1d8c Citations

Crystal structure of Escherichia coli malate synthase G complexed with magnesium and glyoxylate at 2.0 A resolution: mechanistic implications.

Biochemistry 39 3156-68 (2000)
Cited: 60 times
EuropePMC logo PMID: 10715138


The crystal structure of selenomethionine-substituted malate synthase G, an 81 kDa monomeric enzyme from Escherichia coli has been determined by MAD phasing, model building, and crystallographic refinement to a resolution of 2.0 A. The crystallographic R factor is 0.177 for 49 242 reflections observed at the incident wavelength of 1.008 A, and the model stereochemistry is satisfactory. The basic fold of the enzyme is that of a beta8/alpha8 (TIM) barrel. The barrel is centrally located, with an N-terminal alpha-helical domain flanking one side. An inserted beta-sheet domain folds against the opposite side of the barrel, and an alpha-helical C-terminal domain forms a plug which caps the active site. Malate synthase catalyzes the condensation of glyoxylate and acetyl-coenzyme A and hydrolysis of the intermediate to yield malate and coenzyme A, requiring Mg(2+). The structure reveals an enzyme-substrate complex with glyoxylate and Mg(2+) which coordinates the aldehyde and carboxylate functions of the substrate. Two strictly conserved residues, Asp631 and Arg338, are proposed to provide concerted acid-base chemistry for the generation of the enol(ate) intermediate of acetyl-coenzyme A, while main-chain hydrogen bonds and bound Mg(2+) polarize glyoxylate in preparation for nucleophilic attack. The catalytic strategy of malate synthase appears to be essentially the same as that of citrate synthase, with the electrophile activated for nucleophilic attack by nearby positive charges and hydrogen bonds, while concerted acid-base catalysis accomplishes the abstraction of a proton from the methyl group of acetyl-coenzyme A. An active site aspartate is, however, the only common feature of these two enzymes, and the active sites of these enzymes are produced by quite different protein folds. Interesting similarities in the overall folds and modes of substrate recognition are discussed in comparisons of malate synthase with pyruvate kinase and pyruvate phosphate dikinase.

Articles - 1d8c mentioned but not cited (2)

Reviews citing this publication (7)

  1. Indirect use of deuterium in solution NMR studies of protein structure and hydrogen bonding. Tugarinov V. Prog Nucl Magn Reson Spectrosc 77 49-68 (2014)
  2. Biotechnological potential of the ethylmalonyl-CoA pathway. Alber BE. Appl. Microbiol. Biotechnol. 89 17-25 (2011)
  3. Role of magnesium in alleviation of aluminium toxicity in plants. Bose J, Babourina O, Rengel Z. J. Exp. Bot. 62 2251-2264 (2011)
  4. Experimental approaches for NMR studies of side-chain dynamics in high-molecular-weight proteins. Sheppard D, Sprangers R, Tugarinov V. Prog Nucl Magn Reson Spectrosc 56 1-45 (2010)
  5. NMR studies of protein structure and dynamics. Kay LE. J. Magn. Reson. 173 193-207 (2005)
  6. Methyl groups as probes of structure and dynamics in NMR studies of high-molecular-weight proteins. Tugarinov V, Kay LE. Chembiochem 6 1567-1577 (2005)
  7. Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins. Tugarinov V, Hwang PM, Kay LE. Annu. Rev. Biochem. 73 107-146 (2004)

Articles citing this publication (51)

  1. Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Tugarinov V, Kanelis V, Kay LE. Nat Protoc 1 749-754 (2006)
  2. Homology among (betaalpha)(8) barrels: implications for the evolution of metabolic pathways. Copley RR, Bork P. J. Mol. Biol. 303 627-641 (2000)
  3. Solution NMR-derived global fold of a monomeric 82-kDa enzyme. Tugarinov V, Choy WY, Orekhov VY, Kay LE. Proc. Natl. Acad. Sci. U.S.A. 102 622-627 (2005)
  4. Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid. Salsbury FR, Knutson ST, Poole LB, Fetrow JS. Protein Sci. 17 299-312 (2008)
  5. Refined solution structure of the 82-kDa enzyme malate synthase G from joint NMR and synchrotron SAXS restraints. Grishaev A, Tugarinov V, Kay LE, Trewhella J, Bax A. J. Biomol. NMR 40 95-106 (2008)
  6. Crystal structure of LeuA from Mycobacterium tuberculosis, a key enzyme in leucine biosynthesis. Koon N, Squire CJ, Baker EN. Proc. Natl. Acad. Sci. U.S.A. 101 8295-8300 (2004)
  7. Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G. Tugarinov V, Kay LE. J. Mol. Biol. 327 1121-1133 (2003)
  8. Dependence of amino acid side chain 13C shifts on dihedral angle: application to conformational analysis. London RE, Wingad BD, Mueller GA. J. Am. Chem. Soc. 130 11097-11105 (2008)
  9. Structure of the Escherichia coli malate synthase G:pyruvate:acetyl-coenzyme A abortive ternary complex at 1.95 A resolution. Anstrom DM, Kallio K, Remington SJ. Protein Sci. 12 1822-1832 (2003)
  10. L-malyl-coenzyme A/beta-methylmalyl-coenzyme A lyase is involved in acetate assimilation of the isocitrate lyase-negative bacterium Rhodobacter capsulatus. Meister M, Saum S, Alber BE, Fuchs G. J. Bacteriol. 187 1415-1425 (2005)
  11. Ter-dependent stress response systems: novel pathways related to metal sensing, production of a nucleoside-like metabolite, and DNA-processing. Anantharaman V, Iyer LM, Aravind L. Mol Biosyst 8 3142-3165 (2012)
  12. The apparent malate synthase activity of Rhodobacter sphaeroides is due to two paralogous enzymes, (3S)-Malyl-coenzyme A (CoA)/{beta}-methylmalyl-CoA lyase and (3S)- Malyl-CoA thioesterase. Erb TJ, Frerichs-Revermann L, Fuchs G, Alber BE. J. Bacteriol. 192 1249-1258 (2010)
  13. Using reaction mechanism to measure enzyme similarity. O'Boyle NM, Holliday GL, Almonacid DE, Mitchell JB. J. Mol. Biol. 368 1484-1499 (2007)
  14. The product complex of M. tuberculosis malate synthase revisited. Anstrom DM, Remington SJ. Protein Sci. 15 2002-2007 (2006)
  15. An optimized isotopic labelling strategy of isoleucine-γ2 methyl groups for solution NMR studies of high molecular weight proteins. Ayala I, Hamelin O, Amero C, Pessey O, Plevin MJ, Gans P, Boisbouvier J. Chem. Commun. (Camb.) 48 1434-1436 (2012)
  16. Assessment of a rigorous transitive profile based search method to detect remotely similar proteins. Sandhya S, Chakrabarti S, Abhinandan KR, Sowdhamini R, Srinivasan N. J. Biomol. Struct. Dyn. 23 283-298 (2005)
  17. Methyl-detected 'out-and-back' NMR experiments for simultaneous assignments of Alabeta and Ilegamma2 methyl groups in large proteins. Sheppard D, Guo C, Tugarinov V. J. Biomol. NMR 43 229-238 (2009)
  18. Crystal structure and functional analysis of homocitrate synthase, an essential enzyme in lysine biosynthesis. Bulfer SL, Scott EM, Couture JF, Pillus L, Trievel RC. J. Biol. Chem. 284 35769-35780 (2009)
  19. The malate synthase of Paracoccidioides brasiliensis Pb01 is required in the glyoxylate cycle and in the allantoin degradation pathway. Zambuzzi-Carvalho PF, Cruz AH, Santos-Silva LK, Goes AM, Soares CM, Pereira M. Med. Mycol. 47 734-744 (2009)
  20. Brucella abortus depends on pyruvate phosphate dikinase and malic enzyme but not on Fbp and GlpX fructose-1,6-bisphosphatases for full virulence in laboratory models. Zúñiga-Ripa A, Barbier T, Conde-Álvarez R, Martínez-Gómez E, Palacios-Chaves L, Gil-Ramírez Y, Grilló MJ, Letesson JJ, Iriarte M, Moriyón I. J. Bacteriol. 196 3045-3057 (2014)
  21. Kinetic analysis of the effects of monovalent cations and divalent metals on the activity of Mycobacterium tuberculosis alpha-isopropylmalate synthase. de Carvalho LP, Blanchard JS. Arch. Biochem. Biophys. 451 141-148 (2006)
  22. Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins. Mas G, Crublet E, Hamelin O, Gans P, Boisbouvier J. J. Biomol. NMR 57 251-262 (2013)
  23. Evolution of the genetic code by incorporation of amino acids that improved or changed protein function. Francis BR. J. Mol. Evol. 77 134-158 (2013)
  24. Molecular characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, in Euglena gracilis. Nakazawa M, Minami T, Teramura K, Kumamoto S, Hanato S, Takenaka S, Ueda M, Inui H, Nakano Y, Miyatake K. Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 141 445-452 (2005)
  25. Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids. Miyanoiri Y, Takeda M, Okuma K, Ono AM, Terauchi T, Kainosho M. J. Biomol. NMR 57 237-249 (2013)
  26. Discovery of an iron-regulated citrate synthase in Staphylococcus aureus. Cheung J, Murphy ME, Heinrichs DE. Chem. Biol. 19 1568-1578 (2012)
  27. Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids. Nucci NV, Marques BS, Bédard S, Dogan J, Gledhill JM, Moorman VR, Peterson RW, Valentine KG, Wand AL, Wand AJ. J. Biomol. NMR 50 421-430 (2011)
  28. Identification of HN-methyl NOEs in large proteins using simultaneous amide-methyl TROSY-based detection. Guo C, Tugarinov V. J. Biomol. NMR 43 21-30 (2009)
  29. Atomic resolution structures of Escherichia coli and Bacillus anthracis malate synthase A: comparison with isoform G and implications for structure-based drug discovery. Lohman JR, Olson AC, Remington SJ. Protein Sci. 17 1935-1945 (2008)
  30. Properties of R-citramalyl-coenzyme A lyase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus. Friedmann S, Alber BE, Fuchs G. J. Bacteriol. 189 2906-2914 (2007)
  31. Differential expression of a malate synthase gene during the preinfection stage of symbiosis in the ectomycorrhizal fungus Laccaria bicolor. Balasubramanian S, Kim SJ, Podila GK. New Phytol. 154 517-527 (2002)
  32. Estimates of methyl 13C and 1H CSA values (Deltasigma) in proteins from cross-correlated spin relaxation. Tugarinov V, Scheurer C, Brüschweiler R, Kay LE. J. Biomol. NMR 30 397-406 (2004)
  33. Selective 1H- 13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins. Guo C, Tugarinov V. J. Biomol. NMR 46 127-133 (2010)
  34. Structure and mechanism of HpcH: a metal ion dependent class II aldolase from the homoprotocatechuate degradation pathway of Escherichia coli. Rea D, Fülöp V, Bugg TD, Roper DI. J. Mol. Biol. 373 866-876 (2007)
  35. Addressing the overlap problem in the quantitative analysis of two dimensional NMR spectra: application to (15)N relaxation measurements. Tugarinov V, Choy WY, Kupce E, Kay LE. J. Biomol. NMR 30 347-352 (2004)
  36. Kinetic and chemical mechanism of malate synthase from Mycobacterium tuberculosis. Quartararo CE, Blanchard JS. Biochemistry 50 6879-6887 (2011)
  37. The glcB locus of Rhizobium leguminosarum VF39 encodes an arabinose-inducible malate synthase. García-de los Santos A, Morales A, Baldomá L, Clark SR, Brom S, Yost CK, Hernández-Lucas I, Aguilar J, Hynes MF. Can. J. Microbiol. 48 922-932 (2002)
  38. Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-proS and valine-proS methyl groups for the detection of long range NOEs. Kerfah R, Plevin MJ, Pessey O, Hamelin O, Gans P, Boisbouvier J. J. Biomol. NMR 61 73-82 (2015)
  39. Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G. Bracken CD, Neighbor AM, Lamlenn KK, Thomas GC, Schubert HL, Whitby FG, Howard BR. BMC Struct. Biol. 11 23 (2011)
  40. Biochemical characterization of malate synthase G of P. aeruginosa. Roucourt B, Minnebo N, Augustijns P, Hertveldt K, Volckaert G, Lavigne R. BMC Biochem. 10 20 (2009)
  41. Biophysical characterization of the enzyme I of the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system. Hurtado-Gómez E, Fernández-Ballester G, Nothaft H, Gómez J, Titgemeyer F, Neira JL. Biophys. J. 90 4592-4604 (2006)
  42. SbnG, a citrate synthase in Staphylococcus aureus: a new fold on an old enzyme. Kobylarz MJ, Grigg JC, Sheldon JR, Heinrichs DE, Murphy ME. J. Biol. Chem. 289 33797-33807 (2014)
  43. The crystal structures of the tri-functional Chloroflexus aurantiacus and bi-functional Rhodobacter sphaeroides malyl-CoA lyases and comparison with CitE-like superfamily enzymes and malate synthases. Zarzycki J, Kerfeld CA. BMC Struct. Biol. 13 28 (2013)
  44. Equilibrium and kinetics of the unfolding and refolding of Escherichia coli Malate Synthase G monitored by circular dichroism and fluorescence spectroscopy. Maheshwari A, Verma VK, Chaudhuri TK. Biochimie 92 491-498 (2010)
  45. Purification and characterization of malate synthase from the glucose-grown wood-rotting basidiomycete Fomitopsis palustris. Munir E, Hattori T, Shimada M. Biosci. Biotechnol. Biochem. 66 576-581 (2002)
  46. Mycobacterium tuberculosis Malate Synthase Structures with Fragments Reveal a Portal for Substrate/Product Exchange. Huang HL, Krieger IV, Parai MK, Gawandi VB, Sacchettini JC. J. Biol. Chem. 291 27421-27432 (2016)
  47. Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain. Miyanoiri Y, Ishida Y, Takeda M, Terauchi T, Inouye M, Kainosho M. J. Biomol. NMR 65 109-119 (2016)
  48. Comparative analysis of malate synthase G from Mycobacterium tuberculosis and E. coli: role of ionic interaction in modulation of structural and functional properties. Kumar R, Bhakuni V. Int. J. Biol. Macromol. 49 917-922 (2011)
  49. Estimating side-chain order in methyl-protonated, perdeuterated proteins via multiple-quantum relaxation violated coherence transfer NMR spectroscopy. Sun H, Godoy-Ruiz R, Tugarinov V. J. Biomol. NMR 52 233-243 (2012)
  50. Simultaneous measurement of ¹H-¹⁵N and methyl ¹Hm-¹³Cm residual dipolar couplings in large proteins. Liao X, Godoy-Ruiz R, Guo C, Tugarinov V. J. Biomol. NMR 51 191-198 (2011)
  51. Dissociation of Mg(ii) and Zn(ii) complexes of simple 2-oxocarboxylates - relationship to CO2 fixation, and the Grignard and Barbier reactions. Miller GBS, Uggerud E. Org. Biomol. Chem. 15 6813-6825 (2017)