Introduction
Tryptophan synthase (TrpS) is an enzyme mainly found in bacteria, plants, and fungi [1]. TrpS must be functional for pathogenic bacteria in macrophages to survive, and it is used as a way to avoid host immune response. Tryptophan is necessary for bacteria and lower eukaryotic organisms for protein synthesis. Salmonella typhimurium and Escherichia coli serve as model organisms for the understanding of TrpS processes and structure [1].
Structure
TrpS is a pyridoxal 5’-phosphate (PLP)-dependent enzyme. TrpS has an alpha and beta chain that form a linear alpha-beta-beta-alpha heterotetrameric complex [2]. This is termed as the alpha2beta2 complex, and each subunit is also referred to as TrpA and TrpB for the alpha and beta subunits respectively. The alpha active site contains catalytic residues Glu and Asp, and a hydrophobic intramolecular tunnel allows for the transport of indole from the alpha subunit active site to the beta subunit active site (Miles, 2001). The alpha and beta chain are encoded by trpA and trpB genes that are involved in TrpS regulatory operon. In bacteria and plants, the alpha and beta chains are separate, but in fungi the two chains are fused into one protein (Tryptophan synthase, alpha chain, active site). The alpha2beta2 tetramer complex contains pyridoxal-phosphate, glycerol-3-phosphate, Na+ ions. Another key site in tryptophan synthase is the monovalent cation (MVC) site, which is made up of cations like Na+ and K+, along with Cs+ [3]. For regulation, TrpA and TrpB cycle between low-activity open conformation and a high-activity closed state. This is done by the binding of an IGP substrate to TrpA which promotes the high activity closed state which activates the TrpB high activity closed state. The states are reset back to low activity open conformation by the production of L-Trp external aldimine [2].
Function
TrpS main function is to catalyze the last two steps in tryptophan biosynthesis. Reactions take place at the active sites of the alpha and beta subunit. The alpha reaction, or TrpA, catalyzes the reaction involving indole-3-glycerol phosphate (IGP) cleavage, and the products made are indole and D-glyceraldehyde 3-phosphate. The alpha reaction is done through general acid-base catalysis [1]. The TrpA active site is located at the top of the central beta-barrel with two acidic residues, Glu and Asp, involved in the acid-base catalysis [2]. Indole will diffuse through the hydrophobic intramolecular tunnel that leads to the active site of the beta subunit.
TrpB is a type II PLP-dependent enzyme with N- and C-terminal domains, and the TrpB active site is located in a cleft between these two domains that has the PLP cofactor [2].The N-terminal is the communication domain which helps coordinate the activity in TrpA and TrpB active sites. beta reaction condenses the indole with the L-serine substrate to make L-tryptophan and water. The beta reaction is also called pyridoxal-5’-phosphate-dependent reaction [1]. The catalytic cycle is reversible until the C-C bond formation [4]. The MVC site is involved in the catalysis and regulation of substrate channeling through the hydrophobic intramolecular tunnel. For in vitro, this site is involved in equilibrium distribution of the intermediates formed during the serine reaction. Tryptophan synthase is regulated by the propagation of allosteric signals via networds of noncovalent amino acid interactions. TrpS is regulated when the indole product from the alpha reaction is transferred to the beta reaction. A substitution of a surface netowork residue in the alpha reaction enhances TrpS production by controlling the opening of the indole channel and also stimulating the beta reaction [5]. These regulations are necessary for the cell to efficiently use resources since tryptophan biosynthesis is an expensive process.
Relevance
Due to TrpS involvement in defense and biosynthesis of tryptophan for pathogenic bacteria, it is generally used a target for antibiotics. Overexpression studies found that tryptophan synthase is a biological target [6]. Examples of studied bacteria would be Streptococcus pneumoniae, Legionella pneumophila and Francisella tularensis. Research is done into the phylogenetic gaps these organisms have in order to better understand the unique features of TrpS orthologs for each bacteria [2]. One researched bacterium is the Mycobacterium tuberculosis (Mtb) in order to develop a drug that inhibits tryptophan synthase. The main reason is due to how widespread tuberculosis is (Mtb is a causative agent of tuberculosis). Drug research into Mtb is meant to find a way to combat the bacteria and limit its ability to develop resistance due to mutations, and strains are becoming resistant to TB therapy. In order to inhibit tryptophan synthase, sulfolanes and indoline-5-sulfonamides are used. More recent research related to tryptophan inhibition found that 3-amino-3-imino-2-phenyldiazenylpropanamide was a successful inhibitor by binding to the alpha subunit of TrpS. Other research involved in tryptophan synthase is the evolutionary origin of it and why it may have evolved. A study found that tryptophan synthase uses an atypical mechanism in order to achieve substrate specificity. Specifically, TrpS is capable of catalyzing a reaction with L-threonine (Thr), which creates [2S,3S]-beta-methyltryptophan. While Thr does bind efficiently, it decreases the affinity for indol and can disrupt allosteric signaling and regulation for the catalytic cycle. Thr is considered to be a universal and abundant metabolite, so research in seeing if TrpS can catalyze the synthesis of beta-MeTrp and seeing how TrpS can choose between Ser and Thr [4].
