Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

• DOI: 10.1128/jb.184.11.2906-2913.2002
• Publication Date: 2002-07-27T10:09:29Z
• Authors: Keietsu Abe; Fumito Ohnishi; Kyoko Yagi; Tasuku Nakajima; Takeshi Higuchi; Motoaki Sano; Masayuki Machida; Rafiquel I. Sarker; Peter C. Maloney
• Keywords: Alanine; Aspartate carbamoyltransferase

ABSTRACT Tetragenococcus halophila D10 catalyzes the decarboxylation of l -aspartate with nearly stoichiometric release -alanine and CO 2 . This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid putative asp operon consisting two genes, which we designated aspD aspT , encoding an -aspartate-β-decarboxylase (AspD) aspartate-alanine antiporter (AspT), respectively, determined nucleotide sequences. The sequence analysis revealed that genes pD1 were following order: promoter → deduced amino acid AspD showed similarity to sequences known -aspartate-β-decarboxylases from Pseudomonas dacunhae Alcaligenes faecalis Hydropathy analyses suggested gene product encodes hydrophobic protein multiple membrane-spanning regions. was subcloned into Escherichia coli expression vector pTrc99A, cotranscribed resulting pTrcAsp. Expression E. coincided appearance capacity catalyze aspartate alanine. Histidine-tagged (AspDHis) also expressed purified cell extracts. AspDHis clearly exhibited activity -aspartate-β-decarboxylase. Recombinant AspT solubilized membranes reconstituted proteoliposomes. catalyzed self-exchange electrogenic heterologous exchange Thus, confers proton motive metabolic cycle decarboxylase, keeps intracellular levels alanine, countersubstrate for aspartate, high.