title: A new member of the DMATS superfamily from Aspergillus niger catalyzes prenylations of both tyrosine and tryptophan derivatives.
abstract: A putative prenyltransferase gene of the dimethylallyltryptophan synthase (DMATS) family, An13g01840, was identified in the genome sequence of Aspergillus niger. The deduced polypeptide CAK41583 consists of 465 amino acids with a calculated molecular mass of 52.7 kDa. To evaluate gene function, the coding sequence was cloned into pET28a and overexpressed in Escherichia coli. The soluble His6-fusion protein was purified to near homogeneity on Ni-NTA agarose and used for enzyme assays with diverse aromatic substrates in the presence of dimethylallyl diphosphate. HPLC analysis revealed product formation in the incubation mixtures with L-tyrosine and five derivatives thereof. Structure elucidation of the enzyme products by NMR and MS analyses confirmed O-prenylations and proved the identification of a tyrosine O-prenyltransferase (TyrPT). As in the case of SirD from Leptosphaeria maculans, TyrPT also accepted 4-amino-L-phenylalanine for an N-prenylation and L-tryptophan for a C7-prenylation. The K M values of TyrPT for L-tyrosine, L-tryptophan, and dimethylallyl diphosphate (DMAPP) were found to be 0.24, 0.19, and 0.71 mM, respectively. The k cat of L-tyrosine and L-tryptophan reactions were determined at 0.58 and 0.0053 s(-1), respectively. The results presented in this study enhance the relationship of tyrosine O- and tryptophan C7-prenyltranferases and provide meanwhile a new enzyme for production of prenylated derivatives. In comparison to the known tyrosine prenyltransferase SirD, TyrPT showed significantly higher catalytic activity for several substrates, e.g., 4-amino-L-phenylalanine as well as 4- and 5-methyl-DL-tryptophan.
authors: Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037, Marburg, Germany.
authors: Aili
authors: A
authors: Fan
authors: Huizhi
authors: H
authors: Chen
authors: Rui
authors: R
authors: Wu
authors: Hui
authors: H
authors: Xu
authors: Shu-Ming
authors: SM
authors: Li
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008970:Molecular Weight; D054876:Prenylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D014364:Tryptophan; D019883:Alkyl and Aryl Transferases; C032409:tryptophan dimethylallyltransferase