Crystal structure of a putative isochorismatase hydrolase from Oleispira antarctica

  • Anna M. Goral
  • Karolina L. Tkaczuk
  • Maksymilian Chruszcz
  • Olga Kagan
  • Alexei Savchenko
  • Wladek Minor


Isochorismatase-like hydrolases (IHL) constitute a large family of enzymes divided into five structural families (by SCOP). IHLs are crucial for siderophore-mediated ferric iron acquisition by cells. Knowledge of the structural characteristics of these molecules will enhance the understanding of the molecular basis of iron transport, and perhaps resolve which of the mechanisms previously proposed in the literature is the correct one. We determined the crystal structure of the apo-form of a putative isochorismatase hydrolase OaIHL (PDB code: 3LQY) from the antarctic γ-proteobacterium Oleispira antarctica, and did comparative sequential and structural analysis of its closest homologs. The characteristic features of all analyzed structures were identified and discussed. We also docked isochorismate to the determined crystal structure by in silico methods, to highlight the interactions of the active center with the substrate. The putative isochorismate hydrolase OaIHL from O. antarctica possesses the typical catalytic triad for IHL proteins. Its active center resembles those IHLs with a D–K–C catalytic triad, rather than those variants with a D–K–X triad. OaIHL shares some structural and sequential features with other members of the IHL superfamily. In silico docking results showed that despite small differences in active site composition, isochorismate binds to in the structure of OaIHL in a similar mode to its binding in phenazine biosynthesis protein PhzD (PDB code 1NF8).


Iron uptake Isochorismatase hydrolases Structural comparison of isochorismatases Structural genomics 



Putative isochorismatase hydrolase from Oleispira antarctica


Isochorismatase-like hydrolase


Isochorismate/isochorismic acid




Structural classification of proteins


Protein Data Bank


Root mean square deviation


Multiple sequence alignment



The authors thank Andrzej Joachimiak and the members of the Structural Biology Center at the Advanced Photon Source and the Midwest Center for Structural Genomics for help and discussions. The authors also thank Matthew Zimmerman for critically reading the manuscript. The work described in the paper was supported by NIH PSI Grant GM074942. The results shown in this report are derived from work performed at Argonne National Laboratory, at the Structural Biology Center of the Advanced Photon Source. Argonne is operated by the University of Chicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under Contract DE-AC02-06CH11357.


  1. 1.
    Kadi N, Challis GL (2009) Chapter 17. Siderophore biosynthesis a substrate specificity assay for nonribosomal peptide synthetase-independent siderophore synthetases involving trapping of acyl-adenylate intermediates with hydroxylamine. Methods Enzymol 458:431–457PubMedCrossRefGoogle Scholar
  2. 2.
    Neilands JB (1991) A brief history of iron metabolism. Biol Met 4(1):1–6PubMedCrossRefGoogle Scholar
  3. 3.
    Raymond KN, Muller G, Matzanke BF (1984) Complexation of iron by siderophores—a review of their solution and structural chemistry and biological function. Topics Curr Chem 123:49–102CrossRefGoogle Scholar
  4. 4.
    O’Brien IG, Gibson F (1970) The structure of enterochelin and related 2,3-dihydroxy-N-benzoylserine conjugates from Escherichia coli. Biochim Biophys Acta 215(2):393–402PubMedCrossRefGoogle Scholar
  5. 5.
    Pollack JR, Neilands JB (1970) Enterobactin, an iron transport compound from Salmonella typhimurium. Biochem Biophys Res Commun 38(5):989–992PubMedCrossRefGoogle Scholar
  6. 6.
    Raymond KN, Dertz EA, Kim SS (2003) Enterobactin: an archetype for microbial iron transport. Proc Natl Acad Sci USA 100(7):3584–3588PubMedCrossRefGoogle Scholar
  7. 7.
    Harris WR, Carrano CJ, Cooper SR, Sofen SR, Avdeef AE, McArdle JV, Raymond KN (1979) Coordination chemistry of microbial iron transport compounds. 19. Stability-constants and electrochemical behavior of ferric enterobactin and model complexes. J Am Chem Soc 101:6097–6104CrossRefGoogle Scholar
  8. 8.
    Ichihara S, Mizushima S (1978) Identification of an outer membrane protein responsible for the binding of the Fe-enterochelin complex to Escherichia coli cells. J Biochem 83(1):137–140PubMedGoogle Scholar
  9. 9.
    Langman L, Rosenber H, Young IG, Frost GE, Gibson F (1972) Enterochelin system of iron transport in Escherichia coli: mutations affecting ferric-enterochelin esterase. J Bacteriol 112:1142–1149PubMedGoogle Scholar
  10. 10.
    Lee CW, Ecker DJ, Raymond KN (1985) The pH-dependent reduction of ferric enterobactin probed by electrochemical methods and its implications for microbial iron transport. J Am Chem Soc 107:6920–6923CrossRefGoogle Scholar
  11. 11.
    Walsh CT, Liu J, Rusnak F, Sakaitani M (1990) Molecular studies on enzymes in chorismate metabolism and the enterobactin biosynthetic pathway. Chem Rev 90:1105–1129CrossRefGoogle Scholar
  12. 12.
    Caruthers J, Zucker F, Worthey E, Myler PJ, Buckner F, Van Voorhuis W, Mehlin C, Boni E, Feist T, Luft J et al (2005) Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily. Protein Sci 14(11):2887–2894PubMedCrossRefGoogle Scholar
  13. 13.
    Yakimov MM, Giuliano L, Gentile G, Crisafi E, Chernikova TN, Abraham WR, Lunsdorf H, Timmis KN, Golyshin PN (2003) Oleispira antarctica gen. nov., sp. nov., a novel hydrocarbonoclastic marine bacterium isolated from Antarctic coastal sea water. Int J Syst Evol Microbiol 53(Pt 3):779–785PubMedCrossRefGoogle Scholar
  14. 14.
    Head IM, Jones DM, Roling WF (2006) Marine microorganisms make a meal of oil. Nat Rev Microbiol 4(3):173–182PubMedCrossRefGoogle Scholar
  15. 15.
    Yakimov MM, Timmis KN, Golyshin PN (2007) Obligate oil-degrading marine bacteria. Curr Opin Biotechnol 18(3):257–266PubMedCrossRefGoogle Scholar
  16. 16.
    Zhang RG, Skarina T, Katz JE, Beasley S, Khachatryan A, Vyas S, Arrowsmith CH, Clarke S, Edwards A, Joachimiak A et al (2001) Structure of Thermotoga maritima stationary phase survival protein SurE: a novel acid phosphatase. Structure 9(11):1095–1106PubMedCrossRefGoogle Scholar
  17. 17.
    Rosenbaum G, Alkire RW, Evans G, Rotella FJ, Lazarski K, Zhang RG, Ginell SL, Duke N, Naday I, Lazarz J et al (2006) The Structural Biology Center 19ID undulator beamline: facility specifications and protein crystallographic results. J Synchrotron Radiat 13(Pt 1):30–45PubMedGoogle Scholar
  18. 18.
    Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276:307-326 Google Scholar
  19. 19.
    Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr A Found Crystallogr 64(Pt 1):112–122CrossRefGoogle Scholar
  20. 20.
    Otwinowski Z (ed) (1991) MLPHARE: Isomorphous replacement and anomalous scattering. In: Proceedings of the CCP4 SERC, Daresbury LaboratoryGoogle Scholar
  21. 21.
    Cowtan K (1994) ‘dm’: an automated procedure for phase improvement by density modification. Jt CCP4 ESF-EACBM Newsl Protein Crystallogr 31:34–38Google Scholar
  22. 22.
    Terwilliger TC (2002) Automated structure solution, density modification and model building. Acta Crystallogr D Biol Crystallogr 58(Pt 11):1937–1940PubMedCrossRefGoogle Scholar
  23. 23.
    Perrakis A, Morris R, Lamzin VS (1999) Automated protein model building combined with iterative structure refinement. Nat Struct Biol 6(5):458–463PubMedCrossRefGoogle Scholar
  24. 24.
    Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Cryst D60:2126–2132Google Scholar
  25. 25.
    Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53(Pt 3):240–255PubMedCrossRefGoogle Scholar
  26. 26.
    Painter J, Merritt EA (2006) TLSMD web server for the generation of multi-group TLS models. J Appl Crystallogr 39(1):109–111CrossRefGoogle Scholar
  27. 27.
    Lovell SC, Davis IW, Adrendall WB, de Bakker PIW, Word JM, Prisant MG, Richardson JS, Richardson DC (2003) Structure validation by C alpha geometry: phi, psi and C beta deviation. Proteins Struct Funct Genet 50:437–450PubMedCrossRefGoogle Scholar
  28. 28.
    Yang H, Guranovic V, Dutta S, Feng Z, Berman HM, Westbrook JD (2004) Automated and accurate deposition of structures solved by X-ray diffraction to the Protein Data Bank. Acta Crystallogr D Biol Crystallogr 60(Pt 10):1833–1839PubMedCrossRefGoogle Scholar
  29. 29.
    Bernstein FC, Koetzle TF, Williams GJ, Meyer EF Jr, Brice MD, Rodgers JR, Kennard O, Shimanouchi T, Tasumi M (1977) The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol 112(3):535–542PubMedCrossRefGoogle Scholar
  30. 30.
    Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402PubMedCrossRefGoogle Scholar
  31. 31.
    Soding J (2005) Protein homology detection by HMM–HMM comparison. Bioinformatics 21(7):951–960PubMedCrossRefGoogle Scholar
  32. 32.
    Soding J, Remmert M, Biegert A, Lupas AN (2006) HHsenser: exhaustive transitive profile search using HMM–HMM comparison. Nucleic Acids Res 34(Web Server issue):W374–W378Google Scholar
  33. 33.
    Holm L, Rosenstrom P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38(Web Server issue):W545–W549Google Scholar
  34. 34.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797PubMedCrossRefGoogle Scholar
  35. 35.
    Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22(12):2577–2637PubMedCrossRefGoogle Scholar
  36. 36.
    Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372(3):774–797PubMedCrossRefGoogle Scholar
  37. 37.
    Glaser F, Pupko T, Paz I, Bell RE, Bechor-Shental D, Martz E, Ben-Tal N (2003) ConSurf: identification of functional regions in proteins by surface-mapping of phylogenetic information. Bioinformatics 19(1):163–164PubMedCrossRefGoogle Scholar
  38. 38.
    Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14(6):1188–1190PubMedCrossRefGoogle Scholar
  39. 39.
    Schneider TD, Stephens RM (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18(20):6097–6100PubMedCrossRefGoogle Scholar
  40. 40.
    Abagyan RA (1994) Totrov M.M., D.A K: Icm: A New Method For Protein Modeling and Design: Applications To Docking and Structure Prediction From The Distorted Native Conformation. J Comput Chem 15:488–506CrossRefGoogle Scholar
  41. 41.
    Murzin AG, Brenner SE, Hubbard T, Chothia C (1995) SCOP: a structural classification of proteins database for the investigation of sequences and structures. J Mol Biol 247(4):536–540PubMedGoogle Scholar
  42. 42.
    Greene LH, Lewis TE, Addou S, Cuff A, Dallman T, Dibley M, Redfern O, Pearl F, Nambudiry R, Reid A et al (2007) The CATH domain structure database: new protocols and classification levels give a more comprehensive resource for exploring evolution. Nucleic Acids Res 35(Database issue):D291–D297Google Scholar
  43. 43.
    Parsons JF, Calabrese K, Eisenstein E, Ladner JE (2003) Structure and mechanism of Pseudomonas aeruginosa PhzD, an isochorismatase from the phenazine biosynthetic pathway. Biochemistry 42(19):5684–5693PubMedCrossRefGoogle Scholar
  44. 44.
    Kunzler DE, Sasso S, Gamper M, Hilvert D, Kast P (2005) Mechanistic insights into the isochorismate pyruvate lyase activity of the catalytically promiscuous PchB from combinatorial mutagenesis and selection. J Biol Chem 280(38):32827–32834PubMedCrossRefGoogle Scholar
  45. 45.
    Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S, Khanna A, Marshall M, Moxon S, Sonnhammer EL et al (2004) The Pfam protein families database. Nucleic Acids Res 32(Database issue):D138–D141Google Scholar
  46. 46.
    Jabs A, Weiss MS, Hilgenfeld R (1999) Non-proline cis peptide bonds in proteins. J Mol Biol 286(1):291–304PubMedCrossRefGoogle Scholar
  47. 47.
    Weiss MS, Jabs A, Hilgenfeld R (1998) Peptide bonds revisited. Nat Struct Biol 5(8):676PubMedCrossRefGoogle Scholar
  48. 48.
    MacArthur MW, Thornton JM (1991) Influence of proline residues on protein conformation. J Mol Biol 218(2):397–412PubMedCrossRefGoogle Scholar
  49. 49.
    Stewart DE, Sarkar A, Wampler JE (1990) Occurrence and role of cis peptide bonds in protein structures. J Mol Biol 214(1):253–260PubMedCrossRefGoogle Scholar
  50. 50.
    Luo HB, Zheng H, Zimmerman MD, Chruszcz M, Skarina T, Egorova O, Savchenko A, Edwards AM, Minor W (2010) Crystal structure and molecular modeling study of N-carbamoylsarcosine amidase Ta0454 from Thermoplasma acidophilum. J Struct Biol 169(3):304–311PubMedCrossRefGoogle Scholar
  51. 51.
    Colovos C, Cascio D, Yeates TO (1998) The 1.8 angstrom crystal structure of the ycaC gene product from Escherichia coli reveals an octameric hydrolase of unknown specificity. Struct Fold Des 6:1329–1337Google Scholar
  52. 52.
    Du XL, Wang WR, Kim R, Yakota H, Nguyen H, Kim SH (2001) Crystal structure and mechanism of catalysis of a pyrazinamidase from Pyrococcus horikoshii. Biochemistry 40:14166–14172 Google Scholar
  53. 53.
    Romao MJ, Turk D, Gomisruth FX, Huber R, Schumacher G, Mollering H, Russmann L (1992) Crystal-structure analysis, refinement and enzymatic-reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2.0-angstrom resolution, J Mol Biol 226:1111–1130 Google Scholar
  54. 54.
    Zajc A, Romao MJ, Turk B, Huber R (1996) Crystallographic and fluorescence studies of ligand binding to N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. J Mol Biol 263:269–283 Google Scholar
  55. 55.
    Drake EJ, Nicolai DA, Gulick AM (2006) Structure of the EntB multidomain nonribosomal peptide synthetase and functional analysis of its interaction with the EntE adenylation domain. Chem Biol 13:409-419 Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Anna M. Goral
    • 1
    • 3
    • 4
    • 5
  • Karolina L. Tkaczuk
    • 1
    • 3
  • Maksymilian Chruszcz
    • 1
    • 3
  • Olga Kagan
    • 2
    • 3
  • Alexei Savchenko
    • 2
    • 3
  • Wladek Minor
    • 1
    • 3
  1. 1.Department of Molecular Physiology and Biological PhysicsUniversity of VirginiaCharlottesvilleUSA
  2. 2.Banting and Best Department of Medical ResearchUniversity of TorontoTorontoCanada
  3. 3.Midwest Center for Structural Genomics
  4. 4.Inter-Faculty Interdisciplinary Doctoral Studies in Natural Sciences and MathematicsUniversity of WarsawWarsawPoland
  5. 5.Faculty of ChemistryUniversity of WarsawWarsawPoland

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