Characterization of Diverse Natural Variants of CYP102A1 Found Within a Species of Bacillus Megaterium

Overview

Journal AMB Express

Date 2011 Sep 13

PMID 21906327

Citations 5

Authors

Ji-Yeon Kang

So-Young Kim

Dooil Kim

Dong-Hyun Kim

Sun-Mi Shin

Sun-Ha Park

Keon-Hee Kim

Heung-Chae Jung

Jae-Gu Pan

Young Hee Joung

Youn-Tae Chi

Ho Zoon Chae

Taeho Ahn

Chul-Ho Yun

Affiliations

Soon will be listed here.

Abstract

An extreme diversity of substrates and catalytic reactions of cytochrome P450 (P450) enzymes is considered to be the consequence of evolutionary adaptation driven by different metabolic or environmental demands. Here we report the presence of numerous natural variants of P450 BM3 (CYP102A1) within a species of Bacillus megaterium. Extensive amino acid substitutions (up to 5% of the total 1049 amino acid residues) were identified from the variants. Phylogenetic analyses suggest that this P450 gene evolve more rapidly than the rRNA gene locus. It was found that key catalytic residues in the substrate channel and active site are retained. Although there were no apparent variations in hydroxylation activity towards myristic acid (C14) and palmitic acid (C16), the hydroxylation rates of lauric acid (C12) by the variants varied in the range of >25-fold. Interestingly, catalytic activities of the variants are promiscuous towards non-natural substrates including human P450 substrates. It can be suggested that CYP102A1 variants can acquire new catalytic activities through site-specific mutations distal to the active site.

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References

van Vugt-Lussenburg B, Stjernschantz E, Lastdrager J, Oostenbrink C, Vermeulen N, Commandeur J . Identification of critical residues in novel drug metabolizing mutants of cytochrome P450 BM3 using random mutagenesis. J Med Chem. 2007; 50(3):455-61. DOI: 10.1021/jm0609061. View

Kim D, Kim K, Kim D, Liu K, Jung H, Pan J . Generation of human metabolites of 7-ethoxycoumarin by bacterial cytochrome P450 BM3. Drug Metab Dispos. 2008; 36(11):2166-70. DOI: 10.1124/dmd.108.021220. View

Eiben S, Bartelmas H, Urlacher V . Construction of a thermostable cytochrome P450 chimera derived from self-sufficient mesophilic parents. Appl Microbiol Biotechnol. 2007; 75(5):1055-61. DOI: 10.1007/s00253-007-0922-z. View

Guengerich F . Cytochromes P450, drugs, and diseases. Mol Interv. 2004; 3(4):194-204. DOI: 10.1124/mi.3.4.194. View

Khersonsky O, Roodveldt C, Tawfik D . Enzyme promiscuity: evolutionary and mechanistic aspects. Curr Opin Chem Biol. 2006; 10(5):498-508. DOI: 10.1016/j.cbpa.2006.08.011. View

Whitehouse C, Bell S, Yang W, Yorke J, Blanford C, Strong A . A highly active single-mutation variant of P450BM3 (CYP102A1). Chembiochem. 2009; 10(10):1654-6. DOI: 10.1002/cbic.200900279. View

Williams J, Hyland R, Jones B, Smith D, Hurst S, Goosen T . Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab Dispos. 2004; 32(11):1201-8. DOI: 10.1124/dmd.104.000794. View

Stjernschantz E, van Vugt-Lussenburg B, Bonifacio A, de Beer S, van der Zwan G, Gooijer C . Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3. Proteins. 2007; 71(1):336-52. DOI: 10.1002/prot.21697. View

Yun C, Yim S, Kim D, Ahn T . Functional expression of human cytochrome P450 enzymes in Escherichia coli. Curr Drug Metab. 2006; 7(4):411-29. DOI: 10.2174/138920006776873472. View

10.

Kim D, Ahn T, Jung H, Pan J, Yun C . Generation of the human metabolite piceatannol from the anticancer-preventive agent resveratrol by bacterial cytochrome P450 BM3. Drug Metab Dispos. 2009; 37(5):932-6. DOI: 10.1124/dmd.108.026484. View

11.

Li H, Poulos T . The structure of the cytochrome p450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid. Nat Struct Biol. 1997; 4(2):140-6. DOI: 10.1038/nsb0297-140. View

12.

Omura T, Sato R . THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. J Biol Chem. 1964; 239:2379-85. View

13.

Johnson M, Zuo H, Lee K, Trebley J, Rae J, Weatherman R . Pharmacological characterization of 4-hydroxy-N-desmethyl tamoxifen, a novel active metabolite of tamoxifen. Breast Cancer Res Treat. 2004; 85(2):151-9. DOI: 10.1023/B:BREA.0000025406.31193.e8. View

14.

Peters M, Meinhold P, Glieder A, Arnold F . Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3. J Am Chem Soc. 2003; 125(44):13442-50. DOI: 10.1021/ja0303790. View

15.

Kim D, Kim K, Isin E, Guengerich F, Chae H, Ahn T . Heterologous expression and characterization of wild-type human cytochrome P450 1A2 without conventional N-terminal modification in Escherichia coli. Protein Expr Purif. 2007; 57(2):188-200. DOI: 10.1016/j.pep.2007.10.010. View

16.

Leclercq L, Cuyckens F, Mannens G, de Vries R, Timmerman P, Evans D . Which human metabolites have we MIST? Retrospective analysis, practical aspects, and perspectives for metabolite identification and quantification in pharmaceutical development. Chem Res Toxicol. 2009; 22(2):280-93. DOI: 10.1021/tx800432c. View

17.

Lewis D, Watson E, Lake B . Evolution of the cytochrome P450 superfamily: sequence alignments and pharmacogenetics. Mutat Res. 1998; 410(3):245-70. DOI: 10.1016/s1383-5742(97)00040-9. View

18.

Boddupalli S, Estabrook R, Peterson J . Fatty acid monooxygenation by cytochrome P-450BM-3. J Biol Chem. 1990; 265(8):4233-9. View

19.

Gustafsson M, Roitel O, Marshall K, Noble M, Chapman S, Pessegueiro A . Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium. Biochemistry. 2004; 43(18):5474-87. DOI: 10.1021/bi035904m. View

20.

Kitazume T, Haines D, Estabrook R, Chen B, Peterson J . Obligatory intermolecular electron-transfer from FAD to FMN in dimeric P450BM-3. Biochemistry. 2007; 46(42):11892-901. DOI: 10.1021/bi701031r. View