Skip to main content
SpringerLink
Log in

Crystal structure of CmlI, the arylamine oxygenase from the chloramphenicol biosynthetic pathway

  • Original Paper
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

The diiron cluster-containing oxygenase CmlI catalyzes the conversion of the aromatic amine precursor of chloramphenicol to the nitroaromatic moiety of the active antibiotic. The X-ray crystal structures of the fully active, N-terminally truncated CmlIΔ33 in the chemically reduced Fe2+/Fe2+ state and a cis μ-1,2(η 1:η 1)-peroxo complex are presented. These structures allow comparison with the homologous arylamine oxygenase AurF as well as other types of diiron cluster-containing oxygenases. The structural model of CmlIΔ33 crystallized at pH 6.8 lacks the oxo-bridge apparent from the enzyme optical spectrum in solution at higher pH. In its place, residue E236 forms a μ-1,3(η 1:η 2) bridge between the irons in both models. This orientation of E236 stabilizes a helical region near the cluster which closes the active site to substrate binding in contrast to the open site found for AurF. A very similar closed structure was observed for the inactive dimanganese form of AurF. The observation of this same structure in different arylamine oxygenases may indicate that there are two structural states that are involved in regulation of the catalytic cycle. Both the structural studies and single crystal optical spectra indicate that the observed cis μ-1,2(η 1:η 1)-peroxo complex differs from the μ-η 1:η 2-peroxo proposed from spectroscopic studies of a reactive intermediate formed in solution by addition of O2 to diferrous CmlI. It is proposed that the structural changes required to open the active site also drive conversion of the µ-1,2-peroxo species to the reactive form.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Scheme 2

Similar content being viewed by others

Abbreviations

CmlI:

Full length wild-type CmlI enzyme

CmlIΔ33:

33 amino acid N-terminally truncated variant of CmlI used for crystallization

CAM:

Chloramphenicol

NH2-CAM:

d-threo-1-(4-aminophenyl)-2-dichloroacetylamino-1,3-propanediol, the arylamine precursor of CAM and natural CmlI substrate

Fe-AurF:

The functional diiron variant of the aureothin arylamine oxygenase, AurF

Mn-AurF:

The catalytically inactive dimanganese variant of AurF

P :

Diferric peroxo intermediate of CmlI or CmlIΔ33

RNR-R2:

The R2 subunit of ribonucleotide reductase which houses the diiron cluster

MMOH:

Hydroxylase component of the soluble form of methane monooxygenase, MMO

T4moH:

Toluene 4-monooxygenase hydroxylase

References

  1. Winkler R, Hertweck C (2007) ChemBioChem 8:973–977

    Article  CAS  PubMed  Google Scholar 

  2. Ju K-S, Parales RE (2010) Microbiol Mol Biol Rev 74:250–272

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kirner S, Hammer PE, Hill DS, Altmann A, Fischer I, Weislo LJ, Lanahan M, van Pée K-H, Ligon JM (1998) J Bacteriol 180:1939–1943

    CAS  PubMed  PubMed Central  Google Scholar 

  4. He J, Magarvey N, Piraee M, Vining LC (2001) Microbiology 147:2817–2829

    Article  CAS  PubMed  Google Scholar 

  5. He J, Hertweck C (2003) Chem Biol 10:1225–1232

    Article  CAS  PubMed  Google Scholar 

  6. Lee J, Simurdiak M, Zhao H (2005) J Biol Chem 280:36719–36727

    Article  CAS  PubMed  Google Scholar 

  7. Simurdiak M, Lee J, Zhao H (2006) ChemBioChem 7:1169–1172

    Article  CAS  PubMed  Google Scholar 

  8. Lu HG, Chanco E, Zhao HM (2012) Tetrahedron 68:7651–7654

    Article  CAS  Google Scholar 

  9. Doull J, Ahmed Z, Stuttard C, Vining LC (1985) J Gen Microbiol 131:97–104

    CAS  PubMed  Google Scholar 

  10. Makris TM, Chakrabarti M, Münck E, Lipscomb JD (2010) Proc Natl Acad Sci USA 107:15391–15396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fischbach MA, Walsh CT (2006) Chem Rev 106:3468–3496

    Article  CAS  PubMed  Google Scholar 

  12. Walsh CT, Chen H, Keating TA, Hubbard BK, Losey HC, Luo L, Marshall CG, Miller DA, Patel HM (2001) Curr Opin Chem Biol 5:525–534

    Article  CAS  PubMed  Google Scholar 

  13. Pacholec M, Sello JK, Walsh CT, Thomas MG (2007) Org Biomol Chem 5:1692–1694

    Article  CAS  PubMed  Google Scholar 

  14. Piraee M, White RL, Vining LC (2004) Microbiology 150:85–94

    Article  CAS  PubMed  Google Scholar 

  15. Makris TM, Vu VV, Meier KK, Komor AJ, Rivard BS, Münck E, Que L Jr, Lipscomb JD (2015) J Am Chem Soc 137:1608–1617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wallar BJ, Lipscomb JD (1996) Chem Rev 96:2625–2657

    Article  CAS  PubMed  Google Scholar 

  17. Fox BG, Froland WA, Dege JE, Lipscomb JD (1989) J Biol Chem 264:10023–10033

    CAS  PubMed  Google Scholar 

  18. Rosenzweig AC, Frederick CA, Lippard SJ, Nordlund P (1993) Nature 366:537–543

    Article  CAS  PubMed  Google Scholar 

  19. Nordlund P, Sjöberg BM, Eklund H (1990) Nature 345:593–598

    Article  CAS  PubMed  Google Scholar 

  20. Fox BG, Shanklin J, Somerville C, Münck E (1993) Proc Natl Acad Sci USA 90:2486–2490

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lindqvist Y, Huang WJ, Schneider G, Shanklin J (1996) EMBO 15:4081–4092

    CAS  Google Scholar 

  22. Guy JE, Whittle E, Kumaran D, Lindqvist Y, Shanklin J (2007) J Biol Chem 282:19863–19871

    Article  CAS  PubMed  Google Scholar 

  23. Schirmer A, Rude MA, Li X, Popova E, del Cardayre SB (2010) Science 329:559–562

    Article  CAS  PubMed  Google Scholar 

  24. Li N, Chang W-C, Warui DM, Booker SJ, Krebs C, Bollinger JM Jr (2012) Biochemistry 51:7908–7916

    Article  CAS  PubMed  Google Scholar 

  25. Pikus JD, Studts JM, Achim C, Kauffmann KE, Münck E, Steffan RJ, McClay K, Fox BG (1996) Biochemistry 35:9106–9119

    Article  CAS  PubMed  Google Scholar 

  26. Sazinsky MH, Bard J, Di Donato A, Lippard SJ (2004) J Biol Chem 279:30600–30610

    Article  CAS  PubMed  Google Scholar 

  27. Andrews SC (2010) Biochim Biophys Acta 1800:691–705

    Article  CAS  PubMed  Google Scholar 

  28. Skulan AJ, Brunold TC, Baldwin J, Saleh L, Bollinger JM Jr, Solomon EI (2004) J Am Chem Soc 126:8842–8855

    Article  CAS  PubMed  Google Scholar 

  29. Srnec M, Rokob TA, Schwartz JK, Kwak Y, Rulisek L, Solomon EI (2012) Inorg Chem 51:2806–2820

    Article  CAS  PubMed  Google Scholar 

  30. Liu KE, Valentine AM, Qiu D, Edmondson DE, Appelman EH, Spiro TG, Lippard SJ (1995) J Am Chem Soc 117:4997–4998

    Article  CAS  Google Scholar 

  31. Broadwater JA, Ai J, Loehr TM, Sanders-Loehr J, Fox BG (1998) Biochemistry 37:14664–14671

    Article  CAS  PubMed  Google Scholar 

  32. Yun D, Garcia-Serres R, Chicalese BM, An YH, Huynh BH, Bollinger JM Jr (2007) Biochemistry 46:1925–1932

    Article  CAS  PubMed  Google Scholar 

  33. Vu VV, Emerson JP, Martinho M, Kim YS, Münck E, Park MH, Que L Jr (2009) Proc Natl Acad Sci USA 106:14814–14819

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Korboukh VK, Li N, Barr EW, Bollinger JM Jr, Krebs C (2009) J Am Chem Soc 131:13608–13609

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Li N, Korboukh VK, Krebs C, Bollinger JM Jr (2010) Proc Natl Acad Sci USA 107:15722–15727

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Zocher G, Winkler R, Hertweck C, Schulz GE (2007) J Mol Biol 373:65–74

    Article  CAS  PubMed  Google Scholar 

  37. Choi YS, Zhang H, Brunzelle JS, Nair SK, Zhao H (2008) Proc Natl Acad Sci USA 105:6858–6863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Winkler R, Zocher G, Richter I, Friedrich T, Schulz GE, Hertweck C (2007) Angew Chem Int Ed 46:8605–8608

    Article  CAS  Google Scholar 

  39. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) J Appl Crystallogr 40:658–674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Emsley P, Cowtan K (2004) Acta Crystallogr E60:2126–2132

    Google Scholar 

  41. Murshudov GN, Skubak P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Longa F, Vagin AA (2011) Acta Cryst D67:355–367

    Google Scholar 

  42. Holmes MA, Le Trong I, Turley S, Sieker LC, Stenkamp RE (1991) J Mol Biol 218:583–593

    Article  CAS  PubMed  Google Scholar 

  43. Makris TM, Knoot CJ, Wilmot CM, Lipscomb JD (2013) Biochemistry 52:6662–6671

    Article  CAS  PubMed  Google Scholar 

  44. Ookubo T, Sugimoto H, Nagayama T, Masuda H, Sato T, Tanaka K, Maeda Y, Okawa H, Hayashi Y, Uehara A, Suzuki M (1996) J Am Chem Soc 118:701–702

    Article  CAS  Google Scholar 

  45. Kim K, Lippard SJ (1996) J Am Chem Soc 118:4914–4915

    Article  CAS  Google Scholar 

  46. Zhang X, Furutachi H, Fujinami S, Nagatomo S, Maeda Y, Watanabe Y, Kitagawa T, Suzuki M (2005) J Am Chem Soc 127:826–827

    Article  CAS  PubMed  Google Scholar 

  47. Bailey LJ, Fox BG (2009) Biochemistry 48:8932–8939

    Article  CAS  PubMed  Google Scholar 

  48. Han Z, Sakai N, Boettger LH, Klinke S, Hauber J, Trautwein AX, Hilgenfeld R (2015) Structure 23:882–892

    Article  CAS  PubMed  Google Scholar 

  49. Fiedler AT, Shan X, Mehn MP, Kaizer J, Torelli S, Frisch JR, Kodera M, Que L Jr (2008) J Phys Chem A 112:13037–13044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ray WJ, Puvathingal JM (1985) Anal Biochem 146:307–312

    Article  CAS  PubMed  Google Scholar 

  51. Xiong J, Kurtz DM Jr, Ai J, Sanders-Loehr J (2000) Biochemistry 39:5117–5125

    Article  CAS  PubMed  Google Scholar 

  52. Wei P-P, Skulan AJ, Mitić N, Yang Y-S, Saleh L, Bollinger JM Jr, Solomon EI (2004) J Am Chem Soc 126:3777–3788

    Article  CAS  PubMed  Google Scholar 

  53. Banerjee R, Meier KK, Münck E, Lipscomb JD (2013) Biochemistry 52:4331–4342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Rosenzweig AC, Nordlund P, Takahara PM, Frederick CA, Lippard SJ (1995) Chem Biol 2:409–418

    Article  CAS  Google Scholar 

  55. Voegtli WC, Khidekel N, Baldwin J, Ley BA, Bollinger JM Jr, Rosenzweig AC (2000) J Am Chem Soc 122:3255–3261

    Article  CAS  Google Scholar 

  56. Tinberg CE, Lippard SJ (2011) Acc Chem Res 44:280–288

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Kovaleva EG, Lipscomb JD (2007) Science 316:453–457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Kovaleva EG, Lipscomb JD (2008) Biochemistry 47:11168–11170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Knoot CJ, Purpero VM, Lipscomb JD (2015) Proc Natl Acad Sci USA 112:388–393

    Article  CAS  PubMed  Google Scholar 

  60. Wallar BJ, Lipscomb JD (2001) Biochemistry 40:2220–2233

    Article  CAS  PubMed  Google Scholar 

  61. Mitić N, Schwartz JK, Brazeau BJ, Lipscomb JD, Solomon EI (2008) Biochemistry 47:8386–8397

    Article  PubMed  PubMed Central  Google Scholar 

  62. Yang Y-S, Broadwater JA, Pulver SC, Fox BG, Solomon EI (1999) J Am Chem Soc 121:2770–2783

    Article  CAS  Google Scholar 

  63. Schwartz JK, Wei P-P, Mitchell KH, Fox BG, Solomon EI (2008) J Am Chem Soc 130:7098–7109

    Article  CAS  PubMed  Google Scholar 

  64. Liu Y, Nesheim JC, Lee S-K, Lipscomb JD (1995) J Biol Chem 270:24662–24665

    Article  CAS  PubMed  Google Scholar 

  65. Logan DT, Su XD, Aberg A, Regnstrom K, Hajdu J, Eklund H, Nordlund P (1996) Structure 4:1053–1064

    Article  CAS  PubMed  Google Scholar 

  66. Elango N, Radhakrishnan R, Froland WA, Wallar BJ, Earhart CA, Lipscomb JD, Ohlendorf DH (1997) Protein Sci 6:556–568

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Banerjee R, Proshlyakov Y, Lipscomb JD, Proshlyakov DA (2015) Nature 518:431–434

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Lee SK, Lipscomb JD (1999) Biochemistry 38:4423–4432

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Institutes of Health Grants GM 100943 and GM 118030 (to J.D.L.) and NIH graduate traineeship GM 08700 (to C. J. K.). We would like to thank Carrie Wilmot and her research group for many helpful discussions. We also thank Ed Hoeffner at the University of Minnesota Kahlert Structural Biology Laboratory for suggestions in indexing the CmlIΔ33 data and collecting the data sets. We also thank Klaus Lovendahl for generating the expression construct for the truncated enzyme and Anna Komor and Brent Rivard for assistance with biochemical characterization. Diffraction data were collected at Argonne National Laboratory, Structural Biology Center Beamline 19-ID at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. We are thankful for computational resources from the Supercomputing Institute and the facilities at the Kahlert Structural Biology Laboratory at the University of Minnesota.

Author information

Authors and Affiliations

  1. Department of Biochemistry Molecular Biology and Biophysics and the Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN, 55455, USA

    Cory J. Knoot & John D. Lipscomb

  2. Stanford Synchrotron Radiation Lightsource, 2575 Sand Hill Road, Menlo Park, CA, 94025, USA

    Elena G. Kovaleva

Authors
  1. Cory J. Knoot
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elena G. Kovaleva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John D. Lipscomb
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John D. Lipscomb.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 946 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Knoot, C.J., Kovaleva, E.G. & Lipscomb, J.D. Crystal structure of CmlI, the arylamine oxygenase from the chloramphenicol biosynthetic pathway. J Biol Inorg Chem 21, 589–603 (2016). https://doi.org/10.1007/s00775-016-1363-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-016-1363-x

Keywords

Search

Navigation