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J. Biol. Chem., Vol. 283, Issue 23, 16068-16076, June 6, 2008

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Molecular Analysis of a 4-Dimethylallyltryptophan Synthase from Malbranchea aurantiaca^*

Yousong Ding, Robert M. Williams^¶, and David H. Sherman¹

From the Life Sciences Institute and Departments of Medicinal Chemistry, Microbiology & Immunology, and Chemistry, University of Michigan, Ann Arbor, Michigan 48109 and the ^¶Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523 and the University of Colorado Cancer Center, Aurora, Colorado 80045

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 µM. 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.

Received for publication, March 12, 2008

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-Bank^TM/EBI Data Bank with accession number(s) EU420001 [GenBank] and EU420002.

^* This work was supported, in whole or in part, by National Institutes of Health Grant CA070375 (to R. M. W.). This work was also supported by the Hans W. Vahlteich Professorship (to D. H. S.), and an Eli Lilly & Company pre-doctoral fellowship (to Y. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data, Figs. S1-S5, and Tables S1 and S2.

¹ To whom correspondence should be addressed: Life Sciences Institute and Dept. of Medicinal Chemistry, University of Michigan, 210 Wash-tenaw Ave, Ann Arbor, MI 48109-2216. Tel.: 734-615-9907; E-mail: davidhs{at}lsi.umich.edu.

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