Biochemical and Genetic Characterization of the Enterococcus faecalis Oxaloacetate Decarboxylase Complex

Abstract

c 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.T he main activity of lactic acid bacteria (LAB) during fermentation is the catabolism of sugars present in food, producing lactic acid, but these microorganisms also have the capability to metabolize other substrates, such as citrate. Citrate fermentation by LAB leads to the production of aromatic compounds (diacetyl/ acetoin) and CO 2 , which contributes to the formation of so-called eyes (or holes) in cheeses. Thus, the utilization of citrate in milk by LAB has a positive effect on the quality of the end products. Therefore, the interest of the dairy industry in understanding citrate utilization by LAB has promoted research of the proteins involved in this metabolic pathway (1).Citrate fermentation in bacteria involves the intracellular conversion of citrate to oxaloacetate (OAA) and acetate by citrate lyase. OAA then is decarboxylated to pyruvate by oxaloacetate decarboxylase, which is then diverted into different metabolic pathways, depending on the metabolic needs. Oxaloacetate-de-

Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein

“…Deviating apparent molecular weight is also seen in other studies. [ 32 33 34 ] Structural rigidity and kinks caused by high proline content may decrease electrophoretic mobility of the protein. Gel shifting due to high content of proline residue in protein structure has been shown in other studies.…”

Ca 2+ -Citrate Uptake and Metabolism in Lactobacillus casei ATCC 334

“…Several energy-transducing steps may be discriminated in the pathway, including uptake of the Ca 2ϩ -citrate complex, which has been shown to be coupled to the uptake of protons in the case of homologous transporters (3,19,39), excretion of Ca 2ϩ by an unknown transporter that is likely to couple the flux to an antiport with protons, and the decarboxylation of oxaloacetate by the membrane-bound OAD that in the Gram-negative counterparts functions as a Na ϩ pump (6). A very recent study of the OAD complex of the closely related Grampositive bacterium Enterococcus faecalis showed that the soluble OAD-A subunit that presumably couples the decarboxylation reaction to ion pumping by the membrane-bound OAD-B subunit was found for a significant part in the cytoplasm, which potentially could have a negative effect on the free-energy conservation of the reaction (44). At any rate, the net result of the charge and proton movements across the membrane is zero, and the pathway does not seem to contribute to the metabolic energy pool in Lb.…”

Functional Analysis of the Citrate Activator CitO from Enterococcus faecalis Implicates a Divalent Metal in Ligand Binding

“…The cit cluster is encoded in two divergent operons: citHO encoding for the citrate transporter citH , and the regulatory protein citO . The catabolic enzymes are encoded upstream, in the minus strand, in the oadHDB-citCDEFX-oadA-citMG operon (Blancato et al, 2006 , 2008 ; Espariz et al, 2011 ; Repizo et al, 2013 ). There are two binding elements for CitO in the intergenic region, which regulate the expression of genes involved in the transport and degradation of citrate.…”