Effect of site-directed mutagenesis of the conserved aspartate and glutamate on E. coli undecaprenyl pyrophosphate synthase catalysis.
Undecaprenyl pyrophosphate synthase (UPPs) catalyzes condensation of eight molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C(55)-undecaprenyl pyrophosphate. We have mutated the aspartates and glutamates in the five conserved regions (I to V) of UPPs protein sequence to evaluate their effects on substrate binding and catalysis. The mutant enzymes including D26A, E73A, D150A, D190A, E198A, E213A, D218A, and D223A were expressed and purified to great homogeneity. Kinetic analyses of these mutant enzymes indicated that the substitution of D26 in region I with alanine resulted in a 10(3)-fold decrease of k(cat) value compared to wild-type UPPs. Its IPP K(m) value has only minor change. The mutagenesis of D150A has caused a much lower IPP affinity with IPP K(m) value 50-fold larger than that of wild-type UPPs but did not affect the FPP K(m) and the k(cat). The E213A mutant UPPs has a 70-fold increased IPP K(m) value and has a 100-fold decreased k(cat) value compared to wild-type. These results suggest that D26 of region I is critical for catalysis and D150 in region IV plays a significant role of IPP binding. The E213 residue in region V is also important in IPP binding as well as catalysis. Other mutant UPPs enzymes in this study have shown no significant change (<5-fold) of k(cat) with exception of E73A and D218A. Both enzymes have 10-fold lower k(cat) value relative to wild-type UPPs.
Pan
J J
JJ
Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan.
Yang
L W
LW
Liang
P H
PH
D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D018698:Glutamic Acid / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D019883:Alkyl and Aryl Transferases; C023266:undecaprenyl pyrophosphate synthetase