Browse > Article
http://dx.doi.org/10.4014/jmb.1504.04057

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

Rimal, Hemraj (Department of Pharmaceutical Engineering, SunMoon University)
Yu, Sang-Cheol (Department of Pharmaceutical Engineering, SunMoon University)
Jang, Jong Hwa (Department of Dental Hygiene, Hanseo University)
Oh, Tae-Jin (Department of Pharmaceutical Engineering, SunMoon University)
Publication Information
Journal of Microbiology and Biotechnology / v.25, no.9, 2015 , pp. 1417-1424 More about this Journal
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 modeling; in vitro assay; Streptomyces peucetius;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
연도 인용수 순위
1 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.
2 Shrestha P, Oh TJ, Liou K, Sohng JK. 2008. Cytochrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin. Appl. Microbiol. Biotechnol. 79: 555-562.   DOI
3 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.   DOI
4 Sippl MJ. 1993. Recognition of errors in three-dimensional structures of proteins. Proteins 17: 355-362.   DOI
5 The ExPASy (Expert ProteinAnalysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB). Available at http://ca.expasy.org.
6 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.   DOI
7 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.   DOI
8 Venkatachalam CM, Jiang X, Oldeld T, Waldman M. 2003. LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites. J. Mol. Graph. Model. 21: 289-307.   DOI
9 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.
10 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.   DOI
11 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.   DOI
12 Ortiz de Montellano PR. 1995. Cytochrome P450, 2nd Ed. Plenum, NY.
13 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.   DOI
14 Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
15 Sherman DH, Shengying L, Yermalitskaya LV, Kim Y, Smith JA, Waterman MR, Podust LM. 2006. The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae. J. Biol. Chem. 281: 26289-26297.   DOI
16 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.
17 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.   DOI
18 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.
19 Guengerich FP. 1991. Reactions and significance of cytochrome P-450 enzymes. J. Biol. Chem. 266: 10019-10022.
20 Gunsalus IC, Sligar SG. 1978. Advances in Enzymology and Related Areas of Molecular Biology, pp. 1-44. Vol. 47. John Wiley & Sons, NY.
21 Murray RDH, Mendez J, Brown SA. 1982. The Natural Coumarins, Occurrence, Chemistry and Biochemistry. John Wiley & Sons NY.
22 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.   DOI
23 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.   DOI
24 Maiti R, Van Domselaar GH, Zhang H, Wishart DS. 2004. SuperPose: a simple server for sophisticated structural superposition. Nucleic Acids Res. 32: W590-W594.   DOI
25 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.
26 Oriel PJ, Savithiry S, Chang HC. 1997. Process for the preparation of monoterpenes using bacterium containing recombinant DNA. US patent 5,688,673.
27 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.   DOI
28 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.   DOI
29 Bernhardt R. 1996. Cytochrome P450: structure, function, and generation of reactive oxygen species. Rev. Physiol. Biochem. Pharmacol. 127: 137-221.
30 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.   DOI
31 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.   DOI
32 Discovery Studio 3.5. 2012. Accelrys Inc., San Diego, CA, USA. Available from http://www.accelrys.com.
33 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.   DOI