title: Delta1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline.
abstract: Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.
authors.affiliation: Institute of Biotechnology, National Pingtung University of Science & Technology, 912, Pingtung, Taiwan.
authors.forename: Tzou-Chi
authors.initials: TC
authors.lastname: Huang
authors.forename: Yi-Wen
authors.initials: YW
authors.lastname: Huang
authors.forename: Hui-Ju
authors.initials: HJ
authors.lastname: Hung
authors.forename: Chi-Tang
authors.initials: CT
authors.lastname: Ho
authors.forename: Mei-Li
authors.initials: ML
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D011394:Proline Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011758:Pyrroles / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D011758:Pyrroles; D011994:Recombinant Proteins; C015485:delta-1-pyrroline-5-carboxylate; D011394:Proline Oxidase; C426303:2-acetyl-1-pyrroline