A unique hydrophobic cluster near the active site contributes to differences in borrelidin inhibition among threonyl-tRNA synthetases.
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 microm. 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.
Ruan
Benfang
B
Department of Molecular Biophysics, Yale University, New Haven, Connecticut 06520-8114, USA.
Bovee
Michael L
ML
Sacher
Meik
M
Stathopoulos
Constantinos
C
Poralla
Karl
K
Francklyn
Christopher S
CS
Söll
Dieter
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D005233:Fatty Alcohols / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D017421:Sequence Analysis; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013914:Threonine-tRNA Ligase / Q000037:antagonists & inhibitors*
D001426:Bacterial Proteins; D005233:Fatty Alcohols; C005066:borrelidin; D013914:Threonine-tRNA Ligase