Biochemical and genetic characterization of the Enterococcus faecalis oxaloacetate decarboxylase complex

Appl Environ Microbiol. 2013 May;79(9):2882-90. doi: 10.1128/AEM.03980-12. Epub 2013 Feb 22.

Abstract

Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • Carboxy-Lyases / genetics*
  • Carboxy-Lyases / isolation & purification
  • Carboxy-Lyases / metabolism
  • Citric Acid / metabolism
  • Cytoplasm / enzymology
  • Enterococcus faecalis / enzymology*
  • Enterococcus faecalis / genetics
  • Enterococcus faecalis / physiology
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Fermentation
  • Hydrogen-Ion Concentration
  • Multienzyme Complexes / genetics*
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism
  • Oxaloacetic Acid / metabolism
  • Protein Subunits
  • Recombinant Proteins
  • Sequence Deletion
  • Transgenes

Substances

  • Bacterial Proteins
  • Multienzyme Complexes
  • Protein Subunits
  • Recombinant Proteins
  • Citric Acid
  • Oxaloacetic Acid
  • Carboxy-Lyases
  • oxaloacetate decarboxylase