Abstract：
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Miller BG,Raines RTdoi
10.1021/bi0506268subject
Has Abstractpub_date
2005-08-16 00:00:00pages
10776-83issue
32eissn
0006-2960issn
1520-4995journal_volume
44pub_type
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