Occurrence and expression of tricarboxylate synthases in Ralstonia eutropha.
2-Methylcitrate synthase (2-MCS1) and citrate synthase (CS) of Ralstonia eutropha strain H16 were separated by affinity chromatography and analyzed for their substrate specificities. 2-MCS1 used not only the primary substrate propionyl-CoA but also acetyl-CoA and, at a low rate, even butyryl-CoA and valeryl-CoA for condensation with oxaloacetate. The KM values for propionyl-CoA and acetyl-CoA were 0.061 or 0.35 mM, respectively. This enzyme is therefore a competitor for acetyl-CoA during biosynthesis of poly(3-hydroxybutyrate) (PHB) and has to be taken into account if metabolic fluxes are calculated for PHB biosynthesis. In contrast, CS could not use propionyl-CoA as a substrate. The gene-encoding CS (cisY) of R. eutropha was cloned and encodes for a protein consisting of 433 amino acids with a calculated molecular weight of 48,600 Da; it is not truncated in the N-terminal region. Furthermore, a gene encoding a second functionally active 2-methylcitrate synthase (2-MCS2, prpC2) was identified in the genome of R. eutropha. The latter was localized in a gene cluster with genes for an NAD(H)-dependent malate dehydrogenase and a putative citrate lyase. RT-PCR analysis of R. eutropha growing on different carbon sources revealed the transcription of prpC2. In addition, cells of recombinant Escherichia coli strains harboring prpC2 of R. eutropha exhibited high 2-MCS activity of 0.544 U mg-1. A prpC2 knockout mutant of R. eutropha exhibited an identical phenotype as the wild type if grown on different media. 2-MCS2 seems to be dispensable, and a function could not be revealed for this enzyme.
Ewering
Christian
C
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149, Münster, Germany.
Brämer
Christian O
CO
Bruland
Nadine
N
Bethke
Axel
A
Steinbüchel
Alexander
A
D000105:Acetyl Coenzyme A / Q000378:metabolism / Q000493:pharmacokinetics; D000214:Acyl Coenzyme A / Q000378:metabolism / Q000493:pharmacokinetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002244:Carbon; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D003470:Culture Media; C024343:butyryl-coenzyme A; C009061:propionyl-coenzyme A; C044180:valeryl-coenzyme A; D000105:Acetyl Coenzyme A; D002244:Carbon; D002950:Citrate (si)-Synthase; C112399:2-methylcitrate synthase; D007652:Oxo-Acid-Lyases