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J. Biol. Chem., Vol. 279, Issue 6, 4903-4912, February 6, 2004

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Crystal Structure of Octaprenyl Pyrophosphate Synthase from Hyperthermophilic Thermotoga maritima and Mechanism of Product Chain Length Determination^*

Rey-Ting Guo, Chih-Jung Kuo¶, Chia-Cheng Chou¶||, Tzu-Ping Ko¶, Hui-Lin Shr¶||, Po-Huang Liang¶**, and Andrew H.-J. Wang¶||

From the Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan, Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan, and ^¶Institute of Biological Chemistry and ^||Core Facility for Protein X-ray Crystallography, Academia Sinica, Taipei 115, Taiwan

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C₄₀ 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 nine 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 two large residues Phe-52 and Phe-132. The products of the mutant F132A OPPs are predominantly C₅₀, longer than the C₄₀ synthesized by the wild-type and F52A mutant OPPs, suggesting that Phe-132 is the key residue for determining the product chain length. Ala-76 and Ser-77 located close to the FPP binding site and Val-73 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. Ala-76 is the fifth 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. The new structure of the trans-type OPPs, together with the recently determined cis-type UPPs, significantly extends our understanding on the biosynthesis of long chain polyprenyl molecules.

Received for publication, September 12, 2003 , and in revised form, November 7, 2003.

The atomic coordinates and structure factors (code 1V4E for native OPPs, 1V4H for F52A, 1V4J for V73Y, 1V4K for S77F, and 1V4I F132A) have been deposited in the Protein Data Bank, Research laboratory for Structural Bioinformatics, Rutgers University, New wick, NJ (http://www.rcsb.org/).

^* The synchrotron data collection was conducted with the Biological Crystallography Facilities (Taiwan Beamline BL12B2 at SPring-8) supported by the National Science Council (NSC). This work was supported in part by grants from Academia Sinica and National Science Council (NSC91-3112-P-001-019-Y) (to A. H.-J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence may be addressed: Inst. of Biological Chemistry, Academia Sinica, 128 Academia Rd., Taipei 115, Taiwan. Tel.: 886-2-2785-5696 (ext. 6070); Fax: 886-2-2788-9759; E-mail: phliang{at}gate.sinica.edu.tw.

To whom correspondence may be addressed: Inst. of Biological Chemistry, Academia Sinica, 128 Academia Rd., Taipei 115, Taiwan. Tel.: 886-2-2788-1981; Fax: 886-2-2788-2043; E-mail: ahjwang{at}gate.sinica.edu.tw.

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