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| - δ1-Pyrroline-5-carboxylic Acid Formed by ProlineDehydrogenase from the Bacillus subtilis ssp. natto Expressedin Escherichia coli as a Precursor for 2-Acetyl-1-pyrroline
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| - 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 andexpressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-readingframe that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. Thededuced 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 tohomogeneity 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 purifiedrecombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathwayrepresents the first report of a biological mechanism by which 2-AP may be synthesized in vitro byPRODH. Keywords: Proline dehydrogenase; B. subtilis subsp. natto; 2-acetyl-1-pyrroline; δ1-pyrroline-5-carboxylic acid
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