Structure and inhibition of subunit I of the anthranilate synthase complex of Mycobacterium tuberculosis and expression of the active complex

Abstract

The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.

A Three-Ring Circus: Metabolism of the Three Proteogenic Aromatic Amino Acids and Their Role in the Health of Plants and Animals

“…Other bacterial AS such as the fused Streptomyces venezuelae are not only monomeric, but also cannot use ammonia instead of glutamine to aminate chorismate (Ashenafi et al, 2015 ). Among pathogenic bacteria from which crystallographic structures of AS are available, the oligomeric organization differs substantially between Serratia marcrescens and S. typhimurium , where the α 2 β 2 tetramer associates via the AS-I subunits (Spraggon et al, 2001 ; Figure 3Bi ), while in the M. tuberculosis enzyme, AS-I is a homodimer, even when AS-II is present (Bashiri et al, 2015 ; Figure 3Bii ). In the last enzyme, the allosteric binding site for the inhibitor tryptophan is found near the interfacial region.…”

Indole Propionic Acid, an Unusual Antibiotic Produced by the Gut Microbiota, With Anti-inflammatory and Antioxidant Properties

“…Tryptophan biosynthesis is an essential pathway in mycobacteria in vitro as well as in vivo (Zhang et al, 2013;Wellington et al, 2017). The pathway is regulated via a negative feedback loop in which the end product tryptophan acts as allosteric inhibitor of the first committed enzymatic step of the pathway catalyzed by anthranilate synthase TrpE (Bashiri et al, 2015). Thus, we hypothesized that IPA may mimic tryptophan as allosteric inhibitor and block tryptophan synthesis.…”

Gut Microbiota Metabolite Indole Propionic Acid Targets Tryptophan Biosynthesis in Mycobacterium tuberculosis

“…Zhang et al (12) and Wellington et al (13) have demonstrated that the Trp biosynthetic pathway is essential for growth and viability of M. tuberculosis in standard (Trp-free) medium as well as in macrophages and mice. In M. tuberculosis , Trp regulates its own synthesis by acting as a cooperative allosteric inhibitor of anthranilate synthase TrpE, which catalyzes the first committing step in the pathway (14, 15).…”

“…The homodimeric M. tuberculosis protein TrpE (Rv1609), in conjunction with glutamine amidotransferase (TrpG) providing ammonia, catalyzes the formation of anthranilate from chorismate (15, 16). The N-terminal region of TrpE contains the allosteric Trp binding motif 53 LLESX 10 S 67 (15). The 10-amino-acid residue X 10 loops of the two TrpE subunits approach each other at the interface, providing part of the structural framework for the cooperative allosteric inhibitory effect of Trp binding.…”

“…The 10-amino-acid residue X 10 loops of the two TrpE subunits approach each other at the interface, providing part of the structural framework for the cooperative allosteric inhibitory effect of Trp binding. In addition, TrpE residues 170 HHEGT 174 are involved in the cooperative allosteric inhibition mechanism by making interactions with their subunit’s Trp binding motif as well as with the Trp binding motif and the 170 HHEGT 174 residues of the partner subunit (15).…”