Created by: Gloria Pezzella 

          Tryptophan synthase beta subunit (PBD ID: 6AM8) from Pyrococcus furiosus is a biosynthetic protein and a subunit of the system tryptophan synthase, a heterodimeric enzyme which is used as a model for allosteric enzymes and also an important enzyme for synthesizing noncanonical amino acids for biocatalysis. The overall function of the tryptophan synthase beta subunit is to synthesize L-tryptophan from indole and L-serine. The beta subunit of tryptophan synthase was evolved from Pyrococcus furiosus for noncanonical amino acid synthase activity (1). Noncanonical amino acids are outside of the twenty naturally occuring amino acids. Noncanonical amino acids are useful in studying a number of cellular processes such as ligand drug complex interactions and expanding the genetic code (2). The beta subunit of tryptophan synthase from Pyrococcus furiosus, without being bound to the alpha subunit, is used to study the stand-alone properties of TrpB in the catalysis of noncanonical amino acids and as a model of allostery. The tryptophan synthase beta subunit of Pyrococcus furiosus is also known as PfTrpB (1). PfTrpB is highly active at temperatures between 90-100 °C as Pyrococcus furiosus is an extremophile which lives in extremely high temperatures (around 100 °C). Thus, TrpB is less active as an enzyme in this organism at temperatures below 90 °C (3). 

          The method used for macromolecular crystallization of PfTrpB is vapor diffusion sitting drop. PfTrpB is ideally crystallized in PEG3550 and 0.1 M Na HEPES at a pH of 7.85 and at 298 K. Additionally, X-ray diffraction is used to characterize the crystallized structure (4). In the absence of the ligand tryptophan, PfTrpB crystallizes in an open state, meaning that the COMM domain, residues 95-161 mediating communication between the alpha and beta subunits of tryptophan synthase, is more open allowing greater movement among several side chains bordering the active site which together make up 25% of the sequence. When beta-methyl tryptophan is bound, an extended open state is observed as it connects the active site to the solvent. This conformation is crucial for transport of substrate products between the solution and the active site (1). 

          PfTrpB is comprised of four subunit chains, A, B, C, and D, each with a molecular weight of 43327.65 Da making the entire structure's molecular weight 173310.6. The crystal structure of the protein was fully crystallized. Additionally, the isoelectric point, where the protein solubility is minimal as the positive and negative charges among residues are equal, is 7.01 for PfTrpB (5). Each chain or subunit has the same protein sequence, but slightly vary in their local structural conformations. 

          The sequence of PfTrpB contains 1584 residues, all of which are crystallized. Each subunit contains 396 residues. The secondary structure of the A and D chains of PfTrpB consists of 46% alpha helices with 20 helices and 185 residues, 21% beta sheets with 22 strands and 84 residues, and an unknown number of random coils and 3/10 helices. The B and C chains consist of 47% alpha helices with 22 helices and 189 residues, 20% beta sheets with 19 strands and 81 residues, and an unknown number of random coils and 3/10 helices (4). Hydrogen bonding, particularly via the alpha helices increases the stability of the conformation of PfTrpB, specifically Asp-300 hydrogen bonded to Ser-292 and Asp-300 hydrogen bonded to the hydroxyl of the external aldimine intermediate. The alpha helices in each subunit interact, forming hydrogen bonds between charged residues and hydrophobic interactions between folded sheets to encompass a globular protein (1). 

          The beta subunit specifically receives indole released by indole glycerol phosphate from the alpha subunit and uses a pyridoxal phosphate cofactor to affect the beta substitution of a serine hydroxyl group on indole to yield L-tryptophan. The beta substitution reaction proceeds through at least nine steps to facilitate the release of L-tryptophan. This reaction along with other indole analogues can make more than thirty different noncanonical amino acids. This beta substitution reaction, which occurs fully within the beta subunit, is much less efficient when the beta subunit is separate from the alpha as the initial complex when it is the initially isolated. However, several variants of TrpB from Pyrococcus furiosus have high catalytic activity on their own as when mutations were introduced through directed evolution, the rate limiting step could be altered to enable rapid engineering of allosteric enzymes. These variants include PfTrpB2G9, PfTrpB4D11, PfTrpB4G1, and PfTrpB2B9. The first three of these mutations have higher catalytic activity than the entire tryptophan synthase protein, with PfTrpB4D11 more than double the catalytic activity. The original enzyme, PfTrpB has only one third the catalytic activity of the original tryptophan synthase heterodiamer. These different variants increase their catalytic activity by accelerating alpha carbon deprotonation, stabilizing the electrophilic amino acrylate intermediate, and causing a new step to become rate-limiting other than an external aldimine intermediate (1). 

          PfTrpB has several amino acid residues that are important to its function. Lys-82 is a pyrodoxyl phosphate cofactor which binds to the active site through protonated Schiff base linkage in the resting state of PfTrpB. Lys-82 also covalently binds to the cofactor in the open crystallized form of PfTrpB. Ser-274, His-275, Asp-300, and Tyr-301 all help make the active site for tryptophan which is a part of determining the degree of openness of the protein (1). 

          PfTrpB has three ligands, a sodium ion, [3-Hydroxy-2-methyl-5-phosphonooxymethyl-pyridin-4-ylmethyl]-L-Tryptophane, and tryptophan. The sodium ion functions to stabilize the protein structure using ionic like interactions between the carboxyl groups and the ion between Gly-303, Tyr-301, Ser-263, and Ser-265 in each of the A, B, C, and D chains of PfTrpB (3). [3-Hydroxy-2-methyl-5-phosphonooxymethyl-pyridin-4-ylmethyl]-L-Tryptophane is a monomer ligand in the B and C chains of PfTrpB and serves as an enzyme inhibitor (6). Tryptophan is present as a ligand in chains A, B, and D, and binds noncovalently to stabilize the crystal structure. Additionally, through L-peptide linking L-tryptophan can be used to aid sleep and increase serotonin production (4). 

          The Psi-BLAST server is used to find proteins with similar primary structure to a protein query using E values which decrease with greater sequence homology between the query and the subject sequence (7). Additionally, the Dali server is used to find proteins with tertiary structure similarities to a query by measuring a Z-score using a sum-of-pairs method producing a measure of similarity by comparing intramolecular distances. A Z-score greater than two indicates a protein has similar folds (8). The comparison protein used is cysteine synthase, with a Z-score of 32.4 and an E value of 5E-73. 

          The protein Cysteine synthase (PBD ID: 3FCA-A) is found in Thermus thermopilus, a gram positive bacteria which is thermophilic, while PfTrpB is found in Pyrococcus furiosus, which is an extremophile. Cysteine synthase acts as a sensor and modulator of thiol metabolism by responding to changes in nutrient conditions, while PfTrpB is used for biosynthesis of L-tryptophan. Cysteine synthase is comprised of only 2 subunits as opposed to the 4 in PfTrpB. Additionally, cysteine synthase has just one ligand, the zinc ion, whereas PfTrpB has 3 ligands. Cysteine synthase is also comprised of 17% beta sheets and 45% alpha helices, while PfTrpB is comprised of 21% beta sheets and 46% alpha helices. Cysteine synthase only contains zinc ions as a ligand which is a free ion that stabilizes the structure, whereas PfTrpB has several other monomer ligands that function more directly in the enzyme activity (4). 

          Mutations in the TrpB structure in Pyrococcus furiosus can mimic the allostery of the original subunit and enhance catalytic activity. This makes PfTrpB a good model for manipulating the protein structure of the standalone subunit and serves as a precedent for how other allosteric enzymes can be isolated in the same manner. PfTrpB also aids in understanding how noncanonical amino acids can be synthesized by inducing mutations, and these noncanonical amino acids can be used to form drug complexes. In the future, scientists can look at TrpB in organisms that function in standard conditions, such as mesophiles, to note whether the catalytic activity of the protein is influenced by temperature in all capacities. The tryptophan synthase alpha beta beta alpha heterodiamer could additionally be replaced with TrpB in other organisms. Furthermore, enhancing several mutations could be used to understand whether or not this activity can be increased in other organisms other than Pyrococcus furiosus.