Molecular cloning and expression in Escherichia coli of an active fused Zea mays L. D-amino acid oxidase.
D-Amino acid oxidase (DAAO) is an FAD-dependent enzyme that metabolizes D-amino acids in microbes and animals. However, such ability has not been identified in plants so far. We predicted a complete DAAO coding sequence consisting of 1158 bp and encoding a protein of 386 amino acids. We cloned this sequence from the leaf cDNA population of maize plants that could utilize D-alanine as a nitrogen source and grow normally on media containing D-Ala at the concentrations of 100 and 1000 ppm. For more understanding of DAAO ability in maize plant, we produced a recombinant plasmid by the insertion of isolated cDNA into the pMALc2X Escherichia coli expression vector, downstream of the maltose-binding protein coding sequence. The pMALc2X-DAAO vector was used to transform the TB1 strain of E. coli cells. Under normal growth conditions, fused DAAO (with molecular weight of about 78 kDa) was expressed up to 5 mg/liter of bacterial cells. The expressed product was purified by affinity chromatography and subjected to in vitro DAAO activity assay in the presence of five different D-amino acids. Fused DAAO could oxidize D-alanine and D-aspartate, but not D-leucine, D-isoleucine, and D-serine. The cDNA sequence reported in this paper has been submitted to EMBL databases under accession number AM407717.
Gholizadeh
A
A
Research Institute for Fundamental Sciences, University of Tabriz, Iran. aghz_bioch@yahoo.co.in
Kohnehrouz
B B
BB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D013237:Stereoisomerism; D003313:Zea mays / Q000201:enzymology* / Q000254:growth & development
D000596:Amino Acids; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D003605:D-Amino-Acid Oxidase