Structure, mechanism and inhibition of anthranilate phosphoribosyltransferase

Abstract

Anthranilate phosphoribosyltransferase catalyses the second reaction in the biosynthesis of tryptophan from chorismate in microorganisms and plants. The enzyme is homodimeric with the active site located in the hinge region between two domains. A range of structures in complex with the substrates, substrate analogues and inhibitors have been determined, and these have provided insights into the catalytic mechanism of this enzyme. Substrate 5-phospho-d-ribose 1-diphosphate (PRPP) binds to the C-terminal domain and coordinates to Mg²⁺, in a site completed by two flexible loops. Binding of the second substrate anthranilate is more complex, featuring multiple binding sites along an anthranilate channel. This multi-modal binding is consistent with the substrate inhibition observed at high concentrations of anthranilate. A series of structures predict a dissociative mechanism for the reaction, similar to the reaction mechanisms elucidated for other phosphoribosyltransferases. As this enzyme is essential for some pathogens, efforts have been made to develop inhibitors for this enzyme. To date, the best inhibitors exploit the multiple binding sites for anthranilate. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Keywords: AnPRT; PRT; TrpD; tryptophan biosynthesis.

Figure 1.

The PRT family. Overview of the architectures and metabolic role of PRT enzymes type I–IV. I: type I on the example of hypoxanthine PRT from Trypanosoma cruzi [16] (Protein Data Bank (PDB) ascension code: 1TC2). II: type II on the example of quinolinate PRT from M. tuberculosis [17] (PDB ascension code: 1QPR). III: type III on the example of AnPRT from M. tuberculosis [18] (PDB ascension code: 1ZVW). IV: type IV on the example of ATP-PRT from Lactococcus lactis [19] (PDB ascension code: 1Z7N). Representative monomeric structures are displayed as cartoon coloured according to secondary structure elements, helices (red), strands (yellow) and loops (green). The metal ions (green spheres) indicate the active site location. (Online version in colour.)

Figure 2.

AnPRT fold. Three-dimensional structure of AnPRT on the example of PRPP-bound MtuAnPRT (PDB ascension code: 1ZVW) [18] in cartoon representation with secondary structure elements labelled. Ligands PRPP (yellow) and Mg²⁺ (green) are displayed as spheres highlighting the active site location. Heteroatoms are coloured as follows: oxygen (red), phosphorous (orange). (Online version in colour.)

Figure 3.

Proposed reaction mechanisms for AnPRT catalysed reaction, occurring through either a dissociative (top) or an associative (bottom) mechanism.

Figure 4.

PRPP binding site and loop rearrangements associated with PRPP binding. (a) PRPP binding site in MtuAnPRT (PDB 1ZVW [18]). PRPP is displayed with yellow carbon, Mg²⁺ ions are shown as green spheres, loop β1-α5 is coloured in cyan and loop β2-α6 is coloured in magenta. Polar interactions formed with PRPP and Mg²⁺ are displayed as black dashed lines. (b) Superimposition of apoensyme (PDB 2BPQ [18] and 3QR9 [6]) and PRPP-bound (PDB 1ZVW) MtuAnPRT to highlight the conformational changes of loop β1-α5 (light cyan for apoenzyme structures and cyan for PRPP-bound structure) and β2-α6 (light pink for apoenzyme structures and magenta for PRPP-bound structure). (Online version in colour.)

Figure 5.

Anthranilate channel and anthranilate binding sites. Active site loops β1-α5 and β2-α6 are shown in light cyan and magenta, respectively. (a) Anthranilate channel in MtuAnPRT (PDB 4N5V [46]) is displayed with a light blue surface. A PRPP molecule and 4-fluoroanthranilate molecules bound in site 1 and site 3 of MtuAnPRT (PDB 4N5V) are shown with yellow carbon atoms. Anthranilate molecules bound in site 1 and site 2 in SsoAnPRT (PDB 1ZYK [47]) are shown with white carbon atoms. (b) Anthranilate binding site 1 in MtuAnPRT (PDB 4N5V). Polar interactions are displayed as black dashed lines. (c) Anthranilate binding site 2. Superimposed active sites of MtuAnPRT (PDB 3QQS [6]) and SsoAnPRT (green carbon atoms, PDB 2GVQ [47]). The bianthranilate-like inhibitor (ACS172) in MtuAnPRT (yellow carbon atoms) occupies sites 1 and 2 simultaneously. Polar interactions formed by the site 2 anthranilate portion of ACS172 in MtuAnPRT are displayed as black dashed lines, and those formed by site 2 anthranilate in SsoAnPRT are displayed as green dashed lines. (d) Anthranilate binding site 3 in MtuAnPRT (PDB 4N5V). Polar interactions are displayed as black dashed lines. (Online version in colour.)

Figure 6.

(a) The open (PDB 3QR9 [6]), closed (PDB 1ZVW [18]) and folded (PDB 4X5D [35]) conformations of loop β2-α6 in MtuAnPRT (coloured in different shades of magenta). PRPP (from 1ZVW) and one anthranilate bound in site 3 (from 4X5D) are displayed with yellow carbon atoms. (b) Proposed ligand binding orders and active site loop movements for the catalytic cycle of MtuAnPRT by Cookson et al. [35]. The conformations of loop β2-α6 are labelled as open, folded and closed accordingly. (Online version in colour.)

Figure 7.

Structures of anthranilate-like inhibitors ACS172, 2-((2-carboxy-5-methylphenyl)amino) (I) and 2,6-bis((2-carboxy-5-methylphenyl)amino)-3-methylbenzoate (II) [9], as well as phosphonates 2-(2-carboxyphenylamino)-5-(5-phosphonopentyloxy)benzoic acid (III) and 2-(2-carboxyphenylamino)-5-((1-(2-phosphonoethyl)-1H-1,2,3-triazol-4-yl)methoxy)benzoic acid (IV) [53].