Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Abstract

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (K m ϳ 2 M), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ϳ1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg 2؉ ion in a manner that allows it to assist in transition state stabilization. The Mg 2؉ ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg 2؉ ⅐ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg 2؉ coordination in the human cytosolic (H c )TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in H c TrpRS with a yetto-be determined role for Mg 2؉ . Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to H c TrpRS.

Selective inhibition of apicoplast tryptophanyl-tRNA synthetase causes delayed death in Plasmodium falciparum

“…The nature of TrpRS inhibition by indolmycin was explored using the recently determined crystal structure of Pf TrpRS api homologue (5DK4; 33% sequence homology) from Bacillus stearothermophilus with bound indolmycin, ATP, and magnesium 40 superimposed with the structure of the cytosolic Plasmodium TrpRS (4J75; Fig. 5b–d ) with bound charged tryptophan 26 .…”

Regulation of an iron-dependent repressor by tryptophan availability attenuates transcription of the tryptophan salvage genes inChlamydia trachomatis

“…To examine whether ribosome stalling at the WWW motif abrogated translation of YtgR, we designed an expression system for YtgCR in E. coli, where we expressed YtgCR WWW or YtgCR YYF C-terminally tagged with a 3XFLAG epitope from the IPTG-inducible pET151 plasmid. We then took advantage of the competitive inhibitor of prokaryotic tryptophanyl-tRNA synthetase, indolmycin 38,39 , which prevents the incorporation of tryptophan during translation and has been shown to promote ribosome stalling at tryptophan codons 40 . As a control, we included a treatment with the translation inhibitor AN3365, which functions as a noncompetitive inhibitor of prokaryotic leucyl-tRNA synthetase 41,42 .…”

Biosynthetic study of β-methyl amino acid building blocks involved in natural products

“…大多数非天然氨基酸都 是通过非核糖体肽合成酶(NRPS)的催化进入到天然 产物的骨架中, 并对后者发挥重要的生物活性具有 至关重要的作用 [4] . β-位甲基化氨基酸是一种非天然氨基酸, 在一 些活性天然产物的结构及某些初级代谢的通路中也 有发现, 其中一些天然产物已经成为上市药物或重 要的临床药物前体, 如达托霉素(Daptomycin) [5] 、尼可 霉 素 Z(Nikkomycin Z) [6] 、 甘 露 霉 素 (Mannopeptimy-cin) [7] 、Friulimicin B [8] 以及吲哚霉素(Indolmycin) [9] 期 的 研 究 中 , 这 类 酶 被 发 现 存 在 于 一 些 梭 菌 (Clostridium)的L-Glu降解途径 [10] . GLM是一种异源 四聚体蛋白, 由两种亚基δ(15 kDa左右)和ε(55 kDa左…”