A 7-dimethylallyltryptophan synthase from Aspergillus fumigatus: overproduction, purification and biochemical characterization.
A putative prenyltransferase gene, Afu3g12930, was identified in the genome sequence of Aspergillus fumigatus. EAL92290, encoded by Afu3g12930, consists of 472 aa, with a molecular mass of about 53 kDa. The coding sequence of Afu3g12930 was cloned in pQE60, and overexpressed in Escherichia coli. The soluble His(6)-fusion protein was purified to apparent homogeneity, and characterized biochemically. The enzyme was found to catalyse the prenylation of Trp at the C-7 position of the indole moiety, in the presence of dimethylallyl diphosphate (DMAPP); therefore, it functions as a 7-dimethylallyltryptophan synthase (7-DMATS). The structure of the enzymic product was elucidated by NMR and MS analysis. K(m) values were 67 microM for DMAPP, and 137 microM for l-Trp. Geranyl diphosphate was not accepted as prenyl donor, while Trp-containing dipeptides were found to be aromatic substrates of 7-DMATS. 7-DMATS did not need divalent metal ions for its enzymic reaction, although Ca(2+) enhanced the reaction velocity slightly. The enzyme is the second dimethylallyltryptophan synthase identified in A. fumigatus. Interestingly, it shares a sequence identity of only 31 % at the amino acid level with another known dimethylallyltryptophan synthase, FgaPT2, from the same fungus; FgaPT2 prenylates l-Trp at the C-4 position of the indole ring. Afu3g12930 belongs to a putative biosynthetic gene cluster consisting of eight genes. Orthologous clusters were also identified in the genome sequences of Neosartorya fischeri and Aspergillus terreus. The putative roles of the genes in the cluster are discussed.
Kremer
Anika
A
Heinrich-Heine-Universität Düsseldorf, Institut für Pharmazeutische Biologie und Biotechnologie, Universitätsstrasse 1, D-40225 Düsseldorf, Germany.
Westrich
Lucia
L
Eberhard-Karls-Universität Tübingen, Pharmazeutische Biologie, Auf der Morgenstelle 8, D-72076 Tübingen, Germany.
Li
Shu-Ming
SM
Heinrich-Heine-Universität Düsseldorf, Institut für Pharmazeutische Biologie und Biotechnologie, Universitätsstrasse 1, D-40225 Düsseldorf, Germany.
D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism; D004224:Diterpenes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D045782:Hemiterpenes; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D005810:Multigene Family; D009943:Organophosphorus Compounds; D054876:Prenylation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism
D003067:Coenzymes; D004271:DNA, Fungal; D011756:Diphosphates; D004224:Diterpenes; D045782:Hemiterpenes; D008670:Metals; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C511282:geranyl diphosphate; C043060:3,3-dimethylallyl pyrophosphate; D014364:Tryptophan; D019883:Alkyl and Aryl Transferases; C032409:tryptophan dimethylallyltransferase