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J. Biol. Chem., Vol. 276, Issue 30, 28459-28464, July 27, 2001

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Identification of Significant Residues for Homoallylic Substrate Binding of Micrococcus luteus B-P 26 Undecaprenyl Diphosphate Synthase^*

Yugesh Kharel, Yuan-Wei Zhang, Masahiro Fujihashi, Kunio Miki^§, and Tanetoshi Koyama^¶

From the Institute of Multidisciplinary Research for Advanced Materials (formerly the Institute for Chemical Reaction Science), Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan, the  Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan, and the ^§ RIKEN Harima Institute/SPring-8, Koto 1-1-1, Mikazukicho, Sayo-gun, Hyogo 679-5148, Japan

The primary structure of cis-prenyltransferase is totally different from those of trans-prenyltransferases (Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Biol. Chem. 272, 19476-19481). To better understand the molecular mechanism of enzymatic cis-prenyl chain elongation, we selected seven charged residues in the conserved Region V and two of Phe-Ser motif in Region III of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26 for substitutions by site-directed mutagenesis and examined their effects on substrate binding and catalysis. Kinetic studies indicated that replacements of Arg-197 or Arg-203 with Ser, and Glu-216 with Gln resulted in 7-11-fold increases of K_m values for isopentenyl diphosphate and 18-1200-fold decreases of k_cat values compared with those of the wild-type enzyme. In addition, two mutants with respect to the Phe-Ser motif in Region III, F73A and S74A, showed 16-32-fold larger K_m values for isopentenyl diphosphate and 12-16-fold lower k_cat values than those of the wild-type. Furthermore, product analysis indicated that three mutants, F73A, S74A, and E216Q, yielded shorter chain prenyl diphosphates as their main products. These facts together with the protein structural analysis recently carried out (Fujihashi, M., Zhang, Y.-W., Higuchi, Y., Li, X.-Y., Koyama, T., and Miki, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4337-4342) indicated that the diphosphate moiety of homoallylic substrate is electrostatically recognized by the three charged amino acids, Arg-197, Arg-203, and Glu-216, in Region V and the Phe-Ser motif in Region III, also indispensable for homoallylic substrate binding as well as catalytic function. It was suggested that the undecaprenyl diphosphate synthase takes a different mode for the binding of isopentenyl diphosphate from that of trans-prenyl chain elongating enzymes.

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