Molecular cloning and catalytic characterization of a recombinant tropine biosynthetic tropinone reductase from Withania coagulans leaf

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• 作者：Amit K. Kushwaha ; Neelam S. Sangwan ; Sandhya Tripathi ; Rajender S. Sangwan ; ¹ ; ^{dr.sangwan@gmail.com}
• 关键词：WcTR-I ; Withania coagulans tropinone reductase I ; PCR ; Polymerase chain reaction ; RACE ; Rapid amplification of cDNA ends ; ORF ; Open reading frame ; kDa ; kilo Dalton ; pI ; Isoelectric point ; NADPH ; ¦Â-nicotinamide adenine dinucleotide phosphate reduced ; SDRs
• 刊名：Gene
• 出版年：2013
• 出版时间：10 March, 2013
• 年：2013
• 卷：516
• 期：2
• 页码：238-247
• 全文大小：1734 K

文摘

Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34 kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30 % retention of activity at 60 ¡ãC) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species.