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Originally published In Press as doi:10.1074/jbc.M106747200 on October 1, 2001

J. Biol. Chem., Vol. 276, Issue 50, 47474-47482, December 14, 2001
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Mechanism of Product Chain Length Determination and the Role of a Flexible Loop in Escherichia coli Undecaprenyl-pyrophosphate Synthase Catalysis*

Tzu-Ping KoDagger §, Yi-Kai ChenDagger §, Howard Robinson, Pei-Chun TsaiDagger , Yi-Gui Gao, Annie P.-C. Chen||, Andrew H.-J. WangDagger ||**, and Po-Huang LiangDagger ||Dagger Dagger

From the Dagger  Institute of Biological Chemistry, Academia Sinica, Taipei 115 and || Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan

The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the beta A-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four beta -strands (beta A-beta B-beta D-beta C) and two helices (alpha 2 and alpha 3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C70 polymer rather than the C55 polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C30 intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C55 product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C55 product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25-200-fold decrease in kcat values. W75A showed an 8-fold increase of the FPP Km value, and 22-33-fold increases in the IPP Km values were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release.

* This work was supported in part by grants from Academia Sinica (to A. H. J. W. and P. H. L.), by Grant GM41612 from the National Institutes of Health (to A. H. J. W.), and by Grant NSC89-2113-M-001-061 from the Taiwan National Science Council (to P. H. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1JP3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

§ Both authors contributed equally to this work.

Present address: Dept. of Biochemistry, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.

** To whom correspondence may be addressed. Tel.: 886-2-2788-1981; Fax: 886-2-2788-9759; E-mail: ahjwang@gate.sinica.edu.tw.

Dagger Dagger To whom correspondence may be addressed. Tel.: 886-2-2785-5696 (ext. 6070); Fax: 886-2-2788-9759; E-mail: phliang@gate.sinica.edu.tw.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.