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Cryo-crystallography of a true substrate, indole-3-glycerol phosphate, bound to a mutant (alphaD60N) tryptophan synthase alpha2beta2 complex reveals the correct orientation of active site alphaGlu49.

Rhee S, Miles EW, Davies DR
J Biol Chem 273 8553-5 (1998)
Cited: 20 times
EuropePMC logo PMID: 9535826

Abstract

The reversible cleavage of indole-3-glycerol by the alpha-subunit of tryptophan synthase has been proposed to be catalyzed by alphaGlu49 and alphaAsp60. Although previous x-ray crystallographic structures of the tryptophan synthase alpha2beta2 complex showed an interaction between the carboxylate of alphaAsp60 and the bound inhibitor indole-3-propanol phosphate, the carboxylate of alphaGlu49 was too distant to play its proposed role. To clarify the structural and functional roles of alphaGlu49, we have determined crystal structures of a mutant (alphaD60N) alpha2beta2 complex in the presence and absence of the true substrate, indole-3-glycerol phosphate. The enzyme in the crystal cleaves indole-3-glycerol phosphate very slowly at room temperature but not under cryo-conditions of 95 K. The structure of the complex with the true substrate obtained by cryo-crystallography reveals that indole-3-glycerol phosphate and indole-3-propanol phosphate have similar binding modes but different torsion angles. Most importantly, the side chain of alphaGlu49 interacts with 3-hydroxyl group of indole-3-glycerol phosphate as proposed. The movement of the side chain of alphaGlu49 into an extended conformation upon binding the true substrate provides evidence for an induced fit mechanism. Our results demonstrate how cryo-crystallography and mutagenesis can provide insight into enzyme mechanism.

Reviews citing this publication (6)

  1. Divergent evolution of enzymatic function: mechanistically diverse superfamilies and functionally distinct suprafamilies. Gerlt JA, Babbitt PC. Annu Rev Biochem 70 209-246 (2001)
  2. Structure, evolution and action of vitamin B6-dependent enzymes. Jansonius JN. Curr Opin Struct Biol 8 759-769 (1998)
  3. 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)
  4. Tryptophan synthase: a multienzyme complex with an intramolecular tunnel. Miles EW. Chem Rec 1 140-151 (2001)
  5. Biophysical and computational methods to analyze amino acid interaction networks in proteins. O'Rourke KF, Gorman SD, Boehr DD. Comput Struct Biotechnol J 14 245-251 (2016)
  6. New results using Laue diffraction and time-resolved crystallography. Stoddard BL. Curr Opin Struct Biol 8 612-618 (1998)

Articles citing this publication (14)

  1. Functional attributes of the phosphate group binding cup of pyridoxal phosphate-dependent enzymes. Denesyuk AI, Denessiouk KA, Korpela T, Johnson MS. J Mol Biol 316 155-172 (2002)
  2. Exploring the pyridoxal 5'-phosphate-dependent enzymes. Mozzarelli A, Bettati S. Chem Rec 6 275-287 (2006)
  3. 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 352 608-620 (2005)
  4. Tryptophan synthase, an allosteric molecular factory. Barends TR, Dunn MF, Schlichting I. Curr Opin Chem Biol 12 593-600 (2008)
  5. 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)
  6. Common structural elements in the architecture of the cofactor-binding domains in unrelated families of pyridoxal phosphate-dependent enzymes. Denessiouk KA, Denesyuk AI, Lehtonen JV, Korpela T, Johnson MS. Proteins 35 250-261 (1999)
  7. Allosteric communication of tryptophan synthase. Functional and regulatory properties of the beta S178P mutant. Marabotti A, De Biase D, Tramonti A, Bettati S, Mozzarelli A. J Biol Chem 276 17747-17753 (2001)
  8. In silico identification and characterization of 1-aminocyclopropane-1-carboxylate deaminase from Phytophthora sojae. Singh N, Kashyap S. J Mol Model 18 4101-4111 (2012)
  9. Large conformational changes in the Escherichia coli tryptophan synthase beta(2) subunit upon pyridoxal 5'-phosphate binding. Nishio K, Ogasahara K, Morimoto Y, Tsukihara T, Lee SJ, Yutani K. FEBS J 277 2157-2170 (2010)
  10. Millisecond Timescale Motions Connect Amino Acid Interaction Networks in Alpha Tryptophan Synthase. O'Rourke KF, Axe JM, D'Amico RN, Sahu D, Boehr DD. Front Mol Biosci 5 92 (2018)
  11. Mutational scanning of a hairpin loop in the tryptophan synthase beta-subunit implicated in allostery and substrate channeling. Rondard P, Bedouelle H. Biol Chem 381 1185-1193 (2000)
  12. 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)
  13. Distinct conformational dynamics and allosteric networks in alpha tryptophan synthase during active catalysis. O'Rourke KF, D'Amico RN, Sahu D, Boehr DD. Protein Sci 30 543-557 (2021)
  14. 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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