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J. Biol. Chem., Vol. 280, Issue 21, 20762-20774, May 27, 2005

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Crystal Structures of Undecaprenyl Pyrophosphate Synthase in Complex with Magnesium, Isopentenyl Pyrophosphate, and Farnesyl Thiopyrophosphate

ROLES OF THE METAL ION AND CONSERVED RESIDUES IN CATALYSIS^*

Rey-Ting Guo¶||, Tzu-Ping Ko¶||, Annie P.-C. Chen¶, Chih-Jung Kuo¶, Andrew H.-J. Wang¶**, and Po-Huang Liang¶

From the Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan, the ^¶Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, and the Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the consecutive condensation reactions of a farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate that serves as a lipid carrier for peptidoglycan synthesis of bacterial cell wall. The structures of Escherichia coli UPPs were determined previously in an orthorhombic crystal form as an apoenzyme, in complex with Mg²⁺/sulfate/Triton, and with bound FPP. In a further search of its catalytic mechanism, the wild-type UPPs and the D26A mutant are crystallized in a new trigonal unit cell with Mg²⁺/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site. In the wild-type enzyme, Mg²⁺ is coordinated by the pyrophosphate of farnesyl thiopyrophosphate, the carboxylate of Asp²⁶, and three water molecules. In the mutant enzyme, it is bound to the pyrophosphate of IPP. The [Mg²⁺] dependence of the catalytic rate by UPPs shows that the activity is maximal at [Mg²⁺] = 1 mM but drops significantly when Mg²⁺ ions are in excess (50 mM). Without Mg²⁺, IPP binds to UPPs only at high concentration. Mutation of Asp²⁶ to other charged amino acids results in significant decrease of the UPPs activity. The role of Asp²⁶ is probably to assist the migration of Mg²⁺ from IPP to FPP and thus initiate the condensation reaction by ionization of the pyrophosphate group from FPP. Other conserved residues, including His⁴³, Ser⁷¹, Asn⁷⁴, and Arg⁷⁷, may serve as general acid/base and pyrophosphate carrier. Our results here improve the understanding of the UPPs enzyme reaction significantly.

Received for publication, February 24, 2005

The atomic coordinates and structure factors (code 1X06, 1X08, 1X07, and 1X09) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants from Academia Sinica and from National Science Council (NSC91-3112-P-001-019-Y (to A. H.-J. W.) and NSC92-2113-M-001-026 (to P.-H. L.). The National Synchrotron Radiation Research Center, Taiwan, is supported by the National Science Council of Taiwan. 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.

The on-line version of this article (available at http://www.jbc.org) contains two additional figures.

^|| These two authors contributed equally to this work.

^** To whom correspondence may be addressed. Tel.: 886-2-2788-1981; Fax: 886-2-2788-2043; E-mail: ahjwang{at}gate.sinica.edu.tw. To whom correspondence may be addressed. Tel.: 886-2-2785-5696 (ext. 6070); Fax: 886-2-2788-9759; E-mail: phliang{at}gate.sinica.edu.tw.

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