有影响。通过结合G298A-β亚基突变体的动力学测量和吸光度监测的平衡滴定，我们得出结论，其烟酰胺部分在活性位点的定位也发生了改变，从而阻止了还原型FAD和NADP(+)之间稳定的电荷转移复合物的形成。在这项工作过程中，对分别编码GltSβ亚基和α亚基的gltD和gltB基因两侧的巴西固氮螺菌DNA区域进行了测序和分析。尽管巴西固氮螺菌GltS与其他细菌的酶相似，但相应的基因在染色体中的排列和glt操纵子的组成方面似乎有所不同：在巴西固氮螺菌中，与包含gltD和gltB基因的区域相邻的DNA区域中未发现与大肠杆菌和产气克雷伯菌的gltF或枯草芽孢杆菌的gltC相对应的基因，这些基因编码调节蛋白。需要进一步研究以确定这些发现是否也意味着固氮细菌巴西固氮螺菌与肠道细菌在glt基因表达调控方面存在差异。
