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On the structural basis of the catalytic mechanism and the regulation of the alpha subunit of tryptophan synthase from Salmonella typhimurium and BX1 from maize, two evolutionarily related enzymes.

Kulik V, Hartmann E, Weyand M, Frey M, Gierl A, Niks D, Dunn MF, Schlichting I
J Mol Biol 352 608-20 (2005)
Related entries: 1tjp, 1tjr, 1wbj

Cited: 30 times
EuropePMC logo PMID: 16120446

Abstract

Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the alpha-subunit (TSA) of the bifunctional tryptophan synthase (TRPS) catalyzes the cleavage of indole 3-glycerol phosphate (IGP) to indole and d-glyceraldehyde 3-phosphate (G3P). Subsequently, indole diffuses through the connecting tunnel to the beta-active site where it is condensed with serine to form tryptophan and water. The maize enzyme, BX1, a homolog of TSA, also cleaves IGP to G3P and indole, and the indole is further converted to 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, a secondary plant metabolite. BX1 cleaves IGP significantly faster to G3P and indole than does TSA. In line with their different biological functions, these two evolutionary related enzymes differ significantly in their regulatory aspects while catalyzing the same chemistry. Here, the mechanism of IGP cleavage by TSA was analyzed using a novel transition state analogue generated in situ by reaction of 2-aminophenol and G3P. The crystal structure of the complex shows an sp3-hybridized atom corresponding to the C3 position of IGP. The catalytic alphaGlu49 rotates to interact with the sp3-hybridized atom and the 3' hydroxyl group suggesting that it serves both as proton donor and acceptor in the alpha-reaction. The second catalytic residue, alphaAsp60 interacts with the atom corresponding to the indolyl nitrogen, and the catalytically important loop alphaL6 is in the closed, high activity conformation. Comparison of the TSA and TSA-transition state analogue structures with the crystal structure of BX1 suggests that the faster catalytic rate of BX1 may be due to a stabilization of the active conformation: loop alphaL6 is closed and the catalytic glutamate is in the active conformation. The latter is caused by a substitution of the residues that stabilize the inactive conformation in TRPS.

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  1. Functional classification of protein structures by local structure matching in graph representation. Mills CL, Garg R, Lee JS, Tian L, Suciu A, Cooperman GD, Beuning PJ, Ondrechen MJ. Protein Sci 27 1125-1135 (2018)
  2. Quaternary Structure of the Tryptophan Synthase α-Subunit Homolog BX1 from Zea mays. Norris A, Busch F, Schupfner M, Sterner R, Wysocki VH. J Am Soc Mass Spectrom 31 227-233 (2020)
  3. Molecular cloning and functional characterization of BcTSA in the biosynthesis of indole alkaloids in Baphicacanthus cusia. Guo Z, Chen J, Lv Z, Huang Y, Tan H, Zhang L, Diao Y. Front Plant Sci 14 1174582 (2023)


Reviews citing this publication (5)

  1. Benzoxazinoid biosynthesis, a model for evolution of secondary metabolic pathways in plants. Frey M, Schullehner K, Dick R, Fiesselmann A, Gierl A. Phytochemistry 70 1645-1651 (2009)
  2. Tryptophan synthase: the workings of a channeling nanomachine. Dunn MF, Niks D, Ngo H, Barends TR, Schlichting I. Trends Biochem Sci 33 254-264 (2008)
  3. Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex. Dunn MF. Arch Biochem Biophys 519 154-166 (2012)
  4. Tryptophan synthase: a mine for enzymologists. Raboni S, Bettati S, Mozzarelli A. Cell Mol Life Sci 66 2391-2403 (2009)
  5. Allosteric regulation of substrate channeling: Salmonella typhimurium tryptophan synthase. Ghosh RK, Hilario E, Chang CA, Mueller LJ, Dunn MF. Front Mol Biosci 9 923042 (2022)

Articles citing this publication (22)

  1. Protein complexes are under evolutionary selection to assemble via ordered pathways. Marsh JA, Hernández H, Hall Z, Ahnert SE, Perica T, Robinson CV, Teichmann SA. Cell 153 461-470 (2013)
  2. Arabidopsis indole synthase, a homolog of tryptophan synthase alpha, is an enzyme involved in the Trp-independent indole-containing metabolite biosynthesis. Zhang R, Wang B, Ouyang J, Li J, Wang Y. J Integr Plant Biol 50 1070-1077 (2008)
  3. Structure and mechanistic implications of a tryptophan synthase quinonoid intermediate. Barends TR, Domratcheva T, Kulik V, Blumenstein L, Niks D, Dunn MF, Schlichting I. Chembiochem 9 1024-1028 (2008)
  4. Benzoxazinoid biosynthesis in dicot plants. Schullehner K, Dick R, Vitzthum F, Schwab W, Brandt W, Frey M, Gierl A. Phytochemistry 69 2668-2677 (2008)
  5. Tryptophan synthase, an allosteric molecular factory. Barends TR, Dunn MF, Schlichting I. Curr Opin Chem Biol 12 593-600 (2008)
  6. Long-range interactions in the α subunit of tryptophan synthase help to coordinate ligand binding, catalysis, and substrate channeling. Axe JM, Boehr DD. J Mol Biol 425 1527-1545 (2013)
  7. Surface Accessibility and Dynamics of Macromolecular Assemblies Probed by Covalent Labeling Mass Spectrometry and Integrative Modeling. Schmidt C, Macpherson JA, Lau AM, Tan KW, Fraternali F, Politis A. Anal Chem 89 1459-1468 (2017)
  8. Characterisation of the tryptophan synthase alpha subunit in maize. Kriechbaumer V, Weigang L, Fiesselmann A, Letzel T, Frey M, Gierl A, Glawischnig E. BMC Plant Biol 8 44 (2008)
  9. The role of oligomerization and cooperative regulation in protein function: the case of tryptophan synthase. Fatmi MQ, Chang CE. PLoS Comput Biol 6 e1000994 (2010)
  10. H2r: identification of evolutionary important residues by means of an entropy based analysis of multiple sequence alignments. Merkl R, Zwick M. BMC Bioinformatics 9 151 (2008)
  11. H2rs: deducing evolutionary and functionally important residue positions by means of an entropy and similarity based analysis of multiple sequence alignments. Janda JO, Popal A, Bauer J, Busch M, Klocke M, Spitzer W, Keller J, Merkl R. BMC Bioinformatics 15 118 (2014)
  12. Identification of new benzamide inhibitor against α-subunit of tryptophan synthase from Mycobacterium tuberculosis through structure-based virtual screening, anti-tuberculosis activity and molecular dynamics simulations. Naz S, Farooq U, Ali S, Sarwar R, Khan S, Abagyan R. J Biomol Struct Dyn 37 1043-1053 (2019)
  13. Catalytically impaired TrpA subunit of tryptophan synthase from Chlamydia trachomatis is an allosteric regulator of TrpB. Michalska K, Wellington S, Maltseva N, Jedrzejczak R, Selem-Mojica N, Rosas-Becerra LR, Barona-Gómez F, Hung DT, Joachimiak A. Protein Sci 30 1904-1918 (2021)
  14. Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium. Jin Z, Kim JH, Park SU, Kim SU. Plant Cell Rep 35 2449-2459 (2016)
  15. Generation of a Stand-Alone Tryptophan Synthase α-Subunit by Mimicking an Evolutionary Blueprint. Schupfner M, Busch F, Wysocki VH, Sterner R. Chembiochem 20 2747-2751 (2019)
  16. Strategy for cold adaptation of the tryptophan synthase α subunit from the psychrophile Shewanella frigidimarina K14-2: crystal structure and physicochemical properties. Mitsuya D, Tanaka S, Matsumura H, Urano N, Takano K, Ogasahara K, Takehira M, Yutani K, Ishida M. J Biochem 155 73-82 (2014)
  17. Backbone assignments and conformational dynamics in the S. typhimurium tryptophan synthase α-subunit from solution-state NMR. Sakhrani VV, Hilario E, Caulkins BG, Hatcher-Skeers ME, Fan L, Dunn MF, Mueller LJ. J Biomol NMR 74 341-354 (2020)
  18. The tryptophan synthase β-subunit paralogs TrpB1 and TrpB2 in Thermococcus kodakarensis are both involved in tryptophan biosynthesis and indole salvage. Hiyama T, Sato T, Imanaka T, Atomi H. FEBS J 281 3113-3125 (2014)
  19. Fermentative Indole Production via Bacterial Tryptophan Synthase Alpha Subunit and Plant Indole-3-Glycerol Phosphate Lyase Enzymes. Ferrer L, Mindt M, Suarez-Diez M, Jilg T, Zagorščak M, Lee JH, Gruden K, Wendisch VF, Cankar K. J Agric Food Chem 70 5634-5645 (2022)
  20. Computational Analysis on the Allostery of Tryptophan Synthase: Relationship between α/β-Ligand Binding and Distal Domain Closure. Ito S, Yagi K, Sugita Y. J Phys Chem B 126 3300-3308 (2022)
  21. Improving pathway prediction accuracy of constraints-based metabolic network models by treating enzymes as microcompartments. Yang X, Mao Z, Huang J, Wang R, Dong H, Zhang Y, Ma H. Synth Syst Biotechnol 8 597-605 (2023)
  22. PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase. Hilario E, Fan L, Mueller LJ, Dunn MF. J Vis Exp (2020)


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