Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm1021

Citation
Mf. Dunn et al., Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm1021, ARCH MICROB, 176(5), 2001, pp. 355-363
Citations number
47
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
ARCHIVES OF MICROBIOLOGY
ISSN journal
0302-8933 → ACNP
Volume
176
Issue
5
Year of publication
2001
Pages
355 - 363
Database
ISI
SICI code
0302-8933(200111)176:5<355:CACOTP>2.0.ZU;2-E
Abstract
The gene encoding pyruvate carboxylase (pyc) was isolated from a Sinorhizob ium meliloti Rm1021 cosmid bank by complementation of a Rhizobium tropici p yc mutant. PYC-negative mutants of S. meliloti Rm1021 were isolated by tran sposon mutagenesis and were unable to grow with glucose or pyruvate as sole carbon sources, but were symbiotically competent in combination with alfal fa plants. PYC activity assays, pyc::lacZ gene fusion studies and an in viv o biotinylation assay showed that PYC activity in S. meliloti was dependent mainly on biotin availability and not on changes in gene transcription. Th e subunit and holo-enzyme molecular masses of the S. meliloti PYC indicated that the enzyme was an alpha (4) homotetramer. The S. meliloti PYC had a h igh apparent Ka (0.23 mM) for the allosteric activator acetyl-CoA and was p roduct-inhibited by sub-millimolar concentrations of oxaloacetate. In contr ast to other bacterial alpha (4)-PYCS which have been characterized, the S. meliloti enzyme was not strongly inhibited by L-aspartate.