Huang Tzou-Chi, Huang Yi-Wen, Hung Hui-Ju, Ho Chi-Tang, Wu Mei-Li
Institute of Biotechnology, National Pingtung University of Science & Technology, 912, Pingtung, Taiwan.
J Agric Food Chem. 2007 Jun 27;55(13):5097-102. doi: 10.1021/jf0700576. Epub 2007 May 31.
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.
脯氨酸脱氢酶（PRODH）催化Δ1-吡咯啉-5-羧酸（P5C）的生物合成。克隆了枯草芽孢杆菌纳豆芽孢杆菌亚种的脯氨酸脱氢酶基因（BnPRODH）并在大肠杆菌中表达。对该克隆进行核苷酸序列分析，发现一个开放阅读框，其编码一个由302个氨基酸组成的多肽，计算分子量为34.5 kDa。推导的氨基酸序列显示与细菌PRODH和大肠杆菌的PutA具有序列相似性。将BnPRODH基因克隆到pET21b中，并在大肠杆菌BL21(DE3)中高水平表达。表达的蛋白通过镍离子亲和柱层析纯化至均一性后进行表征。纯化的重组BnPRODH用于生产P5C。建立了由P5C和甲基乙二醛组成的模型系统来研究2-乙酰基-1-吡咯啉的形成。我们的数据表明，由纯化的重组PRODH将L-脯氨酸转化而来的P5C可能直接与甲基乙二醛反应形成2-AP。P5C/甲基乙二醛途径代表了首次报道的一种生物学机制，通过该机制PRODH可在体外合成2-AP。
