δ1-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

Tzou Chi Huang, Yi Wen Huang, Hui Ju Hung, Chi-Tang Ho, Mei Li Wu

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Δ1-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.

Original languageEnglish (US)
Pages (from-to)5097-5102
Number of pages6
JournalJournal of agricultural and food chemistry
Volume55
Issue number13
DOIs
StatePublished - Jun 27 2007

All Science Journal Classification (ASJC) codes

  • Chemistry(all)
  • Agricultural and Biological Sciences(all)

Keywords

  • 2-acetyl-1-pyrroline
  • B. subtilis subsp. natto
  • Proline dehydrogenase
  • δ-pyrroline-5-carboxylic acid

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