Dissection of Escherichia coli glutamate 5-kinase: functional impact of the deletion of the PUA domain.
Glutamate 5-kinase (G5K) catalyzes the controlling first step of the synthesis of the osmoprotective amino acid proline, which feed-back inhibits G5K. Microbial G5K generally consists of one amino acid kinase (AAK) and one PUA (named after pseudo uridine synthases and archaeosine-specific transglycosylases) domain. To investigate the role of the PUA domain, we have deleted it from Escherichia coli G5K. We show that wild-type G5K requires free Mg for activity, it is tetrameric, and it aggregates to higher forms in a proline-dependent way. G5K lacking the PUA domain remains tetrameric, active, and proline-inhibitable, but the Mg requirement and the proline-triggered aggregation are greatly diminished and abolished, respectively, and more proline is needed for inhibition. We propose that the PUA domain modulates the function of the AAK domain, opening the way to potential PUA domain-mediated regulation of G5K; and that this domain moves, exposing new surfaces upon proline binding.
Pérez-Arellano
Isabel
I
Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler, 16, Valencia 46013, Spain.
Rubio
Vicente
V
Cervera
Javier
J
D000244:Adenosine Diphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011392:Proline / Q000037:antagonists & inhibitors / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion
D000244:Adenosine Diphosphate; D011392:Proline; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase; D008274:Magnesium