Oxaloacetate decarboxylase of Vibrio cholerae: purification, characterization, and expression of the genes in Escherichia coli

Abstract

The oxaloacetate decarboxylase (OAD) Na + pump consists of subunits a, b, and c, which are expressed from an oadGAB gene cluster present in various anaerobic bacteria. Vibrio cholerae has two copies of oad genes, which are termed oad-1 and oad-2. The oad-2 genes are part of the citrate fermentation operon, while the oad-1 genes are flanked by genes encoding products not involved in a catabolic pathway. The gene sequences of oad-1 and oad-2 of V. cholerae strain O395-N1 were determined. The apparent frameshift in the published sequence of the oadA-2 gene from V. cholerae El Tor N16961 was not present in strain O395-N1. Upon anaerobic growth of V. cholerae on citrate, exclusively the oad-2 genes are expressed. OAD was isolated from these cells by monomeric avidin-Sepharose affinity chromatography. The enzyme was of higher specific activity than that from Klebsiella pneumoniae and was significantly more stable. Decarboxylase activity was Na + dependent, and the activation profile showed strong cooperativity with a Hill coefficient n H =1.8. Oxalate and oxomalonate inhibited the enzyme with half-maximal concentrations of 10 lM and 200 lM, respectively. After reconstitution into proteoliposomes, the enzyme acted as a Na + pump. With size-exclusion chromatography, the enzyme eluted in a symmetrical peak at a retention volume corresponding to an apparent molecular mass of approximately 570 kDa, suggesting a tetrameric structure for OAD-2. The two oad gene clusters were heterologously expressed in Escherichia coli, and the decarboxylases were isolated from the host cells.

Genome of Methylobacillus flagellatus , Molecular Basis for Obligate Methylotrophy, and Polyphyletic Origin of Methylotrophy

“…In addition, a gene was identified encoding a product homologous to the alpha subunit of OAA decarboxylase (Mfla1512), another enzyme potentially capable of converting pyruvate into OAA. However, we were not able to identify genes for the beta and gamma subunits associated with this activity (12). An alternative way of synthe-sizing PEP would be from GAP as shown in Fig.…”

Quaternary Structure of the Oxaloacetate Decarboxylase Membrane Complex and Mechanistic Relationships to Pyruvate Carboxylases

“…1), corresponding to the protein sequences of the OAD complex isoform 2 from Vibrio cholerae (17), were produced in Escherichia coli Rosetta (C43) strain and purified by standard methodologies. Purification of the OAD complex from E. coli membranes was done essentially as described before (18). Further details can be found in supplemental Experimental Procedures.…”

“…Oligomerization State of the OAD Membrane Complex-An obvious question thus was whether this tetramerization is also relevant for the structure of the native membrane complex. To this end, we have done BN-PAGE to analyze the OAD membrane protein complex in Triton X-100-solubilized membranes of E. coli, a detergent already known to maintain the integrity of the complex (18). As shown in Fig.…”

Biochemical and Genetic Characterization of the Enterococcus faecalis Oxaloacetate Decarboxylase Complex

“…Oxaloacetate-decarboxylating enzymes of two different origins have been described (2). In Gram-negative pathogenic microorganisms, such as Klebsiella pneumoniae and Vibrio cholerae, for which the pathway has been extensively studied (3,4), oxaloacetate decarboxylase is a multisubunit Na ϩ pumping membrane-bound complex (termed OAD), while in Gram-positive lactic acid bacteria, such as Lactococcus lactis, the reaction is catalyzed by a soluble, singlesubunit enzyme, named CitM, which is a malic enzyme (ME) (5). In all microorganisms, the genes encoding these enzymes are clustered in the cit locus (Fig.…”

“…1A). In K. pneumoniae and V. cholerae, the expression of the OAD genes is induced together with other enzymes of the citrate fermentation pathway in the presence of citrate and anaerobiosis, conditions during which the tricarboxylic acid (TCA) cycle is not functional (3,6), while in fermentative L. lactis, for instance, the cit cluster is induced by acidification of the medium (7). Genome sequences of selected Gram-positive bacteria of the class Lactobacillales have revealed the presence of genes encoding a membrane-bound complex homologous to the OAD complex of Gram-negative bacteria, but the function of these was never studied (8).…”