Molecular analysis of a 4-dimethylallyltryptophan synthase from Malbranchea aurantiaca.
Prenyltransferases are widely distributed in prokaryotes and eukaryotes and play critical roles in cell signaling, protein trafficking, and elaboration of complex molecules in secondary metabolism. Numerous prenylated natural products have been isolated from diverse microorganisms, including bacteria and fungi. These complex metabolites possess a wide range of biological activities, with some showing promise as medicinal agents. On the other hand, many prenylated secondary metabolites have been described as toxins such as ergot alkaloids that have potent psychotropic activity. We have characterized a new prenyltransferase isolated from genomic DNA of Malbranchea aurentiaca RRC1813. Enzyme specificity was investigated with a series of amino acid substrates revealing its function as a 4-dimethylallyltryptophan synthase. Polypeptide sequence alignment analysis showed that it groups with a new class of prenyltransferase enzymes that lack the typical (N/D)DXXD motif found in these polypeptides. MaPT activity was not dependent on a divalent cation cofactor, although it was reversibly inactivated by 5 mm EDTA. Analysis of kinetic parameters showed reduced enzyme efficiency upon simple modification of l-Trp. Moreover, d-Trp had 0.5% relative activity and functioned as a competitive inhibitor with a K(i) of 40.41 microm. Finally, Thr-105, Asp-179, Lys-189, and Lys-261 in MaPT were serially mutated, and the resulting lesions displayed low or complete loss of activity. This study provides a detailed characterization of a prenyltransferase in Malbranchea species, reveals two enzyme inhibitors, and through site-directed mutagenesis identified several key amino acid residues in catalysis, yielding new insights into this important yet understudied class of natural product biosynthetic enzymes.
Ding
Yousong
Y
Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Williams
Robert M
RM
Sherman
David H
DH
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D004122:Dimethylallyltranstransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004492:Edetic Acid / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D016681:Genome, Fungal / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D020036:Onygenales / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D014364:Tryptophan / Q000737:chemistry
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D014364:Tryptophan; D004492:Edetic Acid; D019883:Alkyl and Aryl Transferases; D004122:Dimethylallyltranstransferase; C032409:tryptophan dimethylallyltransferase