Mycoplasma pneumoniae HPr kinase/phosphorylase.
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. 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, whereas 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, Fru1,6P2 and Pi by fluorescence spectroscopy. In agreement with the previously observed unique regulation, we found a very high affinity for ATP (K(d)=5.4 microM) compared with the HPrK/P proteins from other bacteria. The Kd for Fru1,6P2 was three orders of magnitude higher, which explains why Fru1,6P2 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.
Merzbacher
Matthias
M
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
Detsch
Christian
C
Hillen
Wolfgang
W
Stülke
Jörg
J
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins; D005635:Fructosediphosphates / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D009154:Mutation; D009177:Mycoplasma pneumoniae / Q000201:enzymology* / Q000235:genetics; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D005635:Fructosediphosphates; D010710:Phosphates; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; C110847:HPr kinase; D017346:Protein Serine-Threonine Kinases; C029063:fructose-1,6-diphosphate