Tryptophan synthase: Difference between revisions
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{{Protbox start|Name=Tryptophan synthase|Photo=TryptophanSynthase.png|Caption='''Tryptophan synthase'''.Tryptophan synthase, {PDB: 1WQ5}|Gene=[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Protein&list_uids=13361297&dopt=GenPept] ID#: GI:61680225| |
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| Name = Tryptophan Synthase |
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Structure=[http://www.rcsb.org/pdb/navbarsearch.do?newSearch=yes&isAuthorSearch=no&radioset=Structures&inputQuickSearch=1wq5&image.x=27&image.y=7&image=Search] 1WQ5|Review=[http://www.ncbi.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=4863752&dopt=Abstract] The amino acid sequence of the A protein (alpha subunit) of the tryptophan synthetase of Escherichia coli.}} |
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| EC_number = 4.2.1.20 |
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{{Protbox finish}} |
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| CAS_number = 9014-52-2 |
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| IUBMB_EC_number = 4/2/1/20 |
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| GO_code = 0004834 |
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| image = testing1.png |
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| width = |
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| caption = Subunits: <font color="buff">SdhA</font>, <font color="lime">SdhB</font>, <font color="aqua">SdhC</font> and <font color="orange">SdhD</font> |
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}} |
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'''Tryptophan synthase''' or '''tryptophan synthetase''' is an [[enzyme]] that catalyzes the final two steps in the biosynthesis of [[tryptophan]].<ref name="Tryptophan synthase">{{cite journal | author = Dunn MF, Niks D, Ngo H, Barends TRM, Schlichting I | title = Tryptophan synthase: the workings of a channeling nanomachine | journal = Trends in Biochemical Sciences | volume = 33 | issue = 6 | pages = 254-264 | year = 2008 | month = June | pmid = 18486479 | doi = | url = | issn = }}</ref> It is commonly found in Eubacteria<ref name="Eubacteria">{{cite journal | author = Jablonski P, Jablonski L, Pintado O, Sriranganathan N, Howde C | title = Tryptophan synthase: Identification of Pasteurella multocida tryptophan synthase B-subunit by antisera against strain PI059 | journal = Microbiology | volume = 142 | issue = | pages = 115-121 | year = 1996 | month = September | pmid = 8581158 | doi = | url = | issn = }}</ref>, Archaebacteria<ref name="Archaebacteria">{{cite journal | author = Lazcano A, Diaz-Villgomez E, Mills T, Oro J | title = On the levels of enzymatic substrate specificity: Implications for the early evolution of metabolic pathways | journal = Advances in Space Research | volume = 15 | issue = 3 | pages = 346-356 | year = 1995 | month = March | pmid = 11539248 | doi = | url = | issn = }}</ref>, Protista<ref name="Protista">{{cite journal | author = Anderson I, Watkins R, Samuelson J, Spencer D, Majoros W, Grey M, Loftus B | title = Gene Discovery in the Acanthamoeba castellanii Genome | journal = Protist | volume = 156 | issue = 2 | pages = 203-214 | year = 2005 | month = August | pmid = 16171187 | doi = | url = | issn = }}</ref>, Fungi<ref name="Fungi">{{cite journal | author = Ireland C, Peekhaus N, Lu P, Sangari R, Zhang A, Masurekar P, An Z | title = The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production | journal = Appl Microbiol Biotechnol | volume = 79 | issue = 3 | pages = 451-459 | year = 2008 | month = April | pmid = 18389234 | doi = | url = | issn = }}</ref>, and Plantae<ref name="Plantae">{{cite journal | author = Sanjaya, Hsiao PY, Su RC, Ko SS, Tong CG, Yang RY, Chan MT | title = Overexpression of Arabidopsis thaliana tryptophan synthase beta 1 (AtTSB1) in Arabidopsis and tomato confers tolerance to cadmium stress | journal = Plant Cell Environ | volume = 31 | issue = 8 | pages = 1074-1075 | year = 2008 | month = April | pmid = 18419734 | doi = | url = | issn = }}</ref>. However, it is absent from animalia.<ref name="Animalia">{{cite journal | author = Eckert SC, Kubler E, Hoffmann B, Braus GH | title = The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system | journal = Mol Gen Genet | volume = 263 | issue = 5 | pages = 867-876 | year = 2000 | month = June | pmid = 10905354 | doi = | url = | issn = }}</ref> It is typically found as an α2β2 tetramer. The α subunits catalyze the reversible formation of [[indole]] and [[glyceraldehyde-3-phosphate]] (G3P) from indole-3-glycerol phosphate (IG3). 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 angstrom 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]].<ref name="Overview">{{cite journal | author = Raboni S, Bettati S, Mozzarelli A | title = Tryptophan synthase: a mine for enzymologists | journal = Cell Mol Life Sci | volume = 66 | issue = | pages = 2391-2403 | year = 2009 | month = April | pmid = 19387555 | doi = | url = | issn = }}</ref> The active sites of tryptophan synthase are [[allosterically]] coupled.<ref name="Regulation">{{cite journal | author = Fatmi MQ, Ai R, Chang CA | title = Synergistic regulation and ligand-induced conformational changes of tryptophan synthase | journal = Biochemistry | volume = 48 | issue = | pages = 9921-9931 | year = 2009 | month = September | pmid = 19764814 | doi = | url = | issn = }}</ref> |
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'''Tryptophan synthase''' ({{EC number|4.2.1.20}}), also known as '''tryptophan synthetase''', is an [[enzyme]] found in plants and bacteria, but not in animals, which catalyses the final step in the biosynthesis of [[tryptophan]]. |
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==Enzyme Structure== |
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The enzyme isolated from ''[[E. coli]]'' is an α<sub>2</sub>β<sub>2</sub>-tetramer. The two α-subunits are readily detached from the β<sub>2</sub>-dimer, and the two β-subunits can be separated by treatment with 4 M [[urea]] solution. The association of the subunits is promoted by the presence of [[pyridoxal phosphate]] (PLP) and [[serine]]. Each α-subunit has a [[molecular mass]] of 29500: each β-subunit has a molecular mass of 45000 with one PLP binding site. |
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'''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 2 conformation and a binding site adjacent to the active site for monovalent cations.<ref name="Structure">{{cite journal | author = Grishin NV, Phillips MA, Goldsmith EJ | title = Modeling of the spatial structure of ornithine decarboxylases | journal = Protein Sci | volume = 4 | issue = 7 | pages = 1291-1304 | year = 1995 | month = July | pmid = 7670372 | doi = | url = | issn = }}</ref> 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, the there are interacts between the αGly181 and βSer178 residues.<ref name="Interaction">{{cite journal | author = Schneider TR, Gerhardt E, Lee M, Liang PH, Anderson KS, Schlichting I | title = Loop closure and intersubunit communication in tryptophan synthase | journal = Biochemistry | volume = 37 | issue = 16 | pages = 5394-5406 | year = 1998 | month = April | pmid = 9548921 | doi = | url = | issn = }}</ref> The active sites are regulated allosterically and undergo transitions between open, inactive, and closed, active, states.<ref name="Regulation">{{cite journal | author = Fatmi MQ, Ai R, Chang CA | title = Synergistic regulation and ligand-induced conformational changes of tryptophan synthase | journal = Biochemistry | volume = 48 | issue = | pages = 9921-9931 | year = 2009 | month = September | pmid = 19764814 | doi = | url = | issn = }}</ref> |
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[[Image:Tryptophan synthetase rn.png|500px]] |
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'''Indole-3-glycerol binding site''': See image 3. |
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The different subunits catalyse separate steps in the reactions, as shown in the diagram. Indole 3-glycerolphosphate is converted into [[indole]] and [[glyceraldehyde 3-phosphate]] by the α-subunits, while the β<sub>2</sub>-dimer catalyses the condensation of indole and serine to produce tryptophan. However, the α<sub>2</sub>β<sub>2</sub>-tetramer is 30–100 times more active than the isolated subunits, and does not release free indole during the reaction, since this intermediate is passed between the active sites through a tunnel within the protein, in a process known as [[substrate channeling]].<ref name=Huang>{{cite journal |author=Huang X, Holden HM, Raushel FM |title=Channeling of substrates and intermediates in enzyme-catalyzed reactions |journal=Annu. Rev. Biochem. |volume=70 |pages=149–80 |year=2001 |pmid=11395405 |doi=10.1146/annurev.biochem.70.1.149}}</ref> |
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'''Indole and serine binding site''': See image 3. |
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'''Hydrophobic channel''': The α and β active sites are separated by a 25 angstrom 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. <ref name="Channel">{{cite journal | author = Huang X, Holden HM, Raushel FM | title = Channeling of Substrates and Intermediates in Enzyme-Catalyzes Reactions | journal =Annu Rev Biochem | volume = 70 | issue = | pages = 149-180 | year = 2001 | month = | pmid = 11395405 | doi = | url = | issn = }}</ref> |
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[[Image: Tryptophan Synthase Mechanism 3.gif|thumb|left|alt=caption.|'''Image 1''': Mechanism of Tryptophan Synthase]] |
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==Enzyme Mechanism== |
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'''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.<ref name="Overview">{{cite journal | author = Raboni S, Bettati S, Mozzarelli A | title = Tryptophan synthase: a mine for enzymologists | journal = Cell Mol Life Sci | volume = 66 | issue = | pages = 2391-2403 | year = 2009 | month = April | pmid = 19387555 | doi = | url = | issn = }}</ref> The rate limiting step is the isomerization of IGP.<ref name="a Rate Limiting Step">{{cite journal | author = Anderson KS, Miles EW, Johnson KA | title = Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism. | journal = J Biol Chem | volume = 266 | issue = 13 | pages = 8020-8033 | year = 1991 | month = May | pmid = 1902468 | doi = | url = | issn = }}</ref> See image 1. |
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'''β subunit reaction''': The β subunit catalyzes the β-replacement reaction in which indole and serine condense to form tryptophan. The βLys87, βGlu109, and βSer377 are thought to be directly involved in the catalysis as shown.<ref name="Overview">{{cite journal | author = Raboni S, Bettati S, Mozzarelli A | title = Tryptophan synthase: a mine for enzymologists | journal = Cell Mol Life Sci | volume = 66 | issue = | pages = 2391-2403 | year = 2009 | month = April | pmid = 19387555 | doi = | url = | issn = }}</ref> See image 1. |
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'''Net reaction''': See image 2. |
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[[Image:Tryptophan synthetase rn.png|thumb|right|500px|'''Image 2''': Net Reaction Catalyzed by Tryptophan Synthase]] |
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[[Image: Active Site 1.gif|thumb|right|alt=caption.|'''Image 3''': Active Sites for α (left) and β (right) showing hypothesized catalytic residues]] |
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==Biological Function== |
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Tryptophan synthase is commonly found in Eubacteria, Archaebacteria, Protista, Fungi, and Plantae. It is absent from higher level animals such as humans. Tryptophan is one of the twenty standard [[amino acids]] and one of eight [[essential amino acids]] for humans. |
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==Disease Relevance== |
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As humans do not have this enzyme, it has been explored as a potential [[drug target]] in fighting infectous diseases such as [[tuberculosis]]. It is widely thought that bacteria have alternate mechanisms to produce such amino acids which might make this approach less effective. However, even if the drug only weakens bacteria it would be useful. Additionally, if several such inhibitors were used together, they might be sufficient to kill the bacteria. |
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==Evolution== |
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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 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. |
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==Historical Significance== |
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Tryptophan synthase was the first enzyme identified that had multiple [[catalytic]] capabilities. 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. |
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== See also == |
== See also == |
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*[[Lyase]] |
*[[Lyase]] |
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*{{cite book | author=Conn, E. E.; & Stumpf, P. K. | title=Outlines of Biochemistry | edition=4th | location=New York | publisher=Wiley | year=1976 | isbn=0-471-01772-8}} |
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[[Category:EC 4.2.1]] |
[[Category:EC 4.2.1]] |
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Revision as of 15:54, 18 May 2010
| Tryptophan Synthase | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| File:Testing1.png Subunits: SdhA, SdhB, SdhC and SdhD | |||||||||
| Identifiers | |||||||||
| EC no. | 4.2.1.20 | ||||||||
| CAS no. | 9014-52-2 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||||
| Gene Ontology | AmiGO / QuickGO | ||||||||
| |||||||||
Tryptophan synthase or tryptophan synthetase is an enzyme that catalyzes the final two steps in the biosynthesis of tryptophan.[1] It is commonly found in Eubacteria[2], Archaebacteria[3], Protista[4], Fungi[5], and Plantae[6]. However, it is absent from animalia.[7] It is typically found as an α2β2 tetramer. The α subunits catalyze the reversible formation of indole and glyceraldehyde-3-phosphate (G3P) from indole-3-glycerol phosphate (IG3). 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 angstrom 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.[8] The active sites of tryptophan synthase are allosterically coupled.[9]
Enzyme Structure
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 2 conformation and a binding site adjacent to the active site for monovalent cations.[10] 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, the there are interacts between the αGly181 and βSer178 residues.[11] The active sites are regulated allosterically and undergo transitions between open, inactive, and closed, active, states.[9]
Indole-3-glycerol binding site: See image 3.
Indole and serine binding site: See image 3.
Hydrophobic channel: The α and β active sites are separated by a 25 angstrom 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. [12]
Enzyme Mechanism
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.[8] The rate limiting step is the isomerization of IGP.[13] See image 1.
β subunit reaction: The β subunit catalyzes the β-replacement reaction in which indole and serine condense to form tryptophan. The βLys87, βGlu109, and βSer377 are thought to be directly involved in the catalysis as shown.[8] See image 1.
Net reaction: See image 2.
Biological Function
Tryptophan synthase is commonly found in Eubacteria, Archaebacteria, Protista, Fungi, and Plantae. It is absent from higher level animals such as humans. Tryptophan is one of the twenty standard amino acids and one of eight essential amino acids for humans.
Disease Relevance
As humans do not have this enzyme, it has been explored as a potential drug target in fighting infectous diseases such as tuberculosis. It is widely thought that bacteria have alternate mechanisms to produce such amino acids which might make this approach less effective. However, even if the drug only weakens bacteria it would be useful. Additionally, if several such inhibitors were used together, they might be sufficient to kill the bacteria.
Evolution
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 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.
Historical Significance
Tryptophan synthase was the first enzyme identified that had multiple catalytic capabilities. 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.
References
- ^ Dunn MF, Niks D, Ngo H, Barends TRM, Schlichting I (2008). "Tryptophan synthase: the workings of a channeling nanomachine". Trends in Biochemical Sciences. 33 (6): 254–264. PMID 18486479.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Jablonski P, Jablonski L, Pintado O, Sriranganathan N, Howde C (1996). "Tryptophan synthase: Identification of Pasteurella multocida tryptophan synthase B-subunit by antisera against strain PI059". Microbiology. 142: 115–121. PMID 8581158.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Lazcano A, Diaz-Villgomez E, Mills T, Oro J (1995). "On the levels of enzymatic substrate specificity: Implications for the early evolution of metabolic pathways". Advances in Space Research. 15 (3): 346–356. PMID 11539248.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Anderson I, Watkins R, Samuelson J, Spencer D, Majoros W, Grey M, Loftus B (2005). "Gene Discovery in the Acanthamoeba castellanii Genome". Protist. 156 (2): 203–214. PMID 16171187.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Ireland C, Peekhaus N, Lu P, Sangari R, Zhang A, Masurekar P, An Z (2008). "The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production". Appl Microbiol Biotechnol. 79 (3): 451–459. PMID 18389234.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Sanjaya, Hsiao PY, Su RC, Ko SS, Tong CG, Yang RY, Chan MT (2008). "Overexpression of Arabidopsis thaliana tryptophan synthase beta 1 (AtTSB1) in Arabidopsis and tomato confers tolerance to cadmium stress". Plant Cell Environ. 31 (8): 1074–1075. PMID 18419734.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Eckert SC, Kubler E, Hoffmann B, Braus GH (2000). "The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system". Mol Gen Genet. 263 (5): 867–876. PMID 10905354.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ a b c Raboni S, Bettati S, Mozzarelli A (2009). "Tryptophan synthase: a mine for enzymologists". Cell Mol Life Sci. 66: 2391–2403. PMID 19387555.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ a b Fatmi MQ, Ai R, Chang CA (2009). "Synergistic regulation and ligand-induced conformational changes of tryptophan synthase". Biochemistry. 48: 9921–9931. PMID 19764814.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Grishin NV, Phillips MA, Goldsmith EJ (1995). "Modeling of the spatial structure of ornithine decarboxylases". Protein Sci. 4 (7): 1291–1304. PMID 7670372.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Schneider TR, Gerhardt E, Lee M, Liang PH, Anderson KS, Schlichting I (1998). "Loop closure and intersubunit communication in tryptophan synthase". Biochemistry. 37 (16): 5394–5406. PMID 9548921.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Huang X, Holden HM, Raushel FM (2001). "Channeling of Substrates and Intermediates in Enzyme-Catalyzes Reactions". Annu Rev Biochem. 70: 149–180. PMID 11395405.
{{cite journal}}: Cite has empty unknown parameter:|month=(help)CS1 maint: multiple names: authors list (link) - ^ Anderson KS, Miles EW, Johnson KA (1991). "Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism". J Biol Chem. 266 (13): 8020–8033. PMID 1902468.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link)