Significance of Highly Conserved Aromatic Residues in Micrococcus luteus B-P 26 Undecaprenyl Diphosphate Synthase

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Undecaprenyl diphosphate synthase catalyzes the sequential condensation of eight molecules of isopentenyl diphosphate (IPP) in the cis-configuration into farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of the bacterial cell wall. This cis-type prenyltransferase exhibits a quite different mode of binding of homoallylic substrate IPP from that of trans-type prenyltransferase [Kharel Y. et al. (2001) J. Biol. Chem. 276, 28459-28464]. In order to know the IPP binding mode in more detail, we selected six highly conserved residues in Regions III, IV, and V among nine conserved aromatic residues in Micrococcus luteus B-P 26 UPP synthase for substitution by site-directed mutagenesis. The mutant enzymes were expressed and purified to homogeneity, and then their effects on substrate binding and the catalytic function were examined. All of the mutant enzymes showed moderately similar far-UV CD spectra to that of the wildtype, indicating that none of the replacement of conserved aromatic residues affected the secondary structure of the enzyme. Kinetic analysis showed that the replacement of Tyr-71 with Ser in Region III, Tyr-148 with Phe in Region IV, and Trp-210 with Ala in Region V brought about 10-1, 600-fold decreases in the k_cat/K_m values compared to that of the wild-type but the K_m values for both substrates IPP and FPP resulted in only moderate changes. Substitution of Phe-207 with Ser in Region V resulted in a 13-fold increase in the K_m value for IPP and a 1, 000-2, 000-fold lower k_cat/K_m value than those of the wild-type, although the K_m values for FPP showed about no significant changes. In addition, the W224A mutant as to Region V showed 6-fold and 14-fold increased K_m values for IPP and FPP, respectively, and 100-250-fold decreased k_cat/K_m values as compared to those of the wild-type. These results suggested that these conserved aromatic residues play important roles in the binding with both substrates, IPP and FPP, as well as the catalytic function of undecaprenyl diphosphate synthase.