Acetylaszonalenin biosynthesis in Neosartorya fischeri. Identification of the biosynthetic gene cluster by genomic mining and functional proof of the genes by biochemical investigation.
Based on the structural information of acetylaszonalenin isolated from Neosartorya fischeri, a putative biosynthetic gene cluster was identified in the genome sequence of this fungus by genomic mining. This cluster consists of three genes coding for a putative non-ribosomal peptide synthetase (AnaPS), a prenyltransferase (AnaPT), and an acetyltransferase (AnaAT). The coding sequences of anaPT and anaAT were cloned in pQE70 and pQE60, respectively, and overexpressed in Escherichia coli. The soluble His(6) fusion proteins were purified to near homogeneity and characterized biochemically. The structures of the enzymatic products were elucidated by NMR and mass spectroscopy analysis. AnaPT was found to catalyze the reverse prenylation of (R)-benzodiazepinedione at position C3 of the indole moiety in the presence of dimethylallyl diphosphate, resulting in formation of aszonalenin. AnaAT was found to catalyze the acetylation of aszonalenin at position N1 of the indoline moiety in the presence of acetyl coenzyme A, resulting in formation of acetylaszonalenin. Km values of AnaPT were determined for dimethylallyl diphosphate at 156 microm and for (R)-benzodiazepinedione at 232 microm. Km values of AnaAT were determined for acetyl coenzyme A at 96 microm and for aszonalenin at 61 microm. The turnover numbers of the AnaPT and AnaAT reactions were determined at 1.5 and 0.14 s(-1), respectively.
Yin
Wen-Bing
WB
Philipps-Universita¨t Marburg, Institut fu¨r Pharmazeutische Biologie, Deutschhausstrasse 17A, D-35037 Marburg, Germany.
Grundmann
Alexander
A
Philipps-Universita¨t Marburg, Institut fu¨r Pharmazeutische Biologie, Deutschhausstrasse 17A, D-35037 Marburg, Germany.
Cheng
Jun
J
Philipps-Universita¨t Marburg, Institut fu¨r Pharmazeutische Biologie, Deutschhausstrasse 17A, D-35037 Marburg, Germany.
Li
Shu-Ming
SM
Philipps-Universita¨t Marburg, Institut fu¨r Pharmazeutische Biologie, Deutschhausstrasse 17A, D-35037 Marburg, Germany. Electronic address: shuming.li@staff.uni-marburg.de.
D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004122:Dimethylallyltranstransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016681:Genome, Fungal / Q000502:physiology; D007211:Indoles / Q000737:chemistry* / Q000378:metabolism; D005810:Multigene Family / Q000502:physiology; D055326:Neosartorya / Q000201:enzymology* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054876:Prenylation / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D007211:Indoles; D011994:Recombinant Proteins; D000123:Acetyltransferases; D004122:Dimethylallyltranstransferase; D010453:Peptide Synthases