Substrate and product specificities of cis-type undecaprenyl pyrophosphate synthase

UPPS (undecaprenyl pyrophosphate synthase) catalyses consecutive condensation reactions of FPP (farnesyl pyrophosphate) with eight isopentenyl pyrophosphates to generate C₅₅ UPP, which serves as a lipid carrier for bacterial peptidoglycan biosynthesis. We reported the co-crystal structure of Escherichia coli UPPS in complex with FPP. Its phosphate head-group is bound to positively charged arginine residues and the hydrocarbon moiety interacts with hydrophobic amino acids including L85, L88 and F89, located on the α3 helix of UPPS. We now show that the monophosphate analogue of FPP binds UPPS with an eight times lower affinity (K_d = 4.4 μM) compared with the pyrophosphate analogue, a result of a larger dissociation rate constant (k _off = 192 s^-1). Farnesol (1 mM) lacking the pyrophosphate does not inhibit the UPPS reaction. GGPP (geranylgeranyl pyrophosphate) containing a larger C₂₀ hydrocarbon tail is an equally good substrate (K_m = 0.3 μM and k_cat = 2.1 s^-1) compared with FPP. The shorter C₁₀ GPP (geranyl pyrophosphate) displays a 90-fold larger K_m value (36.0 ± 0.1 μM) but similar k _cat value (1.7 ± 0.1 s^-1) compared with FPP. Replacement of L85, L88 or F89 with Ala increases FPP and GGPP K_m values by the same amount, indicating that these amino acids are important for substrate binding, but do not determine substrate specificity. With GGPP as a substrate, UPPS still catalyses eight isopentenyl pyrophosphate condensation reactions to synthesize C₆₀ product. Computer modelling suggests that the upper portion of the active-site tunnel, where cis double bonds of the product reside, may be critical for determining the final product chain length.