Download Crystal Structure of Octaprenyl Pyrophosphate Synthase from

Crystal Structure of Octaprenyl Pyrophosphate Synthase from
Hyperthermophilic Thermotoga maritima and Mechanism of Product Chain
Length Determination
Rey-Ting Guo (郭瑞庭)1, Chih-Jung Kuo (郭致榮)2, Tzu-Ping Ko (柯子平)2,
Chia-Cheng Chou (周家丞)2, Po-Huang Liang (梁博煌)1, 2, and Andrew H.-J. Wang (王惠鈞)1, 2
1
Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan
2
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of
farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C 40 octaprenyl
pyrophosphate (OPP) which constitutes the side chain of bacterial ubiquinone or menaquinone. In
this study, the first structure of long-chain C40-OPPs from Thermotoga maritima has been
determined to 2.28 Å resolution. OPPs is composed entirely of -helices joined by connecting loops
and is arranged with 9 core helices around a large central cavity. An elongated hydrophobic tunnel
between D and F -helices contains two DDxxD motifs on the top for substrate binding and is
occupied at the bottom with one large residue F132. The products of the mutant F132A OPPs are
predominantly C50, longer than the C40 synthesized by the wild type OPPs, suggesting that F132 is
the key residue for determining the product chain length. A76 and S77 located close to the FPP
binding site and V73 positioned further down the tunnel were individually mutated to larger amino
acids. A76Y and S77F mainly produce C20 indicating that the mutated large residues in the vicinity
of the FPP site limit the substrate chain elongation. A76 is the 5th amino acid upstream from the first
DDxxD motif on helix D of OPPs and its corresponding amino acid in FPPs is Tyr. In contrast,
V73Y mutation led to additional accumulation of C30 intermediate. Based on the structure, we have
sequentially mutated the large amino acids including F132, L128, I123, and D62 to small Ala
underneath the tunnel. The products of the mutant F132A/L128A/I123A/D62A reach C95, beyond
the largest chain length generated by all known trans-prenyltransferases. Further modifications of
enzyme reaction conditions may allow the preparation of high molecular weight polyprenyl
products resembling rubber molecule. The new structure of the trans-type OPPs significantly
extends our understanding on the biosynthesis of long chain polyprenyl molecules.
References:
[1]. R. T. Guo, T. P. Ko, C. C. Chou, H. L. Shr, H. M. Chu, Y. H. Tsai, P. H. Liang, and A. H.-J.
Wang, Acta Crystallogr. Sect. D Biol. Crystallogr. 59, 2265-2268. (2003)
[2]. R. T. Guo, C. J. Kuo, C. C. Chou, T. P. Ko, H. L. Shr, P. H. Liang**, and A. H.-J. Wang**. J.
Biol. Chem. 279, 4903-4912. (2004)
[3]. R. T. Guo*, C. J. Kuo*, T. P. Ko, C. C. Chou, P. H. Liang**, and A. H.-J. Wang**. Biochemistry.
43, 7678-7686. (2004)