Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm1021.
The gene encoding pyruvate carboxylase (pyc) was isolated from a Sinorhizobium meliloti Rm1021 cosmid bank by complementation of a Rhizobium tropici pyc mutant. PYC-negative mutants of S. meliloti Rm1021 were isolated by transposon mutagenesis and were unable to grow with glucose or pyruvate as sole carbon sources, but were symbiotically competent in combination with alfalfa plants. PYC activity assays, pyc::lacZ gene fusion studies and an in vivo biotinylation assay showed that PYC activity in S. meliloti was dependent mainly on biotin availability and not on changes in gene transcription. The subunit and holo-enzyme molecular masses of the S. meliloti PYC indicated that the enzyme was an alpha4 homotetramer. The S. meliloti PYC had a high apparent Ka (0.23 mM) for the allosteric activator acetyl-CoA and was product-inhibited by sub-millimolar concentrations of oxaloacetate. In contrast to other bacterial alpha4-PYCs which have been characterized, the S. meliloti enzyme was not strongly inhibited by L-aspartate.
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Programa de Ingeniería Metabólica, Centro de Investigación sobre Fijación de Nitrógeno, Universidad Nacional Autónoma de México, Apartado Postal 565-A, Cuernavaca, Morelos, Mexico. mike@cifn.unam.mx
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D001710:Biotin / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D000455:Medicago sativa / Q000382:microbiology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic / Q000235:genetics; D011766:Pyruvate Carboxylase / Q000235:genetics* / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D017422:Sequence Analysis, DNA; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013559:Symbiosis
D003470:Culture Media; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D001710:Biotin; D019289:Pyruvic Acid; D011766:Pyruvate Carboxylase; D005947:Glucose