Ergot alkaloid biosynthesis in Aspergillus fumigatus: conversion of chanoclavine-I to chanoclavine-I aldehyde catalyzed by a short-chain alcohol dehydrogenase FgaDH.
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).
Wallwey
Christiane
C
Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037, Marburg, Germany.
Matuschek
Marco
M
Li
Shu-Ming
SM
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D001232:Aspergillus fumigatus / Q000201:enzymology / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004873:Ergolines / Q000378:metabolism*; D004876:Ergot Alkaloids / Q000096:biosynthesis* / Q000737:chemistry; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism; D016133:Polymerase Chain Reaction
D004873:Ergolines; D004876:Ergot Alkaloids; D005656:Fungal Proteins; D009243:NAD; C006959:chanoclavine; D000426:Alcohol Dehydrogenase