Identifying latent enzyme activities: Substrate ambiguity within modern bacterial sugar kinases
Identifying latent enzyme activities: Substrate ambiguity within modern bacterial sugar kinases
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DOI：
10.1021/bi049424m
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发表时间：
2004-06-01
期刊：
影响因子：
2.9
通讯作者：
Raines, RT
中科院分区：
文献类型：
--
作者：
Miller, BG;Raines, RT
酶催化多种结构相关底物转化的能力可以使新催化功能的自然进化成为可能。然而，现代催化剂中这种底物模糊性的普遍性在很大程度上是未知的。为了寻找不明确的糖激酶，我们产生了一种不能进行糖酵解途径的第一步，即葡萄糖磷酸化的细菌。这种生物不能以葡萄糖作为唯一的碳源生存。在它的基因组中，我们发现三个DNA序列，当从一个强大的染色体外启动子转录时，可以补充生物体的营养缺陷型。这些序列包含nanK、yajF和ycjX基因。在体外，NanK、YajF和YcfX蛋白作为具有模糊底物特异性的基本葡萄糖激酶发挥功能，显示葡萄糖磷酸化的k（cat）/K-m值比内源性细菌葡萄糖激酶的k（cat）/K-m值低10（4）倍。我们的研究结果表明，现代基因组窝藏了丰富的潜在酶的活动和现存的代谢途径是模棱两可的，与他们通常的描述。
The ability of enzymes to catalyze the transformation of multiple, structurally related substrates could empower the natural evolution of new catalytic functions. The prevalence of such substrate ambiguity in modern catalysts, however, is largely unknown. To search for ambiguous sugar kinases, we generated a bacterium incapable of performing the first step of the glycolytic pathway, the phosphorylation of glucose. This organism cannot survive with glucose as its sole source of carbon. Within its genome, we find three DNA sequences that, when transcribed from a powerful extrachromosomal promoter, can complement the auxotrophy of the organism. These sequences contain the nanK, yajF, and ycjX genes. In vitro, the NanK, YajF, and YcfX proteins function as rudimentary glucokinases with ambiguous substrate specificites, displaying k(cat)/K-m values for the phosphorylation of glucose that are 10(4)-fold lower than the k(cat)/K-m value of endogenous bacterial glucokinase. Our findings suggest that modern genomes harbor a wealth of latent enzyme activities and that extant metabolic pathways are equivocal, in contrast to their usual depiction.