Characterization of diverse natural variants of CYP102A1 found within a species of Bacillus megaterium

Ji-Yeon Kang 0 So-Young Kim 0 Dooil Kim 0 Dong-Hyun Kim 0 Sun-Mi Shin 0 Sun-Ha Park 0 Keon-Hee Kim 0 Heung-Chae Jung Jae-Gu Pan Young Hee Joung 0 Youn-Tae Chi 0 Ho Zoon Chae 0 Taeho Ahn Chul-Ho Yun 0 0 School of Biological Sciences and Technology, Chonnam National University , Gwangju 500-757, Republic of Korea 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. - Introduction Cytochrome P450s (EC 1.14.14.1; P450 or CYP) are remarkably diverse oxygenation catalysts that are found throughout all classes of life. Although over 11,200 genes of P450s have been found in archaea, bacteria, fungi, plants, and animals (the Cytochrome P450 homepage, http://drnelson.uthsc.edu/P450.statsfile.html), their evolution is not clear. An extreme diversity of substrates and catalytic reactions is characteristic of P450s (Guengerich 2001) and is considered to be the consequence of evolutionary adaptation driven by different metabolic or environmental demands in different organisms. Although most bacterial P450s do not seem to be essential to basic metabolism, they have important roles in the production of secondary metabolites and in detoxication (Kelly et al. 2005). P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase as it is fused to its redox partner, an eukaryotic-like diflavin reductase. Interestingly, sequence analysis for the P450 phylogenetic tree suggested that the CYP102A1 clusters with the eukaryotic P450s but not with other prokaryotic P450s (Lewis et al. 1998). The natural substrates of CYP102A1 are long chain fatty acids (C12 to C20), which are exclusively hydroxylated at the subterminal positions (-1 to -3) (Boddupalli et al. 1990). Furthermore, this enzyme exhibits the highest catalytic activity ever detected among P450 monooxygenase (Boddupalli et al. 1990). Engineered CYP102A1 mutants derived by directed evolution and rational design could oxidize several nonnatural substrates, including pharmaceuticals, shortchain hydrocarbons, and environmental chemicals (Yun et al. 2007; Stjernschantz et al. 2008; Seifert et al. 2009). The potential of engineered CYP102A1 for biotechnological applications has been recognized (Bernhardt 2006). Recently, it was reported that CYP102A1 can be developed as a potentially versatile biocatalyst for the generation of human P450 drug metabolites (Yun et al. 2007; Kim et al. 2009, 2010; Park et al. 2010; Sawayama et al. 2009; Whitehouse et al. 2009; Kim et al. 2011). Human P450 enzymes are responsible for the metabolism of about 75% of drugs used clinically (Williams et al. 2004; Guengerich 2003). Human drug metabolites are very useful in evaluating a drugs efficacy, toxicity, and pharmacokinetics (Johnson et al. 2004; Atrakchi 2009; Leclercq et al. 2009). They can also be used as starting materials for drug candidates. By using a systematic screening strategy, we found a number of natural variants of CYP102A1. Although there were no apparent variations in hydroxylation activity towards myristic acid (C14) and palmitic acid (C16), the oxidation rates of lauric acid (C12) by the variants varied in the range of >25-fold. Some of the natural variants showed catalytic promiscuity towards non-natural substrates, particularly human P450 drug substrates. This study shows that diverse mutations are present in the gene of CYP102A1. Several specific residues for frequent mutations were found and the mutational frequency of reductase domains was much higher than that of heme domains. Materials and methods Materials Isopropyl-b-D-thiogalactopyranoside (IPTG), glucose-6phosphate, glucose-6-phosphate dehydrogenase, aminolevulinic acid (-ALA), reduced b-nicotinamide adenine dinucleotide phosphate (NADPH), fatty acids, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), ferricyanide, phenacetin, acetaminophen, chlorzoxazone, coumarin, 7-ethoxycoumarin, and cytochrome c were obtained from Sigma-Aldrich (St. Louis, MO). Bacterial strains Strains of B. megaterium used in this study were obtained from Korean Culture Center of Microorganisms (KCCM), Korean Collection for Type Cultures (KCTC), American Type Microbiology (ATCC), and the Institute of Fermentation, Osaka (IFO) (Table 1). PCR and cloning of CYP102A1 natural variants For DNA preparations, cells were grown in nutrient broth. After overnight growth at 37C, the cells were centrifuged, washed, lysed, and enzymatically treated to remove RNA and protein. The DNA preparation was then treated with phenol-chloroform (50:50) and ethanol-precipitated. The purity was evaluated by measuring UV absorbance. The variant genes from B. megaterium were amplified by polymerase chain reaction (PCR) using oligonucleotide primers and B. megaterium chromosomal DNA template. First, PCR was carried out in a 50 l reaction mixture containing template plasmid, forward primer BamHI-F (5- AGCGGATCCATGACAATTAAAGAAATGCCTC-3) and reverse primer SacI-R (5-ATCGAGCTCGTAGTTTGTAT-3), dNTPs, and pfu polymerase. The PCR was carried out for 30 cycles consisting of 45 s of denaturation at 94C, 45 s of annealing at 52C, and 90 s of extension at 72C. Next, PCR was Table 1 Bacillus megaterium strains used in this study, and GenBank accession numbers for CYP102A1 variants, 16S rRNA, and ITS sequences between 16S-23S sequencesa aGenBank accession numbers (except J04832) were assigned to nucleotide sequences determined in this study. The corresponding CYP102A1 variant gene for each strain is listed. bThe CYP102A1 variants were named based on the amino acid similarity (Fig. 1a and Table 2). cPreviously known as the nucleotide sequence of P450 BM3 (CYP102A1) from B. megaterium (Ruettinger et al. 1989). dGenetic Information regarding the CYP102A1 variant of B. megaterium QM B1551 (ATCC 12872) was obtained from the Whole Genome Sequencing of B. megaterium http://www.bios.niu.edu/b_megaterium/ and the variant was designated as QM B1551. We only used its geneti (...truncated)