title: Structure-function analysis of glutamine synthetase adenylyltransferase (ATase, EC 2.7.7.49) of Escherichia coli.
abstract: Glutamine synthetase adenylyltransferase (ATase) regulates the activity of glutamine synthetase by adenylylation and deadenylylation in response to signals of nitrogen and carbon status: glutamine, alpha-ketoglutarate, and the uridylylated and unmodified forms of the PII signal transduction protein. ATase consists of two conserved nucleotidyltransferase (NT) domains linked by a central region of approximately 200 amino acids. Here, we study the activities and regulation of mutated and truncated forms of ATase. Our results indicate the following. (i) The N-terminal NT domain contained the adenylyl-removing (AR) active site, and the C-terminal NT domain contained the adenylyltransferase (AT) active site. (ii) The enzyme contained a glutamine binding site, and glutamine increased the affinity for PII. (iii) The enzyme appeared to contain multiple sites for the binding of PII and PII-UMP. (iv) Truncated versions of ATase missing the C-terminal (NT) domain lacked both AT and AR activity, suggesting a role for the C-terminal NT domain in both activities. (v) The purified C-terminal NT domain and larger polypeptides containing this domain had significant basal AT activity, which was stimulated by glutamine. These polypeptides were indifferent to PII and PII-UMP, or their ATase activity was inhibited by either PII or PII-UMP. (vi) Certain point mutations in the central region or an internal deletion removing most of this part of the protein eliminated the AR activity and eliminated activation of the AT activity by PII, while not eliminating the binding of PII or PII-UMP. That is, these mutations in the central region appeared to destroy the communication between the PII and PII-UMP binding sites and the AT and AR active sites. (vii) Certain mutations in the central region of ATase appeared to dramatically improve the binding of glutamine to the enzyme. (viii) While the isolated AT and AR domains of ATase bound poorly to PII and PII-UMP, these domains bound PII and PII-UMP significantly better when linked to the central region of ATase. Together, our results indicate a highly coordinated enzyme, in which the AT and AR domains participate in each other's regulation and distant regulatory sites are in communication with each other. A model for the regulation of ATase by glutamine, PII, and PII-UMP consistent with all data is presented.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Jiang
authors.forename: Augen A
authors.initials: AA
authors.lastname: Pioszak
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Ninfa
mesh_terms: D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000187:drug effects / Q000235:genetics; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014542:Uridine Monophosphate / Q000378:metabolism
chemical_list: D051922:PII Nitrogen Regulatory Proteins; D014542:Uridine Monophosphate; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase; C022515:regulatory protein uridylyltransferase