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References

  1. Agnew CR, Warrilow AG, Burton NM, Lamb DC, Kelly SL, Brady RL. 2012. An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae. Antimicrob. Agents Chemother. 56: 391-402.
    Pubmed PMC CrossRef
  2. Baudry J, Rupasinghe S, Schuler MA. 2006. Class dependent sequence alignment strategy improves the structural and functional modeling of P450s. Protein Eng. Des. Selec. 19:345-353.
    Pubmed CrossRef
  3. Bernhardt R. 1996. Cytochrome P450: structure, function, and generation of reactive oxygen species. Rev. Physiol. Biochem. Pharmacol. 127: 137-221.
    Pubmed CrossRef
  4. Bhattarai S, Liou K, Oh TJ. 2012. Homology modeling and docking studies of Streptomyces peucetius CYP147F1 as limonene hydroxylase. J. Microbiol. Biotechnol. 22: 917-922.
    Pubmed CrossRef
  5. Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M. 1983. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comp. Chem. 4: 187-217.
    CrossRef
  6. Discovery Studio 3.5. 2012. Accelrys Inc., San Diego, CA, USA. Available from http://www.accelrys.com.
  7. Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J. 1995. Structure and function of cytochromes P450: a comparative analysis of three crystal structures. Structure 3: 41-62.
    CrossRef
  8. Hatae T, Hara S, Yokoyama C, Yabuki T, Inoue H, Ullrich V, Tanabe T. 1996. Site-directed mutagenesis of human prostacyclin synthase: alteration of Cys441 of the Cys pocket, and Glu347 and Arg350 of the ExxR motif. FEBS Lett. 389:268-272.
    CrossRef
  9. Gotoh O. 1992. Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J. Biol. Chem. 267: 83-90.
    Pubmed
  10. Guengerich FP. 1991. Reactions and significance of cytochrome P-450 enzymes. J. Biol. Chem. 266: 10019-10022.
    Pubmed
  11. Gunsalus IC, Sligar SG. 1978. Advances in Enzymology and Related Areas of Molecular Biology, pp. 1-44. Vol. 47. John Wiley & Sons, NY.
    Pubmed
  12. Lamb DC, Skaug T, Song HL, Jackson CJ, Podust LM, Waterman MR, et al. 2002. The cytochrome P450 complement (CYPome) of Streptomyces coelicolor A3(2). J. Biol. Chem. 277:24000-24005.
    Pubmed CrossRef
  13. Lovell SC, Davis IQ, Arendall III WB, De Bakker PI, Word JM, Prisant MG, et al. 2003. Structure validation by C α geometry: phi, psi and C beta deviation. Proteins 50: 437-450.
    Pubmed CrossRef
  14. Maiti R, Van Domselaar GH, Zhang H, Wishart DS. 2004. SuperPose: a simple server for sophisticated structural superposition. Nucleic Acids Res. 32: W590-W594.
    Pubmed PMC CrossRef
  15. Murray RDH, Mendez J, Brown SA. 1982. The Natural Coumarins, Occurrence, Chemistry and Biochemistry. John Wiley & Sons NY.
  16. Omura T, Sato R. 1964. The carbon monoxide-binding pigment of liver microsomes: 1. Evidence for its hemoprotein nature. J. Biol. Chem. 239: 2370-2378.
    Pubmed
  17. Oriel PJ, Savithiry S, Chang HC. 1997. Process for the preparation of monoterpenes using bacterium containing recombinant DNA. US patent 5,688,673.
  18. Ortiz de Montellano PR. 1995. Cytochrome P450, 2nd Ed. Plenum, NY.
    CrossRef
  19. Ortiz de Montellano PR. 2004. Cytochrome P450 structure, mechanism, and biochemistry, pp. 183-247. In Ortiz de Montellano PR, Voss De JJ (eds.). Substrate Oxidation by Cytochrome P450 Enzymes. Kluwer Academic/PlenumPublishers, NY.
  20. Peterson JA, Lorence MC, Amarneh B. 1990. Putidaredoxin reductase and putidaredoxin: cloning, sequence determination, and heterologous expression of the proteins. J. Biol. Chem. 265: 6066-6073.
    Pubmed
  21. Ravichandran KG, Boddupalli SS, Hasermann CA, Peterson JA, Deisenhofer J. 1993. Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450’s. Science 261: 731-736.
    Pubmed CrossRef
  22. Rupasinghe S, Schuler MA, Kagawa N, Yuan H, Lei L, Zhao B, et al. 2006. The cytochrome P450 gene family CYP157 does not contain EXXR in the K-helix reducing the absolute conserved P450 residues to a single cysteine. FEBS Lett. 580:6338-6342.
    Pubmed CrossRef
  23. Sali A, Pottertone L, Yuan F, Van Vlijmen H, Karplus M. 1995. Evaluation of comparative protein modeling by MODELLER. Prot. Struct. Funct. Genet. 23: 318-326.
    Pubmed CrossRef
  24. Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  25. Sherman DH, Shengying L, Yermalitskaya LV, Kim Y, Smith JA, Waterman M R, P odust LM. 2 006. T he struc tural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae. J. Biol. Chem. 281: 26289-26297.
    Pubmed PMC CrossRef
  26. Shimizu T, Tateishi T, Hatano M, Fujii-Kuriyama Y. 1991. Probing the role of lysines and arginines in the catalytic function of cytochrome P450d by site-directed mutagenesis. Interaction with NADPH-cytochrome P450 reductase. J. Biol. Chem. 266: 3372-3375.
    Pubmed
  27. Shrestha P , Oh TJ, L iou K, S ohng J K. 2 008. C ytoc hrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin. Appl. Microbiol. Biotechnol. 79: 555-562.
    Pubmed CrossRef
  28. Shrestha P, Oh TJ, Niraula NP, Liou K, Yoo JC, Sohng JK. 2010. Characterization of CYP166B1 and its electron transfer system in Streptomyces peucetius var. caesius ATCC27952. Enzyme Microb. Technol. 46: 372-377.
    CrossRef
  29. Sippl MJ. 1993. Recognition of errors in three-dimensional structures of proteins. Proteins 17: 355-362.
    Pubmed CrossRef
  30. The ExPASy (Expert ProteinAnalysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB). Available at http://ca.expasy.org.
  31. Ueno M, Yamashita M, Hashimoto M, Hino M, Fujie A. 2005. Oxidative activities of heterologously expressed CYP107B1 and CYP105D1 in whole-cell biotransformation using Streptomyces lividans TK24. J. Biosci. Bioeng. 100: 567-572.
    Pubmed CrossRef
  32. Uno T, Okamoto S, Masuda S, Itoh A, Uno Y, Nakamura M, et al. 2008. Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli. Biotechnol. Appl. Biochem. 50: 165-171.
    Pubmed CrossRef
  33. Venkatachalam CM, Jiang X, Oldeld T, Waldman M. 2003. LigandFit: a novel method for the shape-directed rapid doc king o f ligands to p rotein a ctive s ites. J. Mol. Graph. Model. 21: 289-307.
    CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2015; 25(9): 1417-1424

Published online September 28, 2015 https://doi.org/10.4014/jmb.1504.04057

Copyright © The Korean Society for Microbiology and Biotechnology.

Homology Modeling and In Vitro Analysis for Characterization of Streptomyces peucetius CYP157C4

Hemraj Rimal 1, Sang-Cheol Yu 1, Jong Hwa Jang 2 and Tae-Jin Oh 1*

1Department of Pharmaceutical Engineering, SunMoon University, Asan 336-708, Republic of Korea, 2Department of Dental Hygiene, Hanseo University, Seosan 356-706, Republic of Korea

Received: April 22, 2015; Accepted: June 17, 2015

Abstract

In this study, we tried to characterize Streptomyces peucetius CYP157C4 with homology
modeling using three cytochrome P450 (CYP) structures (CYP157C1, CYP164A2, and
CYP107L1), having discovered that CYP157C4 lacks the ExxR motif that was considered
invariant in all CYPs. We used Discovery Studio 3.5 to build our model after first assessing the
stereochemical quality and side-chain environment, and a 7-ethoxycoumarin substrate was
docked into the final model. The model-substrate complex allowed us to identify functionally
important residues and validate the active-site architecture. We found a distance of 4.56 Å
between the 7-ethoxycoumarin and the active site of the heme, and cloning and an in vitro
assay of the CYP157C4 showed the dealkylation of the substrate. Since the details regarding
this group of CYP structures are still unknown, the findings of this study may provide
elucidation to assist with future efforts to find a legitimate substrate.

Keywords: Cytochrome P450, 7-ethoxycoumarin, Homology modelling, in vitro assay, Streptomyces peucetius

References

  1. Agnew CR, Warrilow AG, Burton NM, Lamb DC, Kelly SL, Brady RL. 2012. An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae. Antimicrob. Agents Chemother. 56: 391-402.
    Pubmed KoreaMed CrossRef
  2. Baudry J, Rupasinghe S, Schuler MA. 2006. Class dependent sequence alignment strategy improves the structural and functional modeling of P450s. Protein Eng. Des. Selec. 19:345-353.
    Pubmed CrossRef
  3. Bernhardt R. 1996. Cytochrome P450: structure, function, and generation of reactive oxygen species. Rev. Physiol. Biochem. Pharmacol. 127: 137-221.
    Pubmed CrossRef
  4. Bhattarai S, Liou K, Oh TJ. 2012. Homology modeling and docking studies of Streptomyces peucetius CYP147F1 as limonene hydroxylase. J. Microbiol. Biotechnol. 22: 917-922.
    Pubmed CrossRef
  5. Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M. 1983. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comp. Chem. 4: 187-217.
    CrossRef
  6. Discovery Studio 3.5. 2012. Accelrys Inc., San Diego, CA, USA. Available from http://www.accelrys.com.
  7. Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J. 1995. Structure and function of cytochromes P450: a comparative analysis of three crystal structures. Structure 3: 41-62.
    CrossRef
  8. Hatae T, Hara S, Yokoyama C, Yabuki T, Inoue H, Ullrich V, Tanabe T. 1996. Site-directed mutagenesis of human prostacyclin synthase: alteration of Cys441 of the Cys pocket, and Glu347 and Arg350 of the ExxR motif. FEBS Lett. 389:268-272.
    CrossRef
  9. Gotoh O. 1992. Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J. Biol. Chem. 267: 83-90.
    Pubmed
  10. Guengerich FP. 1991. Reactions and significance of cytochrome P-450 enzymes. J. Biol. Chem. 266: 10019-10022.
    Pubmed
  11. Gunsalus IC, Sligar SG. 1978. Advances in Enzymology and Related Areas of Molecular Biology, pp. 1-44. Vol. 47. John Wiley & Sons, NY.
    Pubmed
  12. Lamb DC, Skaug T, Song HL, Jackson CJ, Podust LM, Waterman MR, et al. 2002. The cytochrome P450 complement (CYPome) of Streptomyces coelicolor A3(2). J. Biol. Chem. 277:24000-24005.
    Pubmed CrossRef
  13. Lovell SC, Davis IQ, Arendall III WB, De Bakker PI, Word JM, Prisant MG, et al. 2003. Structure validation by C α geometry: phi, psi and C beta deviation. Proteins 50: 437-450.
    Pubmed CrossRef
  14. Maiti R, Van Domselaar GH, Zhang H, Wishart DS. 2004. SuperPose: a simple server for sophisticated structural superposition. Nucleic Acids Res. 32: W590-W594.
    Pubmed KoreaMed CrossRef
  15. Murray RDH, Mendez J, Brown SA. 1982. The Natural Coumarins, Occurrence, Chemistry and Biochemistry. John Wiley & Sons NY.
  16. Omura T, Sato R. 1964. The carbon monoxide-binding pigment of liver microsomes: 1. Evidence for its hemoprotein nature. J. Biol. Chem. 239: 2370-2378.
    Pubmed
  17. Oriel PJ, Savithiry S, Chang HC. 1997. Process for the preparation of monoterpenes using bacterium containing recombinant DNA. US patent 5,688,673.
  18. Ortiz de Montellano PR. 1995. Cytochrome P450, 2nd Ed. Plenum, NY.
    CrossRef
  19. Ortiz de Montellano PR. 2004. Cytochrome P450 structure, mechanism, and biochemistry, pp. 183-247. In Ortiz de Montellano PR, Voss De JJ (eds.). Substrate Oxidation by Cytochrome P450 Enzymes. Kluwer Academic/PlenumPublishers, NY.
  20. Peterson JA, Lorence MC, Amarneh B. 1990. Putidaredoxin reductase and putidaredoxin: cloning, sequence determination, and heterologous expression of the proteins. J. Biol. Chem. 265: 6066-6073.
    Pubmed
  21. Ravichandran KG, Boddupalli SS, Hasermann CA, Peterson JA, Deisenhofer J. 1993. Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450’s. Science 261: 731-736.
    Pubmed CrossRef
  22. Rupasinghe S, Schuler MA, Kagawa N, Yuan H, Lei L, Zhao B, et al. 2006. The cytochrome P450 gene family CYP157 does not contain EXXR in the K-helix reducing the absolute conserved P450 residues to a single cysteine. FEBS Lett. 580:6338-6342.
    Pubmed CrossRef
  23. Sali A, Pottertone L, Yuan F, Van Vlijmen H, Karplus M. 1995. Evaluation of comparative protein modeling by MODELLER. Prot. Struct. Funct. Genet. 23: 318-326.
    Pubmed CrossRef
  24. Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  25. Sherman DH, Shengying L, Yermalitskaya LV, Kim Y, Smith JA, Waterman M R, P odust LM. 2 006. T he struc tural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae. J. Biol. Chem. 281: 26289-26297.
    Pubmed KoreaMed CrossRef
  26. Shimizu T, Tateishi T, Hatano M, Fujii-Kuriyama Y. 1991. Probing the role of lysines and arginines in the catalytic function of cytochrome P450d by site-directed mutagenesis. Interaction with NADPH-cytochrome P450 reductase. J. Biol. Chem. 266: 3372-3375.
    Pubmed
  27. Shrestha P , Oh TJ, L iou K, S ohng J K. 2 008. C ytoc hrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin. Appl. Microbiol. Biotechnol. 79: 555-562.
    Pubmed CrossRef
  28. Shrestha P, Oh TJ, Niraula NP, Liou K, Yoo JC, Sohng JK. 2010. Characterization of CYP166B1 and its electron transfer system in Streptomyces peucetius var. caesius ATCC27952. Enzyme Microb. Technol. 46: 372-377.
    CrossRef
  29. Sippl MJ. 1993. Recognition of errors in three-dimensional structures of proteins. Proteins 17: 355-362.
    Pubmed CrossRef
  30. The ExPASy (Expert ProteinAnalysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB). Available at http://ca.expasy.org.
  31. Ueno M, Yamashita M, Hashimoto M, Hino M, Fujie A. 2005. Oxidative activities of heterologously expressed CYP107B1 and CYP105D1 in whole-cell biotransformation using Streptomyces lividans TK24. J. Biosci. Bioeng. 100: 567-572.
    Pubmed CrossRef
  32. Uno T, Okamoto S, Masuda S, Itoh A, Uno Y, Nakamura M, et al. 2008. Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli. Biotechnol. Appl. Biochem. 50: 165-171.
    Pubmed CrossRef
  33. Venkatachalam CM, Jiang X, Oldeld T, Waldman M. 2003. LigandFit: a novel method for the shape-directed rapid doc king o f ligands to p rotein a ctive s ites. J. Mol. Graph. Model. 21: 289-307.
    CrossRef