Mechanism of Product Chain Length Determination and the Role of a Flexible Loop in Escherichia coliUndecaprenyl-pyrophosphate Synthase Catalysis*

The Escherichia coliundecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the βA-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four β-strands (βA-βB-βD-βC) and two helices (α2 and α3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C70polymer rather than the C55polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C30intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C55product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C55product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25–200-fold decrease in kcatvalues. W75A showed an 8-fold increase of the FPP Kmvalue, and 22–33-fold increases in the IPP Kmvalues were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release.