Miller Brian G, Raines Ronald T
Department of Biochemistry, University of Wisconsin, Madison 53706-1544, USA.
Biochemistry. 2005 Aug 16;44(32):10776-83. doi: 10.1021/bi0506268.
During a recent investigation of the persistence of substrate ambiguity in contemporary enzymes, we identified three distinct ambiguous sugar kinases embedded within the modern 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.
在最近一项关于当代酶中底物模糊性持续性的研究中，我们在现代大肠杆菌基因组中鉴定出三种不同的模糊糖激酶[米勒，B.G.，和雷恩斯，R.T.（2004年）《生物化学》43卷，6387 - 6392页]。这些催化剂是YajF、YcfX和NanK多肽，它们都具有基本的葡萄糖激酶活性。在此，我们报告发现了第四种具有模糊底物特异性的细菌激酶。AlsK磷酸化葡萄糖差向异构体D - 阿洛糖，催化常数与米氏常数的比值（k(cat)/K(m)）为6.5×10⁴ M⁻¹ s⁻¹。AlsK也能磷酸化D - 葡萄糖，其k(cat)/K(m)值比天然大肠杆菌葡萄糖激酶的k(cat)/K(m)值低10⁵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，表明源自模糊催化剂的微弱酶活性可为生物体提供精细的代谢能力。为了探究模糊催化剂是如何被招募以提供新功能的，我们将葡萄糖激酶缺陷型细菌置于以葡萄糖为代价进行生长选择的条件下。在这些条件下，该细菌在yajF基因的假定启动子区域发生自发突变，该位点先前已被证明编码一种底物特异性较宽松的糖激酶。该点突变重新生成了一个共有σ⁷⁰启动子序列，导致yajF表达水平增加94倍。这种增加为细菌营养缺陷型中失效的糖酵解途径的重建提供了足够的葡萄糖激酶活性。我们目前的研究结果表明，模糊酶活性在新代谢途径的进化中继续发挥重要作用，并为在自然选择期间促进此类催化剂招募的分子机制提供了见解。
