Substrate and product specificities of cis-type undecaprenyl pyrophosphate synthase

Annie P.C. Chen, Sing Yang Chang, Yu Chung Lin, Yang Sheng Sun, Chao Tsen Chen, Andrew H.J. Wang, Po Huang Liang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

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 (Kd = 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 (Km = 0.3 μM and kcat = 2.1 s-1) compared with FPP. The shorter C10 GPP (geranyl pyrophosphate) displays a 90-fold larger Km 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 Km 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.

Original languageEnglish
Pages (from-to)169-176
Number of pages8
JournalBiochemical Journal
Volume386
Issue number1
DOIs
StatePublished - 15 Feb 2005

Keywords

  • Computer modelling
  • Prenyltransferase
  • Product specificity
  • Site-directed mutagenesis
  • Substrate specificity
  • Undecaprenyl pyrophosphate synthase

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