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J. Biol. Chem., Vol. 280, Issue 1, 571-577, January 7, 2005

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A Unique Hydrophobic Cluster Near the Active Site Contributes to Differences in Borrelidin Inhibition among Threonyl-tRNA Synthetases^*

Benfang Ruan, Michael L. Bovee, Meik Sacher, Constantinos Stathopoulos¶, Karl Poralla||, Christopher S. Francklyn**, and Dieter Söll

From the Departments of Molecular Biophysics and Biochemistry and Chemistry, Yale University, New Haven, Connecticut 06520-8114, Department of Biochemistry, The University of Vermont Health Sciences Complex, Burlington, Vermont 05405-0068, and ^||Institut für Mikrobiologie, Eberhardt-Karls-Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany

Borrelidin, a compound with anti-microbial and anti-angiogenic properties, is a known inhibitor of bacterial and eukaryal threonyl-tRNA synthetase (ThrRS). The inhibition mechanism of borrelidin is not well understood. Archaea contain archaeal and bacterial genre ThrRS enzymes that can be distinguished by their sequence. We explored species-specific borrelidin inhibition of ThrRSs. The activity of ThrRS from Sulfolobus solfataricus and Halobacterium sp. NRC-1 was inhibited by borrelidin, whereas ThrRS enzymes from Methanocaldococcus jannaschii and Archaeoglobus fulgidus were not. In Escherichia coli ThrRS, borrelidin binding induced a conformational change, and threonine was not activated as shown by ATP-PP_i exchange and a transient kinetic assay measuring intrinsic tryptophan fluorescence changes. These assays further showed that borrelidin is a noncompetitive tight binding inhibitor of E. coli ThrRS with respect to threonine and ATP. Genetic selection of borrelidin-resistant mutants showed that borrelidin binds to a hydrophobic region (Thr-307, His-309, Cys-334, Pro-335, Leu-489, Leu-493) proximal to the zinc ion at the active site of the E. coli ThrRS. Mutating residue Leu-489 Trp reduced the space of the hydrophobic cluster and resulted in a 1500-fold increase of the K_i value from 4 nM to 6 µM. An alignment of ThrRS sequences showed that this cluster is conserved in most organisms except for some Archaea (e.g. M. jannaschii, A. fulgidus) and some pathogens (e.g. Helicobacter pylori). This study illustrates how one class of natural product inhibitors affects aminoacyl-tRNA synthetase function, providing potentially useful information for structure-based inhibitor design.

Received for publication, September 27, 2004 , and in revised form, October 26, 2004.

^* This work was supported by grants from the National Institute of General Medical Sciences (to D. S. and C. S. F.) and the Department of Energy (to D. S.). 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.

^¶ Current address: Dept. of Biochemistry and Biotechnology, University of Thessaly, 41221 Larissa, Greece.

^** To whom correspondence may be addressed: Dept. of Biochemistry, University of Vermont Health Sciences Complex, Burlington, VT 05405-0068. Tel.: 802-656-8450; Fax: 802-862-8229; E-mail: franck{at}emba.uvm.edu.

To whom correspondence may be addressed: Dept. of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114. Tel.: 203-432-6200; Fax: 203-432-6202; E-mail: soll{at}trna.chem.yale.edu.

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