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Protein Science (2004), 13:971-978. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Substrate binding mode and reaction mechanism of undecaprenyl pyrophosphate synthase deduced from crystallographic studies

Sing-Yang Chang1, Tzu-Ping Ko1, Annie P.-C. Chen2, Andrew H.-J. Wang1,2 and Po-Huang Liang1,2

1 Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan
2 Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan

(RECEIVED November 18, 2003; FINAL REVISION December 22, 2003; ACCEPTED December 23, 2003)



Abstract

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to form a 55-carbon long-chain product. We previously reported the crystal structure of the apo-enzyme from Escherichia coli and the structure of UPPs in complex with sulfate ions (resembling pyrophosphate of substrate), Mg2+, and two Triton molecules (product-like). In the present study, FPP substrate was soaked into the UPPs crystals, and the complex structure was solved. Based on the crystal structure, the pyrophosphate head group of FPP is bound to the backbone NHs of Gly29 and Arg30 as well as the side chains of Asn28, Arg30, and Arg39 through hydrogen bonds. His43 is close to the C2 carbon of FPP and may stabilize the farnesyl cation intermediate during catalysis. The hydrocarbon moiety of FPP is bound with hydrophobic amino acids including Leu85, Leu88, and Phe89, located on the {alpha}3 helix. The binding mode of FPP in cis-type UPPs is apparently different from that of trans-type and many other prenyltransferases which utilize Asprich motifs for substrate binding via Mg2+. The new structure provides a plausible mechanism for the catalysis of UPPs.

Abbreviations: UPPs, undecaprenyl pyrophosphate synthase • IPP, iso-pentenyl pyrophosphate • FPP, farnesyl pyrophosphate • UPP, undecaprenyl pyrophosphate • FPPs, farnesyl pyrophosphate synthase • GPP, geranyl pyrophosphate • TLC, thin-layer chromatography • NiNTA, nickel nitrilotriacetic acid • HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid • EDTA, ethylenediaminetetraacetic acid • FTase, farnesyltransferase

Keywords: prenyltransferase; farnesyl pyrophosphate; isopentenyl pyrophosphate; crystal structure; substrate binding; metal ion

Reprint requests to: Po-Huang Liang or Andrew H.-J. Wang, Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan; e-mail: phliang{at}gate.sinica.edu.tw or ahjwang{at}gate.sinica.edu.tw; fax: 886-2-2788-9759 or 886-2-2788-2043.

Supplemental material: see www.proteinscience.org

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03519904.


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