Wallwey Christiane, Matuschek Marco, Li Shu-Ming
Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037, Marburg, Germany.
Arch Microbiol. 2010 Feb;192(2):127-34. doi: 10.1007/s00203-009-0536-1. Epub 2009 Dec 29.
Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaDH has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaDH comprises 261 amino acids with a molecular mass of about 27.8 kDa and contains the conserved motifs of classical short-chain dehydrogenases/reductases (SDRs), but shares no worth mentioning sequence similarity with SDRs and other known proteins. The coding region of fgaDH consisting of two exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE60 and overexpressed in E. coli. The soluble tetrameric His(6)-FgaDH was purified to apparent homogeneity and characterized biochemically. It has been shown that FgaDH catalyzes the oxidation of chanoclavine-I in the presence of NAD(+) resulting in the formation of chanoclavine-I aldehyde, which was unequivocally identified by NMR and MS analyzes. Therefore, FgaDH functions as a chanoclavine-I dehydrogenase and represents a new group of short-chain dehydrogenases. K (M) values for chanoclavine-I and NAD(+) were determined at 0.27 and 1.1 mM, respectively. The turnover number was 0.38 s(-1).
麦角生物碱是毒素，也是通过生物技术大规模生产的重要药物。在棒麦角菌素C（一种棒麦角碱型麦角生物碱）的生物合成基因簇中已鉴定出一个假定基因fgaDH。推导的基因产物FgaDH由261个氨基酸组成，分子量约为27.8 kDa，包含经典短链脱氢酶/还原酶（SDR）的保守基序，但与SDR和其他已知蛋白质没有值得一提的序列相似性。通过PCR从烟曲霉的cDNA文库中扩增出由两个外显子组成的fgaDH编码区，克隆到pQE60中并在大肠杆菌中过表达。可溶性四聚体His(6)-FgaDH被纯化至表观均一，并进行了生化表征。结果表明，FgaDH在NAD(+)存在下催化棒麦角菌素-I的氧化，生成棒麦角菌素-I醛，通过NMR和MS分析明确鉴定了该产物。因此，FgaDH作为棒麦角菌素-I脱氢酶发挥作用，代表了一组新的短链脱氢酶。棒麦角菌素-I和NAD(+)的K (M)值分别测定为0.27和1.1 mM。周转数为0.38 s(-1)。
