Among the few regulatory proteins encoded by Mycoplasma pneumoniae is HPr kinase/phosphatase (HPrK/P), the key regulator of carbon metabolism in low-GC Gram-positive bacteria. The corresponding gene, hprK, and the gene encoding the target protein HPr, ptsH, were overexpressed. In vitro analysis of the purified proteins confirmed ATP-dependent phosphorylation of HPr by HPrK/P. In contrast to HPrK/P of Bacillus subtilis, which is by default a phosphatase and needs high ATP concentrations for kinase activity, the M. pneumoniae enzyme exhibits kinase activity at very low ATP concentrations and depends on P i for phosphatase activity. This inverted control of enzymic activity may result from the adaptation to very different ecological niches. While the standard activities of HPrK/P from M. pneumoniae and other Grampositive bacteria differ, they are both modulated by the concentration of ATP, P i and glycolytic intermediates. Site-directed mutagenesis of a potential ATPbinding site and of the HPrK/P signature sequence resulted in four different activity classes : (i) inactive proteins, (ii) enzymes with reduced kinase and phosphatase activities, (iii) enzymes that had lost phosphatase, but not kinase activity, and (iv) enzymes that exhibited increased phosphatase activity.
HPr kinase/phosphatase (HPrK/P) is the key protein in regulation of carbon metabolism in Bacillus subtilis and many other Gram-positive bacteria. Whether this enzyme acts as a kinase or phosphatase is determined by the nutrient status of the cell. Mutational analysis of residues in a Walker A box nucleotide-binding motif revealed that it is not only important for kinase but is also involved in phosphatase activity. In addition, a signature sequence specifically conserved among HPrK/P orthologues is required for phosphatase activity and may be involved in interaction with HPr/HPr-(Ser46)-P. Carbon catabolite repression was abolished in a B. subtilis strain expressing a mutant form of HPrK/P deficient in kinase and phosphatase activities. The growth characteristics of this strain were similar to those of the wild-type. In contrast, B. subtilis strains expressing HPrK/P with partial kinase and no phosphatase activities showed growth impairment but exhibited catabolite repression.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.