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HPr kinase/phosphorylase (HPrK/P) is the key regulator of carbon metabolism in many Gram-positive bacteria. It phosphorylates/dephosphorylates the HPr protein of the bacterial phosphotransferase system on a regulatory serine residue in response to the nutrient status of the cell. In Mycoplasma pneumoniae,HPrK/P is one of the very few regulatory proteins encoded in the genome. | Eur. J. Biochem. 271 367-374 2004 FEBS 2003 doi 10.1046 j.1432-1033.2003.03935.e Mycoplasma pneumoniae HPr kinase phosphorylase Assigning functional roles to the P-loop and the HPr kinase phosphorylase signature sequence motif Matthias Merzbacher Christian Detsch Wolfgang Hillen and Jorg Stiilke Lehrstuhl fur Mikrobiologie Institut fur Mikrobiologie Biochemie und Genetik der Friedrich-Alexander-Universitat Erlangen-Nurnberg Germany HPr kinase phosphorylase HPrK P is the key regulator of carbon metabolism in many Gram-positive bacteria. It phosphorylates dephosphorylates the HPr protein of the bacterial phosphotransferase system on a regulatory serine residue in response to the nutrient status of the cell. In Mycoplasma pneumoniae HPrK P is one of the very lew regulatory proteins encoded in the genome. The regulation of this enzyme by metabolites is unique among HPrK P proteins studied so far it is active as a kinase at low ATP concentrations ehereas the proteins from other bacteria need high ATP concentrations as an indicator of a good nutrient supply for kinase activity. We studied the interaction of M. pneumoniae HPrK P with ATP Fl ll 1.6P2 and Pi by fluorescence spectroscopy. In agreement with the previously observed unique regulation we found a very high affinity for ATP Kd 5.4 M compared with the HPrK P proteins from other bacteria. The Ki for Fru1 6P2 was three orders of magnitude higher which explains w- hy Fml 6P2 has only a weak regulatory effect on M. pneumoniae HPrK P. Mutations of two important regions in the active site of HPrK P the nucleotide binding P-loop and the HPrK P family signature sequence had different effects. P-loop region mutations strongly affect ATP binding and thus all enzymatic functions whereas the signature sequence motif seems to be important for the catalytic mechanism rather than for nucleotide binding. Keywords Gram-positive bacteria HPr kinase phosphory-lase Mycoplasma pneumoniae nutrients regulation. All organisms need to .
