Plasmid-encoded asp operon confers a proton motive metabolic cycle catalyzed by an aspartate-alanine exchange reaction.
Tetragenococcus halophila D10 catalyzes the decarboxylation of L-aspartate with nearly stoichiometric release of L-alanine and CO(2). This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an L-aspartate-beta-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter --> aspD --> aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known L-aspartate-beta-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of L-aspartate-beta-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.
Abe
Keietsu
K
Laboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan. kabe@biochem.tohoku.ac.jp
Ohnishi
Fumito
F
Yagi
Kyoko
K
Nakajima
Tasuku
T
Higuchi
Takeshi
T
Sano
Motoaki
M
Machida
Masayuki
M
Sarker
Rafiquel I
RI
Maloney
Peter C
PC
D000409:Alanine / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D017920:Antiporters / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D007777:Lactobacillaceae / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011510:Proteolipids / Q000378:metabolism; D018892:Proton-Motive Force; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid
C411647:A1CF protein, human; D017920:Antiporters; D011510:Proteolipids; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C020485:proteoliposomes; D001224:Aspartic Acid; D006639:Histidine; D002262:Carboxy-Lyases; C019747:aspartate 4-decarboxylase; D000409:Alanine