Tryptophan synthase or tryptophan synthetase is an enzyme (EC4.2.1.20) that catalyzes the final two steps in the biosynthesis of tryptophan.[1][2] It is commonly found in Eubacteria,[3]Archaebacteria,[4]Protista,[5]Fungi,[6] and Plantae.[7] However, it is absent from Animalia.[8] It is typically found as an α2β2 tetramer.[9][10] The α subunits catalyze the reversible formation of indole and glyceraldehyde-3-phosphate (G3P) from indole-3-glycerol phosphate (IGP). The β subunits catalyze the irreversible condensation of indole and serine to form tryptophan in a pyridoxal phosphate (PLP) dependent reaction. Each α active site is connected to a β active site by a 25 Ångstrom long hydrophobic channel contained within the enzyme. This facilitates the diffusion of indole formed at α active sites directly to β active sites in a process known as substrate channeling.[11] The active sites of tryptophan synthase are allosterically coupled.[12]
Enzyme structure
Active sites for α and β subunits showing hypothesized catalytic residues
Subunits
Tryptophan synthase typically exists as an α-ββ-α complex. The α and β subunits have molecular masses of 27 and 43 kDa respectively. The α subunit has a TIM barrel conformation. The β subunit has a fold type II conformation and a binding site adjacent to the active site for monovalent cations.[13] Their assembly into a complex leads to structural changes in both subunits resulting in reciprocal activation. There are two main mechanisms for intersubunit communication. First, the COMM domain of the β-subunit and the α-loop2 of the α-subunit interact. Additionally, there are interactions between the αGly181 and βSer178 residues.[14] The active sites are regulated allosterically and undergo transitions between open, inactive, and closed, active, states.[12]
Hydrophobic channel
The α and β active sites are separated by a 25 Ångstrom long hydrophobic channel contained within the enzyme allowing for the diffusion of indole. If the channel did not exist, the indole formed at an α active site would quickly diffuse away and be lost to the cell as it is hydrophobic and can easily cross membranes. As such, the channel is essential for enzyme complex function.[15]
Enzyme mechanism
Proposed mechanism of tryptophan synthase
The net reaction of tryptophan synthase turns indole-3-glycerol phosphate and serine into glyceraldehyde-3-phosphate, tryptophan and water. The reaction happens in two steps, each catalyzed by one of the subunits:
Reaction catalyzed by tryptophan synthase
α subunit reaction
The α subunit catalyzes the formation of indole and G3P from a retro-aldol cleavage of IGP. The αGlu49 and αAsp60 are thought to be directly involved in the catalysis as shown.[11] The rate limiting step is the isomerization of IGP.[16] See image 2.
β subunit reaction
The β subunit catalyzes the β-replacement reaction in which indole and serine condense to form tryptophan in a PLP dependent reaction. The βLys87, βGlu109, and βSer377 are thought to be directly involved in the catalysis as shown.[11] Again, the exact mechanism has not been conclusively determined. See image 2.
Biological function
Tryptophan synthase is commonly found in Eubacteria, Archaebacteria, Protista, Fungi, and Plantae. It is absent from animals such as humans. Tryptophan is one of the twenty standard amino acids and one of nine essential amino acids for humans. As such, tryptophan is a necessary component of the human diet.
Substrate scope
Tryptophan synthetase is also known to accept indole analogues, e.g., fluorinated or methylated indoles, as substrates, generating the corresponding tryptophan analogues.[17]
Disease relevance
As humans do not have tryptophan synthase, this enzyme has been explored as a potential drug target.[18] However, it is thought that bacteria have alternate mechanisms to produce amino acids which might make this approach less effective. In either case, even if the drug only weakens bacteria, it might still be useful as the bacteria are already vulnerable in the hostile host environment. As such, the inhibition of tryptophan synthase along with other PLP-enzymes in amino acid metabolism has the potential to help solve medical problems.[19]
Inhibition of tryptophan synthase and other PLP-enzymes in amino acid metabolism has been suggested for:
It is thought that early in evolution the trpB2 gene was duplicated. One copy entered the trp operon as trpB2i allowing for its expression with trpA. TrpB2i formed transient complexes with TrpA and in the process activated TrpA unidirectionally. The other copy remained outside as trpB2o, and fulfilled an existing role or played a new one such as acting as a salvage protein for indole. TrpB2i evolved into TrpB1, which formed permanent complexes with trpA resulting in bidirectional activation. The advantage of the indole salvage protein declined and the TrpB gene was lost. Finally, the TrpB1 and TrpA genes were fused resulting in the formation the bifunctional enzyme.[22]
Historical significance
Tryptophan synthase was the first enzyme identified that had two catalytic capabilities that were extensively studied. It was also the first identified to utilize substrate channeling. As such, this enzyme has been studied extensively and is the subject of great interest.[11]
^Dunn MF, Niks D, Ngo H, Barends TR, Schlichting I (June 2008). "Tryptophan synthase: the workings of a channeling nanomachine". Trends in Biochemical Sciences. 33 (6): 254–64. doi:10.1016/j.tibs.2008.04.008. PMID18486479.
^Miles EW (1991). "Structural basis for catalysis by tryptophan synthase". Advances in Enzymology and Related Areas of Molecular Biology. Advances in Enzymology and Related Areas of Molecular Biology. Vol. 64. pp. 93–172. doi:10.1002/9780470123102.ch3. ISBN9780470123102. PMID2053470.
^Lazcano A, Diaz-Villgomez E, Mills T, Oro J (March 1995). "On the levels of enzymatic substrate specificity: Implications for the early evolution of metabolic pathways". Advances in Space Research. 15 (3): 345–56. doi:10.1016/S0273-1177(99)80106-9. PMID11539248.
^Anderson I, Watkins R, Samuelson J, Spencer D, Majoros W, Grey M, Loftus B (August 2005). "Gene Discovery in the Acanthamoeba castellanii Genome". Protist. 156 (2): 203–14. doi:10.1016/j.protis.2005.04.001. PMID16171187.
^Ireland C, Peekhaus N, Lu P, Sangari R, Zhang A, Masurekar P, An Z (April 2008). "The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production". Appl Microbiol Biotechnol. 79 (3): 451–9. doi:10.1007/s00253-008-1440-3. PMID18389234. S2CID7230896.
^Sanjaya, Hsiao PY, Su RC, Ko SS, Tong CG, Yang RY, Chan MT (April 2008). "Overexpression of Arabidopsis thaliana tryptophan synthase beta 1 (AtTSB1) in Arabidopsis and tomato confers tolerance to cadmium stress". Plant Cell Environ. 31 (8): 1074–85. doi:10.1111/j.1365-3040.2008.01819.x. PMID18419734.{{cite journal}}: CS1 maint: multiple names: authors list (link)
^Eckert SC, Kubler E, Hoffmann B, Braus GH (June 2000). "The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system". Mol Gen Genet. 263 (5): 867–76. doi:10.1007/s004380000250. PMID10905354. S2CID22836208.
^Schneider TR, Gerhardt E, Lee M, Liang PH, Anderson KS, Schlichting I (April 1998). "Loop closure and intersubunit communication in tryptophan synthase". Biochemistry. 37 (16): 5394–406. doi:10.1021/bi9728957. PMID9548921.
^Huang X, Holden HM, Raushel FM (2001). "Channeling of Substrates and Intermediates in Enzyme-Catalyzes Reactions". Annu Rev Biochem. 70: 149–80. doi:10.1146/annurev.biochem.70.1.149. PMID11395405.
^Becker D, Selbach M, Rollenhagen C, Ballmaier M, Meyer TF, Mann M, Bumann D (March 2006). "Robust Salmonella metabolism limits possibilities for new antimicrobials". Nature. 440 (7082): 303–7. doi:10.1038/nature04616. PMID16541065. S2CID4426157.
^Kulik V, Hartmann E, Weyand M, Frey M, Gierl A, Niks D, Dunn MF, Schlichting I (September 2005). "On the structural basis of the catalytic mechanism and the regulation of the α-subunit of tryptophan synthase from Salmonella typhimurium and BXI from maize, two evolutionarily related enzymes". J Mol Biol. 352 (3): 608–20. doi:10.1016/j.jmb.2005.07.014. PMID16120446.
^Leopoldseder S, Hettwer S, Sterner R (November 2006). "Evolution of Multi-Enzyme Complexes: The Case of Tryptophan Synthase". Biochemistry. 45 (47): 14111–9. doi:10.1021/bi061684b. PMID17115706.
