Kharel Yugesh, Zhang Yuan-Wei, Fujihashi Masahiro, Miki Kunio, Koyama Tanetoshi
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577.
J Biochem. 2003 Dec;134(6):819-26. doi: 10.1093/jb/mvg207.
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 wild-type, 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 kcat/Km values compared to that of the wild-type but the Km 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 Km value for IPP and a 1,000-2,000-fold lower kcat/Km value than those of the wild-type, although the Km values for FPP showed about no significant changes. In addition, the W224A mutant as to Region V showed 6-fold and 14-fold increased Km values for IPP and FPP, respectively, and 100-250-fold decreased kcat/Km 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.
十一异戊烯基二磷酸合酶催化八个顺式构型的异戊烯基二磷酸（IPP）分子依次缩合形成法尼基二磷酸（FPP），进而生成十一异戊烯基二磷酸（UPP），UPP对于细菌细胞壁的生物合成不可或缺。这种顺式型异戊二烯基转移酶与反式型异戊二烯基转移酶相比，对同烯丙基底物IPP的结合模式有很大不同[Kharel Y.等人（2001年）《生物化学杂志》276卷，28459 - 28464页]。为了更详细地了解IPP的结合模式，我们在藤黄微球菌B - P 26 UPP合酶的九个保守芳香族残基中的区域III、IV和V中选择了六个高度保守的残基，通过定点诱变进行替换。对突变酶进行表达并纯化至同质，然后检测它们对底物结合和催化功能的影响。所有突变酶的远紫外圆二色光谱与野生型的光谱适度相似，这表明保守芳香族残基的任何替换都没有影响酶的二级结构。动力学分析表明，区域III中用Ser替换Tyr - 71、区域IV中用Phe替换Tyr - 148以及区域V中用Ala替换Trp - 210，与野生型相比，kcat/Km值降低了10 - 至1600倍，但底物IPP和FPP的Km值仅产生适度变化。区域V中用Ser替换Phe - 207导致IPP的Km值增加了13倍，kcat/Km值比野生型低1000 - 2000倍，尽管FPP的Km值没有明显变化。此外，区域V的W224A突变体显示IPP和FPP的Km值分别增加了6倍和14倍，与野生型相比，kcat/Km值降低了100 - 250倍。这些结果表明，这些保守芳香族残基在与底物IPP和FPP的结合以及十一异戊烯基二磷酸合酶的催化功能中发挥着重要作用。
