Properties of NAD(+)- and NADP(+)-dependent malic enzymes of Rhizobium (Sinorhizobium) meliloti and differential expression of their genes in nitrogen-fixing bacteroids.
The wild-type NAD(+)-dependent malic enzyme (dme) gene of Rhizobium (now Sinorhizobium) meliloti was cloned and localized to a 3.1 kb region isolated on the cosmid pTH69. This cosmid complemented the symbiotic nitrogen fixation (Fix-) phenotype of R. meliloti dme mutants. The dme gene was mapped by conjugation to between the cys-11 and leu-53 markers on the R. meliloti chromosome. beta-Galactosidase activities measured in bacterial strains carrying either dme-lacZ or tme-lacZ gene fusions (the tme gene encodes NADP(+)-dependent malic enzyme) indicated that the dme gene was expressed constitutively in free-living cells and in N2-fixing bacteroids whereas expression of the tme gene was repressed in bacteroids. The R. meliloti dme gene product (DME) was overexpressed in and partially purified from Escherichia coli. The properties of this enzyme, together with those of the NADP(+)-dependent malic enzyme (TME) partially purified from R. meliloti dme mutants, were determined. Acetyl-CoA inhibited DME but not TME activity. This result supports the hypothesis that DME, together with pyruvate dehydrogenase, forms a pathway in which malate is converted to acetyl-CoA.
Driscoll
Brian T
BT
Department of Biology, McMaster University, 1280 Main St W, Hamilton, Ontario, Canada L8S 4K1.
Finan
Turlough M
TM
Department of Biology, McMaster University, 1280 Main St W, Hamilton, Ontario, Canada L8S 4K1.
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003360:Cosmids; D004794:Enzyme Repression; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D009586:Nitrogen Fixation / Q000235:genetics*; D015183:Restriction Mapping; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics; D013559:Symbiosis
D002413:Cations, Divalent; D008293:Malates; D000105:Acetyl Coenzyme A; C030298:malic acid; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+); C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)