Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

Abstract

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C.

Fig. 1

Phylogenetic tree of the cytochrome P450 monooxygenases from T. fusca and their closest homologs together with known hyperthermostable P450s. The multiple sequence alignment was made using ClustalX version 2.0.12. The phylogenetic tree was constructed by the neighbor-joining method and was schematically presented using the ClustalX output and MEGA version 4 (Tamura et al. 2007). Prefixes in front of the CYP number abbreviate the genus and species of the respective bacterial organism where the enzyme originates from (Tf T. fusca, Nd Nocardiopsis dassonvillei, Nf Nocardia farcinica, Sal Streptomyces albaduncus, Sco Streptomyces coelicolor, Sgr Streptomyces griseoflavus, Shy Streptomyces hygroscopicus, Ssp Streptomyces sp., Sso Sulfolobus solfataricus, Sto Sulfolobus tokodaii, Pt Picrophilus torridus, Tt Thermus thermophilus). P450s from T. fusca are shown in red; hyperthermostable P450s are given in blue. The bar in the lower left corner represents 0.1 amino acid substitutions per amino acid for the branch length

Scheme 1

Conversion of ethylbenzene (1), propylbenzene (4), and styrene (8) by CYP154H1 from T. fusca. CamA (PdR) and CamB (Pdx) served as electron transfer components

Scheme 2

Conversion of indole derivatives 11, 16, and 19 by CYP154H1 from T. fusca. CamA (PdR) and CamB (Pdx) served as electron transfer components

Scheme 3

Oxidation of sulfides 20a–d by CYP154H1 from T. fusca. CamA (PdR) and CamB (Pdx) served as electron transfer components