The domains carrying the opposing activities in adenylyltransferase are separated by a central regulatory domain.
Adenylyltransferase is a bifunctional enzyme that controls the enzymatic activity of dodecameric glutamine synthetase in Escherichia coli by reversible adenylylation and deadenylylation. Previous studies showed that the two similar but chemically distinct reactions are carried out by separate domains within adenylyltransferase. The N-terminal domain carries the deadenylylation activity, and the C-terminal domain carries the adenylylation activity [Jaggi R, van Heeswijk WC, Westerhoff HV, Ollis DL & Vasudevan SG (1997) EMBO J16, 5562-5571]. In this study, we further map the domain junctions of adenylyltransferase on the basis of solubility and enzymatic analysis of truncation constructs, and show for the first time that adenylyltransferase has three domains: the two activity domains and a central, probably regulatory (R), domain connected by interdomain Q-linkers (N-Q1-R-Q2-C). The various constructs, which have the opposing domain and or central domain removed, all retain their activity in the absence of their respective nitrogen status indicator, i.e. PII or PII-UMP. A panel of mAbs to adenylyltransferase was used to demonstrate that the cellular nitrogen status indicators, PII and PII-UMP, probably bind in the central regulatory domain to stimulate the adenylylation and deadenylylation reactions, respectively. In the light of these results, intramolecular signaling within adenylyltransferase is discussed.
Clancy
Paula
P
Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Australia.
Xu
Yibin
Y
van Heeswijk
Wally C
WC
Vasudevan
Subhash G
SG
Ollis
David L
DL
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018604:Epitope Mapping; D004926:Escherichia coli / Q000201:enzymology; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins / Q000378:metabolism
D009097:Multienzyme Complexes; D051922:PII Nitrogen Regulatory Proteins; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase; C022515:regulatory protein uridylyltransferase; D005974:Glutamate-Ammonia Ligase