The Substrate Capture Mechanism of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Provides a Mode for Inhibition

Abstract

Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5'-phosphoribosyl 1'-pyrophosphate (PRPP) and anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5-119 μM, with the strongest inhibition displayed by a bianthranilate compound that makes hydrogen bond and salt bridge contacts with Mtb-AnPRT via its carboxyl groups. Our results reveal how the substrate capture mechanism of AnPRT can be exploited to inhibit the enzyme's activity and provide a scaffold for the design of improved Mtb-AnPRT inhibitors that may ultimately form the basis of new antituberculosis drugs with a novel mode of action.

Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture Site that Is a Target for Drug Discovery

“…As far as we are aware, this is the first indication of a substrate-capture mechanism in a small-molecule kinase. Indeed, the only precedent that we have found in the literature for a dedicated substrate capture site on any enzyme is that observed for certain microbial anthranilate phosphoribosyl-transferases (Castell et al, 2013; Marino et al, 2006). Our structural, biochemical and mutagenic data have also led us to conclude that the stimulation of ATPase activity of PPIP5K by inositol phosphate analogues is associated with their occupation of the substrate capture site, not the catalytic site.…”

“…Instead, the proximity to the active site of the second ligand binding site (Figure 4) suggests the latter plays a role in catalysis of natural substrates, by facilitating their capture from the bulk phase. A precedent for such a phenomenon has been reported for microbial anthranilate phosphoribosyltransferases (Castell et al, 2013; Marino et al, 2006); anthranilate substrate is first captured at a surface-mounted binding-site, before delivery to a proximal catalytic site (Castell et al, 2013). A similar substrate transfer is feasible in PPIP5K2 KD , and our structural data provide some atomic-level insight into such a phenomenon.…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…The two-domain structure consists of a smaller N-terminal domain of six helices (the ␣-domain) and a larger C-terminal domain of six parallel ␤-sheets and one antiparallel ␤-sheet, as well as eight or more helices (the ␣/␤-domain). Although the five-stranded parallel ␤-sheet characteristic of type I phosphoribosyltransferases is recognizable, anthranilate phosphoribosyltransferase contains additional structural features and thus constitutes a separate fold sometimes designated type IV phosphoribosyltransferase (287). The enzyme is active as a homodimer; the ␣-domain is responsible for the dimerization.…”

“…Since the organism's host, humans, are unable to synthesize tryptophan, a search for inhibitors of tryptophan biosynthetic enzymes, including for anthranilate phosphoribosyltransferase, has been conducted in hopes of identifying an antimicrobial agent (287,290,292).…”

Anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Thermococcus kodakarensis shows maximum activity with zinc and forms a unique dimeric structure

“…Class III has a unique domain structure and includes ATP‐PRTase. Class IV PRTs are limited to TrpD and exhibit a homodimeric structure and a novel PRT fold, consisting of a small N‐terminal α‐helical domain connected to a large C‐terminal α/β domain by a hinge region . The X‐ray structures of TrpD enzymes from Sulfolobus solfataricus ( Ss TrpD; PDB entry http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2GVQ) , Pectobacterium carotovorum ( Pc TrpD; PDB entry http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1KHD) , Mycobacterium tuberculosis ( Mtb TrpD; PDB entry http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4X5B) , Thermus thermophilus ( Tt TrpD; PDB entry http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1V8G; Shimizu and Kunishima, 2004, RIKEN Structural Genomics/Proteomics Initiative, unpublished), Acinetobacter sp.…”