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首页> 外文期刊>Biochemistry >Reconstitution of a defunct glycolytic pathway via recruitment of ambiguous sugar kinases
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Reconstitution of a defunct glycolytic pathway via recruitment of ambiguous sugar kinases

机译：通过募集不明确的糖激酶来重构已失效的糖酵解途径

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During a recent investigation of the persistence of substrate ambiguity in contemporary enzymes, we identified three distinct ambiguous sugar kinases embedded within the modem Escherichia coli genome [Miller, B. G., and Raines, R. T. (2004) Biochemistry 43, 6387-6392]. These catalysts are the YajF, YcfX, and NanK polypeptides, all of which possess rudimentary glucokinase activities. Here, we report on the discovery of a fourth bacterial kinase with ambiguous substrate specificity. AlsK phosphorylates the glucose epimer, D-allose, with a k(cat)/K-m value of 6.5 x 10(4) M-1 s(-1). AlsK also phosphorylates D-glucose, with a k(cat)/K-m value that is 10(5)-fold lower than the k(cat)/K-m value displayed by native E. coli glucokinase. Overexpression of the alsK gene relieves the auxotrophy of a glucokinase-deficient bacterium, demonstrating that weak enzymatic activities derived from ambiguous catalysts can provide organisms with elaborated metabolic capacities. To explore how ambiguous catalysts are recruited to provide new functions, we placed the glucokinase-deficient bacterium under selection for growth at the expense of glucose. Under these conditions, the bacterium acquires a spontaneous mutation in the putative promoter region of the yajF gene, a locus previously shown to encode a sugar kinase with relaxed substrate specificity. The point mutation regenerates a consensus sigma(70) promoter sequence that leads to a 94-fold increase in the level of yajF expression. This increase provides sufficient glucokinase activity for reconstitution of the defunct glycolytic pathway of the bacterial auxotroph. Our current findings indicate that ambiguous enzymatic activities continue to play an important role in the evolution of new metabolic pathways, and provide insight into the molecular mechanisms that facilitate the recruitment of such catalysts during periods of natural selection.
机译：在对现代酶中底物歧义的持久性的最新研究中，我们鉴定了嵌入在现代大肠杆菌基因组内的三种不同的歧义糖激酶[Miller，B. G.，and Raines，R. T.（2004）Biochemistry 43，6387-6392]。这些催化剂是YajF，YcfX和NanK多肽，它们均具有基本的葡萄糖激酶活性。在这里，我们报告发现具有歧义底物特异性的第四个细菌激酶。 AlsK磷酸化葡萄糖差向异构体D-阿洛糖，其k（cat）/ K-m值为6.5 x 10（4）M-1 s（-1）。 AlsK还使D-葡萄糖磷酸化，其k（cat）/ K-m值比天然大肠杆菌葡萄糖激酶显示的k（cat）/ K-m值低10（5）倍。 alsK基因的过表达缓解了葡萄糖激酶缺陷型细菌的营养缺陷，表明源自歧义催化剂的弱酶活性可以为生物体提供精心设计的代谢能力。为了探索如何招募模棱两可的催化剂来提供新功能，我们选择了葡萄糖激酶缺陷型细菌进行生长选择，但要以葡萄糖为代价。在这些条件下，细菌在yajF基因的推定启动子区域获得了自发突变，该位点先前显示为编码具有松弛底物特异性的糖激酶的基因座。点突变可再生一个共有的sigma（70）启动子序列，从而导致yajF表达水平增加94倍。这种增加提供了足够的葡萄糖激酶活性，用于重构细菌营养缺陷型的已失效的糖酵解途径。我们目前的发现表明，模棱两可的酶活性在新的代谢途径的进化中继续发挥重要作用，并提供了在自然选择期间促进此类催化剂募集的分子机制的见解。

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