SUMMARY Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the elongation of farnesyl diphosphate (FPP) to C55 undecaprenyl pyrophosphate (UPP) via sequential condensation reactions of eight isopentenyl pyrophosphate (IPP) units. The E. coli UPPs structure has been solved using single-wavelength anomalous diffraction method. The putative active site is located near the end of the beta-A strand with Asp26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel, surrounded by 4 beta-strands (beta A-B-D-C) and 2 helices (alpha 2 and 3) and lined at the bottom with large residues I62, L137, V105 and H103, is found. The product distributions formed by 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 predominant formation of the C70 polymer rather than the C55 polymer. A69 and A143 are located near the top of the tunnel. In contrast to A143V reaction, the C30 intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small-sized side chain of A69 is required for rapid elongation to the C55 product, while the large-sized hydrophobic side chain of L137 is required to limit the elongation to the C55 product. The roles of residues S71, E73, N74, W75, R77 and E81 located on a flexible loop attached to alpha 3 helix were investigated. The S71A, N74A, or R77A mutants displayed 25¡V200-fold decrease in kcat values. W75A showed a 8-fold increase of FPP Km value and 22¡V33-fold increases in IPP Km values 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.