Conservation of the structure and function of bacterial tryptophan synthases

Abstract

Tryptophan biosynthesis is one of the most characterized processes in bacteria, in which the enzymes from Salmonella typhimurium and Escherichia coli serve as model systems. Tryptophan synthase (TrpAB) catalyzes the final two steps of tryptophan biosynthesis in plants, fungi and bacteria. This pyridoxal 5′-phosphate (PLP)-dependent enzyme consists of two protein chains, α (TrpA) and β (TrpB), functioning as a linear αββα heterotetrameric complex containing two TrpAB units. The reaction has a complicated, multistep mechanism resulting in the β-replacement of the hydroxyl group of L-serine with an indole moiety. Recent studies have shown that functional TrpAB is required for the survival of pathogenic bacteria in macrophages and for evading host defense. Therefore, TrpAB is a promising target for drug discovery, as its orthologs include enzymes from the important human pathogens Streptococcus pneumoniae, Legionella pneumophila and Francisella tularensis, the causative agents of pneumonia, legionnaires' disease and tularemia, respectively. However, specific biochemical and structural properties of the TrpABs from these organisms have not been investigated. To fill the important phylogenetic gaps in the understanding of TrpABs and to uncover unique features of TrpAB orthologs to spearhead future drug-discovery efforts, the TrpABs from L. pneumophila, F. tularensis and S. pneumoniae have been characterized. In addition to kinetic properties and inhibitor-sensitivity data, structural information gathered using X-ray crystallography is presented. The enzymes show remarkable structural conservation, but at the same time display local differences in both their catalytic and allosteric sites that may be responsible for the observed differences in catalysis and inhibitor binding. This functional dissimilarity may be exploited in the design of species-specific enzyme inhibitors.

3D domain swapping in the TIM barrel of the α subunit ofStreptococcus pneumoniaetryptophan synthase

“…The Streptococcus pneumoniae subunit (SpTrpA) studied in the present work is a 258residue protein which in its monomeric form assumes the canonical (/) 8 TIM-barrel fold with three additional helices (Supplementary Fig. S1; Michalska et al, 2019). The TIM barrel represents one of the most abundant and versatile protein folds in nature, and is found in all kingdoms of life and in viruses.…”

Allosteric inhibitors of Mycobacterium tuberculosis tryptophan synthase

“…In general, the mechanism of L‐Trp synthesis is preserved in all known tryptophan synthases (TrpAB) and majority of features are conserved (the α site, the β site, the tunnel connecting active sites and allosteric regulation). At the same time, some differences were reported between TrpAB enzymes suggesting that they can be exploited to design species‐specific inhibitors . This was specifically proposed for the allosteric sites.…”

“…Tryptophan synthase was studied extensively biochemically, structurally and computationally for the past 60 years, but most research focused on a model system from Salmonella typhimurium and orthologs from Escherichia coli and Pyrococcus furiosus . Only recently the field has expanded to include TrpAB from other organisms . In M. tuberculosis TrpAB research the major breakthrough involved developing recombinant coexpression system in E. coli , purification of fully active αββα complex and obtaining crystals that diffracted to high resolution and permitted detailed visualization of enzyme inhibitor interactions and insight into the catalytic mechanism .…”

“…Only recently the field has expanded to include TrpAB from other organisms. 4,19,31 In M. tuberculosis TrpAB research the major breakthrough involved developing recombinant coexpression system in E. coli, purification of fully active αββα complex and obtaining crystals that diffracted to high resolution and permitted detailed visualization of enzyme inhibitor interactions and insight into the catalytic mechanism. 4 In general, the mechanism of L-Trp synthesis is preserved in all known tryptophan synthases (TrpAB) and majority of features are conserved (the α site, the β site, the tunnel connecting active sites and allosteric regulation).…”

“…At the same time, some differences were reported between TrpAB enzymes suggesting that they can be exploited to design species-specific inhibitors. 31 This was specifically proposed for the allosteric sites. We have previously reported an inhibitor BRD4592 that selectively targets MtTrpAB and binds to a new allosteric site with high affinity.…”