In vitro studies of the uridylylation of the three PII protein paralogs from Rhodospirillum rubrum: the transferase activity of R. rubrum GlnD is regulated by alpha-ketoglutarate and divalent cations but not by glutamine.
P(II) proteins have been shown to be key players in the regulation of nitrogen fixation and ammonia assimilation in bacteria. The mode by which these proteins act as signals is by being in either a form modified by UMP or the unmodified form. The modification, as well as demodification, is catalyzed by a bifunctional enzyme encoded by the glnD gene. The regulation of this enzyme is thus of central importance. In Rhodospirillum rubrum, three P(II) paralogs have been identified. In this study, we have used purified GlnD and P(II) proteins from R. rubrum, and we show that for the uridylylation activity of R. rubrum GlnD, alpha-ketoglutarate is the main signal, whereas glutamine has no effect. This is in contrast to, e.g., the Escherichia coli system. Furthermore, we show that all three P(II) proteins are uridylylated, although the efficiency is dependent on the cation present. This difference may be of importance in understanding the effects of the P(II) proteins on the different target enzymes. Furthermore, we show that the deuridylylation reaction is greatly stimulated by glutamine and that Mn(2+) is required.
Jonsson
Anders
A
Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Nordlund
Stefan
S
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D020536:Enzyme Activators / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005973:Glutamine / Q000494:pharmacology; D007656:Ketoglutaric Acids / Q000494:pharmacology*; D051922:PII Nitrogen Regulatory Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D014166:Transferases / Q000302:isolation & purification / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000378:metabolism*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D020536:Enzyme Activators; D007656:Ketoglutaric Acids; D051922:PII Nitrogen Regulatory Proteins; D005973:Glutamine; D014542:Uridine Monophosphate; D014166:Transferases