Structures of <i>Mycobacterium tuberculosis</i> Anthranilate Phosphoribosyltransferase Variants Reveal the Conformational Changes That Facilitate Delivery of the Substrate to the Active Site

Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg²⁺-dependent reaction between 5′-phosphoribosyl-1′-pyrophosphate (PRPP) and anthranilate. The roles of residues predicted to be involved in anthranilate binding have been tested by the analysis of six <i>Mtb</i>-AnPRT variant proteins. Kinetic analysis showed that five of six variants were active and identified the conserved residue R193 as being crucial for both anthranilate binding and catalytic function. Crystal structures of these <i>Mtb</i>-AnPRT variants reveal the ability of anthranilate to bind in three sites along an extended anthranilate tunnel and expose the role of the mobile β2−α6 loop in facilitating the enzyme’s sequential reaction mechanism. The β2−α6 loop moves sequentially between a “folded” conformation, partially occluding the anthranilate tunnel, via an “open” position to a “closed” conformation, which supports PRPP binding and allows anthranilate access via the tunnel to the active site. The return of the β2−α6 loop to the “folded” conformation completes the catalytic cycle, concordantly allowing the active site to eject the product PRA and rebind anthranilate at the opening of the anthranilate tunnel for subsequent reactions. Multiple anthranilate molecules blocking the anthranilate tunnel prevent the β2−α6 loop from undergoing the conformational changes required for catalysis, thus accounting for the unusual substrate inhibition of this enzyme

The Substrate Capture Mechanism of <i>Mycobacterium tuberculosis</i> Anthranilate Phosphoribosyltransferase Provides a Mode for Inhibition

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 <i>Mycobacterium tuberculosis</i> AnPRT (<i>Mtb</i>-AnPRT), along with crystal structures of <i>Mtb</i>-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in <i>Mtb</i>-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 <i>K</i>_i values of 1.5–119 μM, with the strongest inhibition displayed by a bianthranilate compound that makes hydrogen bond and salt bridge contacts with <i>Mtb</i>-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 <i>Mtb</i>-AnPRT inhibitors that may ultimately form the basis of new antituberculosis drugs with a novel mode of action