Cloning, expression, characterization and engineering of cytochrome P450 CYP116B3 from Rhodococcus ruber DSM 44319

The Cytochrome P450 monooxygenases are ubiquitous heme-containing proteins, which catalyze regio- and stereo-selective oxidations of non-activated hydrocarbon. Bacterial P450 enzymes are in most cases not membrane-associated, water soluble and exhibit relatively high stability. Several bacterial strains have been tested for their monooxygenase activity. During the in vivo screening, Rhodoccocus ruber DSM 44319 and Rhodococcus erythropolis DSM 43066 strains exhibited oxidation activity towards cyclohexane. Because the genome DNA sequence of the two Rhodococcus strains has not been sequenced, a homology search was applied using known P450 gene sequences from other Rhodococcus strains. A set of degenerate primers was designed to the most similar regions, identified through the DNA sequence alignment of P450RhF from Rhodococcus sp. NCIMB 9784 and P450 CYP116 from Rhodococcus sp. NI86/21. A P450-like gene fragment with 740 base pairs was amplified from Rhodococcus ruber DSM 44319 by PCR using these degenerate primers. The flanking regions of the P450-like DNA fragment were explored by directional genome walking using PCR combined TA-cloning. The entire P450 gene with 2313 bp was isolated. It encodes a protein of 771 amino acids. The primary protein structure suggests that it is a natural self-sufficient fusion protein consisting of a P450 monooxygenase domain, a flavin-containing reductase domain, and a [2Fe2S] ferredoxin domain. This new P450 gene from Rhodococcus ruber DSM 44319 was named by P450 nomenclature committee CYP116B3 and can be considered as a new member of class IV of cytochrome P450 monooxygenases. The cytochrome P450 monooxygenase CYP116B3 was successfully cloned into vector pET28a(+), and expressed in Escherichia coli BL21(DE3). Subsequently, the enzyme was purified using immobilized metal affinity chromatography with a total yield of 18.6 mg l-1 and purity of 85%. Thin layer chromatography detected only FMN within the reductase domain, as the sequence alignment had predicted. The reductase activity was determined using an exogenous electron acceptor cytochrome c. The reductase domain of this P450 CYP116B3 demonstrated a strong preference for NADPH over NADH. As well as P450RhF, P450 CYP116B3 catalyzed O-dealkylation of 7-ethoxycoumarin gave product 7-hydroxycoumarin. Furthermore, in the presence of NADPH, the P450 CYP116B3 demonstrated hydroxylation activity towards aromatic hydrocarbons, naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene and ethyl benzene. The conversion of naphthalene, acenaphthene and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene and ethyl benzene were hydroxylated exclusively at the side chains. The highest turnover rate catalyzed by wild-type P450 CYP116B3 is about 1 nmol of 7-ethoxyxoumarin converted per 1 nmol of P450 CYP116B3 per minute. Compare to P450 BM-3, the activity of P450 CYP116B3 is very low. In order to investigate the structure-function relationship, a structure model of P450 CYP116B3 was designed based on the known homologous structures. The position 109 was identified which is located on the ceiling of the substrate binding pocket. The substitution of alanine residue at position 109 by phenylalanine may promote binding and oxidation of smaller alkyl substrates, such as cyclohexane or alpha-pinene. However after the substitution no oxidation activity towards smaller substrates was detected. Only an increase of activity towards 7-ethoxycoumarin and PAHs was observed. Directed evolution provides a useful tool to explore enzyme functions without structural information. Therefore, directed evolution was carried out in this study to improve the enzyme activity. The mutagenesis was limited to the monooxygenase domain of CYP116B3. A mutation library was generated by error-prone PCR. A high throughput screening system was developed based on the O-dealkylation of 7-ethoxycoumarin using whole E. coli cells, which expressed the enzyme in 96-well microtiter plates. Finally, the two best mutants, 70A08 (A86T/T91S/A109F/I179F/I267L) and 74H10 (T91S/A109L/I179F/I267L) were identified after four error-prone PCR rounds with 100-fold increased O-dealkylation activity towards 7-ethoxycoumarin. To investigate the alteration of the substrate spectrum of the two mutants, a wide rang of potential substrates was tested, including known substrates of the wild-type CYP116B3. However, 70A08 and 74H10 did not show increased activity towards any substrate besides 7-ethoxycoumarin.