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MetaCyc Enzyme: tryptophan synthase, α subunit

Gene: trpA Accession Numbers: EG11024 (MetaCyc), b1260, ECK1254

Synonyms: try, tryp, α subunit, TSase α, A protein

Species: Escherichia coli K-12 substr. MG1655

Component of: tryptophan synthase (summary available)

Summary:
The TrpA polypeptide (TSase α) functions as the α subunit of the tetrameric (α22) tryptophan synthase complex [Miles77]. As a purified protein, the α subunit is a monomer. TSase α contains the binding site for indole-3-glycerol-phosphate (InGP) and can carry out the cleavage reaction of InGP to indole and glyceraldehyde-3-phosphate, also termed the α reaction. Within the physiological complex with the β subunit, the reaction rate is increased by 1-2 orders of magnitude (in [Kirschner91]).

TrpA has been shown to lack tryptophan residues [Henning62]. Numerous TrpA mutant studies have examined structure-function relationships in this protein. Mutations that affect catalytic activity [Hiraga96, Sarker95, Lim91, Milton86, Yutani87, Yee96a, Yanofsky93, Lim91a], subunit interactions [Swift92, Lim91a], conformational stability [Hiraga96a, Lim92] and folding [Kim01b, Lim91a] have been identified.

The crystal structure of the wild-type TrpA protein has been reported at 2.8 Å resolution [Jeong04, Jeong04a], 2.5 Å resolution [Jeong05] and 2.3 Å resolution [Nishio05]. The crystal structure of a double mutant TrpA protein has been reported at 1.8 Å resolution [Jeong04a, Jeong05].

The TrpA protein has been structurally classified as a (beta alpha)(8) TIM barrel protein, a member of the common TIM barrel superfamily. Nuclear magnetic resonance spectroscopic techniques have been used to investigate its equilibrium folding mechanism in order to obtain insights into the development of structure and stability [Vadrevu08]. Many previous studies of the folding mechanism of recombinant wild-type and mutant TrpA proteins using various biophysical techniques identified intermediates in the folding pathway, for example [Beasty86, Choi95, Gualfetti99, Jeong03, Wintrode05, Wu05, Wu07a].

Locations: cytosol

Map Position: [1,314,440 <- 1,315,246]

Molecular Weight of Polypeptide: 28.724 kD (from nucleotide sequence), 30.0 kD (experimental) [Gschwind79 ]

pI: 5.54

Unification Links: ASAP:ABE-0004232 , CGSC:74 , DIP:DIP-35957N , DisProt:DP00252 , EchoBASE:EB1017 , EcoGene:EG11024 , EcoliWiki:b1260 , ModBase:P0A877 , OU-Microarray:b1260 , PortEco:trpA , PR:PRO_000024117 , Pride:P0A877 , Protein Model Portal:P0A877 , RefSeq:NP_415776 , RegulonDB:EG11024 , SMR:P0A877 , String:511145.b1260 , UniProt:P0A877

Relationship Links: InterPro:IN-FAMILY:IPR002028 , InterPro:IN-FAMILY:IPR011060 , InterPro:IN-FAMILY:IPR013785 , InterPro:IN-FAMILY:IPR018204 , PDB:Structure:1V7Y , PDB:Structure:1WQ5 , PDB:Structure:1XC4 , PDB:Structure:1XCF , Pfam:IN-FAMILY:PF00290 , Prosite:IN-FAMILY:PS00167

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0000162 - tryptophan biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, UniProtGOA11a, GOA06, Yanofsky93]
GO:0009073 - aromatic amino acid family biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Yanofsky93]
GO:0006568 - tryptophan metabolic process Inferred by computational analysis [GOA01a]
GO:0008152 - metabolic process Inferred by computational analysis [GOA01a]
GO:0008652 - cellular amino acid biosynthetic process Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0016829 - lyase activity Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Kirschner91]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01a]
GO:0004834 - tryptophan synthase activity Inferred by computational analysis [GOA06, GOA01, GOA01a]
GO:0030170 - pyridoxal phosphate binding Inferred by computational analysis [Gaudet10]
Cellular Component: GO:0005737 - cytoplasm Inferred from experiment [Murphy69]
GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05]

MultiFun Terms: metabolism biosynthesis of building blocks amino acids tryptophan

Credits:
Imported from EcoCyc 02-Jun-2015 by Paley S , SRI International


Enzymatic reaction of: indoleglycerol phosphate aldolase (tryptophan synthase, α subunit)

EC Number: 4.1.2.8

(1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate <=> indole + D-glyceraldehyde 3-phosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

This reaction is reversible. [Yutani87]

In Pathways: superpathway of chorismate metabolism , superpathway of aromatic amino acid biosynthesis , superpathway of L-tryptophan biosynthesis , L-tryptophan biosynthesis

Credits:
Imported from EcoCyc 02-Jun-2015 by Paley S , SRI International

Summary:
This partial reaction catalyzed by the α subunit alone is reversible, while the overall physiological reaction catalyzed by the α2β2 tryptophan synthase complex is not (in [Lane91]).

The reverse partial reaction (indoleglycerol phosphate synthesis) catalyzed by either the α2β2 complex [Weischet76], or the α subunit [Weischet76a], has been subjected to steady-state kinetic analysis to define the reaction mechanism.

Inhibitors (Competitive): indolepropanol phosphate [Kirschner75]


Subunit of: tryptophan synthase

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of tryptophan synthase = [TrpA]2[(TrpB)2]
         tryptophan synthase, α subunit = TrpA (extended summary available)
         tryptophan synthase, β subunit dimer = (TrpB)2 (extended summary available)
                 tryptophan synthase, β subunit = TrpB

Summary:
The physiologically active form of tryptophan synthase is a tetrameric α22 complex consisting of two α subunits (the protein product of the trpA gene) and a dimer of two β subunits (the protein product of the trpB gene). This complex catalyzes the last two steps in the biosynthesis of tryptophan [Lane91].

Although the α22 complex from Escherichia coli has been well studied, the purified α22 complex from Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella typhimurium) provided crystals suitable for X-ray crystallography. Thus, the complex from this species has been studied in greater detail (reviewed in [Miles01, Dunn08]).

Molecular Weight: 146.5 kD (experimental) [Adachi74]

Credits:
Imported from EcoCyc 02-Jun-2015 by Paley S , SRI International


Enzymatic reaction of: tryptophan synthase

Synonyms: tryptophan desmolase, tryptophan synthetase

EC Number: 4.2.1.20

(1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate + L-serine <=> L-tryptophan + D-glyceraldehyde 3-phosphate + H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is irreversible in the direction shown. [Kirschner91]

In Pathways: superpathway of chorismate metabolism , superpathway of aromatic amino acid biosynthesis , superpathway of L-tryptophan biosynthesis , L-tryptophan biosynthesis

Credits:
Imported from EcoCyc 02-Jun-2015 by Paley S , SRI International

Summary:
The overall tryptophan synthase reaction consists of a sequence of two partial reactions. The α subunit of the complex carries out the aldol cleavage of indole-3-glycerol phosphate to indole + glyceraldehyde-3-phosphate. The β subunit is responsible for the synthesis of L-tryptophan from indole + L-serine. The α22 complex, which alone catalyzes the overall reaction, proceeds at two independent α/β sites via catalysis of the α reaction on the α subunit component, channeling the product (indole) to the pyridoxal 5'-phosphate site on the β component, where, in the presence of L-serine, it is converted to tryptophan [Lane83, Dunn90] and reviewed in [Miles99]. Indole does not appear in solution and is not a free intermediate [Crawford58]. There is apparent subunit communication mediated by transduced conformational changes between the subunits, whereby the rates of the α and β reactions are strongly enhanced by, respectively, the β and α subunits [Lim91a, Kirschner91].

The partial reaction catalyzed by the α subunit is reversible [Yutani87], whereas the partial reaction catalyzed by the β subunit and the overall reaction catalyzed by the α22 complex are considered to be practically irreversible (in [Kirschner91]).

Cofactors or Prosthetic Groups: pyridoxal 5'-phosphate [Miles77]

Cofactor Binding Comment: Pyridoxal phosphate derivatives are much more strongly bound to the complex than to the beta subunit. This finding indicates that strong binding forces, in addition to the Schiff base linkage, exist in the complex, but not in the beta subunit. When this bond is broken during the formation of a derivative, the derivative is only weakly bound to the beta subunit, but is stongly bound to the complex by additional forces.[Miles77]

T(opt): 40 °C [Bang83, BRENDA14, Zhao11a]

pH(opt): 8 [BRENDA14, Zhao11a]


Sequence Features

Feature Class Location Citations Comment
Active-Site 49
[UniProt10a]
UniProt: Proton acceptor; Non-Experimental Qualifier: by similarity;
Active-Site 60
[UniProt10a]
UniProt: Proton acceptor; Non-Experimental Qualifier: by similarity;
Extrinsic-Sequence-Variant 209 -> 224
[UniProt15]
UniProt: In mutant TrpA46-Asp-PR3..

History:
10/20/97 Gene b1260 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11024; confirmed by SwissProt match.


References

Adachi74: Adachi O, Kohn LD, Miles EW (1974). "Crystalline alpha2 beta2 complexes of tryptophan synthetase of Escherichia coli. A comparison between the native complex and the reconstituted complex." J Biol Chem 249(24);7756-63. PMID: 4609974

Bang83: Bang WG, Lang S, Sahm H, Wagner F (1983). "Production L-tryptophan by Escherichia coli cells." Biotechnol Bioeng 25(4);999-1011. PMID: 18548715

Beasty86: Beasty AM, Hurle MR, Manz JT, Stackhouse T, Onuffer JJ, Matthews CR (1986). "Effects of the phenylalanine-22----leucine, glutamic acid-49----methionine, glycine-234----aspartic acid, and glycine-234----lysine mutations on the folding and stability of the alpha subunit of tryptophan synthase from Escherichia coli." Biochemistry 25(10);2965-74. PMID: 2872918

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Choi95: Choi SG, Hardman JK (1995). "Unfolding properties of tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase." J Biol Chem 270(47);28177-82. PMID: 7499309

Crawford58: Crawford IP, Yanofsky C (1958). "On the separation of the tryptophan synthetase of Escherichia coli into two protein components." Proc Natl Acad Sci U S A 44(12);1161-70. PMID: 16590328

Crawford60: CRAWFORD IP (1960). "Identification of the triose phosphate formed in the tryptophan synthetase reaction." Biochim Biophys Acta 45;405-7. PMID: 13696310

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Dunn08: Dunn MF, Niks D, Ngo H, Barends TR, Schlichting I (2008). "Tryptophan synthase: the workings of a channeling nanomachine." Trends Biochem Sci 33(6);254-64. PMID: 18486479

Dunn90: Dunn MF, Aguilar V, Brzovic P, Drewe WF, Houben KF, Leja CA, Roy M (1990). "The tryptophan synthase bienzyme complex transfers indole between the alpha- and beta-sites via a 25-30 A long tunnel." Biochemistry 29(37);8598-607. PMID: 2271543

Gaudet10: Gaudet P, Livstone M, Thomas P (2010). "Annotation inferences using phylogenetic trees." PMID: 19578431

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Gschwind79: Gschwind HP, Gschwind U, Paul CH, Kirschner K (1979). "Affinity chromatography of tryptophan synthase from Escherichia coli. Systematic studies with immobilized tryptophanol phosphate." Eur J Biochem 96(2);403-16. PMID: 378665

Gualfetti99: Gualfetti PJ, Bilsel O, Matthews CR (1999). "The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli." Protein Sci 8(8);1623-35. PMID: 10452606

Henning62: HENNING U, HELINSKI DR, CHAO FC, YANOFSKY C (1962). "The A protein of the tryptophan synthetase of Escherichia coli. Purification, crystallization, and composition studies." J Biol Chem 237;1523-30. PMID: 13906502

Hiraga96: Hiraga K, Yutani K (1996). "Study of cysteine residues in the alpha subunit of Escherichia coli tryptophan synthase. 2. Role in enzymatic function." Protein Eng 9(5);433-8. PMID: 8795043

Hiraga96a: Hiraga K, Yutani K (1996). "Study of cysteine residues in the alpha subunit of Escherichia coli tryptophan synthase. 1. Role in conformational stability." Protein Eng 9(5);425-31. PMID: 8795042

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Jeong03: Jeong JK, Shin HJ, Kim JW, Lee CH, Kim HD, Lim WK (2003). "Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli." Biochem Biophys Res Commun 300(1);29-35. PMID: 12480516

Jeong04: Jeong MS, Jeong JK, Park KS, Kim HT, Lee KM, Lim WK, Jang SB (2004). "Crystallization and preliminary X-ray analysis of tryptophan synthase alpha-subunits from Escherichia coli." Acta Crystallogr D Biol Crystallogr 60(Pt 1);132-4. PMID: 14684907

Jeong04a: Jeong MS, Jeong JK, Lim WK, Jang SB (2004). "Structures of wild-type and P28L/Y173F tryptophan synthase alpha-subunits from Escherichia coli." Biochem Biophys Res Commun 323(4);1257-64. PMID: 15451433

Jeong05: Jeong MS, Jang SB (2005). "Crystallization and X-ray crystallographic studies of wild-type and mutant tryptophan synthase alpha-subunits from Escherichia coli." Mol Cells 19(2);219-22. PMID: 15879705

Kim01b: Kim JW, Kim EY, Park HH, Jung JE, Kim HD, Shin HJ, Lim WK (2001). "Homodimers of mutant tryptophan synthase alpha-subunits in Escherichia coli." Biochem Biophys Res Commun 289(2);568-72. PMID: 11716512

Kirschner75: Kirschner K, Wiskocil RL, Foehn M, Rezeau L (1975). "The tryptophan synthase from Escherichia coli. An improved purification procedure for the alpha-subunit and binding studies with substrate analogues." Eur J Biochem 60(2);513-23. PMID: 1107044

Kirschner91: Kirschner K, Lane AN, Strasser AW (1991). "Reciprocal communication between the lyase and synthase active sites of the tryptophan synthase bienzyme complex." Biochemistry 1991;30(2);472-8. PMID: 1899027

Lane83: Lane AN, Kirschner K (1983). "The catalytic mechanism of tryptophan synthase from Escherichia coli. Kinetics of the reaction of indole with the enzyme--L-serine complexes." Eur J Biochem 129(3);571-82. PMID: 6402362

Lane91: Lane AN, Kirschner K (1991). "Mechanism of the physiological reaction catalyzed by tryptophan synthase from Escherichia coli." Biochemistry 30(2);479-84. PMID: 1899028

Lim91: Lim WK, Sarkar SK, Hardman JK (1991). "Enzymatic properties of mutant Escherichia coli tryptophan synthase alpha-subunits." J Biol Chem 266(30);20205-12. PMID: 1939081

Lim91a: Lim WK, Shin HJ, Milton DL, Hardman JK (1991). "Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subunits." J Bacteriol 1991;173(6);1886-93. PMID: 2001993

Lim92: Lim WK, Brouillette C, Hardman JK (1992). "Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits." Arch Biochem Biophys 292(1);34-41. PMID: 1727648

LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532

Miles01: Miles EW (2001). "Tryptophan synthase: a multienzyme complex with an intramolecular tunnel." Chem Rec 1(2);140-51. PMID: 11893063

Miles77: Miles EW, Moriguchi M (1977). "Tryptophan synthase of Escherichia coli. Removal of pyridoxal 5'-phosphate and separation of the alpha and beta2 subunits." J Biol Chem 1977;252(19);6594-9. PMID: 330534

Miles99: Miles EW, Rhee S, Davies DR (1999). "The molecular basis of substrate channeling." J Biol Chem 274(18);12193-6. PMID: 10212181

Milton86: Milton DL, Napier ML, Myers RM, Hardman JK (1986). "In vitro mutagenesis and overexpression of the Escherichia coli trpA gene and the partial characterization of the resultant tryptophan synthase mutant alpha-subunits." J Biol Chem 261(35);16604-15. PMID: 3023357

Murphy69: Murphy TM, Mills SE (1969). "Immunochemical and enzymatic comparisons of the tryptophan synthase alpha subunits from five species of Enterobacteriaceae." J Bacteriol 97(3);1310-20. PMID: 4887511

Nishio05: Nishio K, Morimoto Y, Ishizuka M, Ogasahara K, Tsukihara T, Yutani K (2005). "Conformational changes in the alpha-subunit coupled to binding of the beta 2-subunit of tryptophan synthase from Escherichia coli: crystal structure of the tryptophan synthase alpha-subunit alone." Biochemistry 44(4);1184-92. PMID: 15667212

Sarker95: Sarker KD, Hardman JK (1995). "Affinities of phosphorylated substrates for the E. coli tryptophan synthase alpha-subunit: roles of Ser-235 and helix-8' dipole." Proteins 21(2);130-9. PMID: 7777488

Swift92: Swift S, Kuhn J, Stewart GS (1992). "Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit." Mol Gen Genet 233(1-2);129-35. PMID: 1603055

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Vadrevu08: Vadrevu R, Wu Y, Matthews CR (2008). "NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein." J Mol Biol 377(1);294-306. PMID: 18234216

Weischet76: Weischet WO, Kirschner K (1976). "The mechanism of the synthesis of indoleglycerol phosphate catalyzed by tryptophan synthase from Escherichia coli. Steady-state kinetic studies." Eur J Biochem 65(2);365-73. PMID: 949971

Weischet76a: Weischet WO, Kirschner K (1976). "Steady-state kinetic studies of the synthesis of indoleglycerol phosphate catalyzed by the alpha subunit of tryptophan synthase from Escherichia coli. Comparison with the alpha2 beta2-complex." Eur J Biochem 65(2);375-85. PMID: 949972

Wintrode05: Wintrode PL, Rojsajjakul T, Vadrevu R, Matthews CR, Smith DL (2005). "An obligatory intermediate controls the folding of the alpha-subunit of tryptophan synthase, a TIM barrel protein." J Mol Biol 347(5);911-9. PMID: 15784252

Wu05: Wu Y, Vadrevu R, Yang X, Matthews CR (2005). "Specific structure appears at the N terminus in the sub-millisecond folding intermediate of the alpha subunit of tryptophan synthase, a TIM barrel protein." J Mol Biol 351(3);445-52. PMID: 16023136

Wu07a: Wu Y, Vadrevu R, Kathuria S, Yang X, Matthews CR (2007). "A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the alpha subunit of tryptophan synthase, a TIM barrel protein." J Mol Biol 366(5);1624-38. PMID: 17222865

Yanofsky93: Yanofsky C, Yee MC, Horn V (1993). "Partial revertants of tryptophan synthetase alpha chain active site mutant Asp60-->Asn." J Biol Chem 268(11);8213-20. PMID: 8463331

Yee96a: Yee MC, Horn V, Yanofsky C (1996). "On the role of helix 0 of the tryptophan synthetase alpha chain of Escherichia coli." J Biol Chem 271(25);14754-63. PMID: 8662916

Yutani87: Yutani K, Ogasahara K, Tsujita T, Kanemoto K, Matsumoto M, Tanaka S, Miyashita T, Matsushiro A, Sugino Y, Miles EW (1987). "Tryptophan synthase alpha subunit glutamic acid 49 is essential for activity. Studies with 19 mutants at position 49." J Biol Chem 262(28);13429-33. PMID: 2888759

Zhao11a: Zhao G, Liu J, Dong K, Zhang F, Zhang H, Liu Q, Jiao Q (2011). "Enzymatic synthesis of L-tryptophan from hair acid hydrolysis industries wastewater with tryptophan synthase." Bioresour Technol 102(3);3554-7. PMID: 20884203


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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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