P450 enzymes are the major heme-containing catalysts involved in the oxidation of various substrates (Ortiz de Montellano, 2015). They are found in nearly all organisms, from bacteria to plants and mammals (Guengerich, 2008). Genome sequencing projects continue to reveal new genes for P450 enzymes from microorganisms including bacteria, archaea, and fungi. To date, more than 21,000 P450 genes have been reported across all classes of organism (
P450 enzymes from species in the genus
Two CYP158A enzymes (CYP158A1 and CYP158A2) have been found in
Glucose-6-phosphate, glucose-6-phosphate dehydrogena se, NADP+, and 2-OH NQ were purchased from Sigma (St. Louis, MO, USA). Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was purchased from Anatrace (Maumee, OH, USA). All the chemicals were of the highest grade commercially available.
Our general approach has been described previously (Lim
The expression and purification of CYP158A3 were carried out as previously described, with some modifications (Lim
Sodium dithionite was added to reduce the ferric-form purified CYP158A3. CO-ferrous CYP158A3 complexes were generated by passing CO gas through solutions of ferrous CYP158A3. The UV-visible spectra were collected on a CARY Varian spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) in 100 mM potassium phosphate buffer (pH 7.4) at room temperature.
Purified CYP158A3 was diluted to 1 μM in 100 mM potassium phosphate buffer (pH 7.4) and divided equally into two glass cuvettes. The spectra were recorded between 350–500 nm on a CARY Varian spectrophotometer while titrating with ligandsmyristic acid or 2-OH NQ (Yun
Hydroxylation of 2-OH NQ by CYP158A3 was analyzed by LC-mass spectrometry. The reaction mixture contained 2 nmol purified CYP158A3 enzyme, and 200 mM
Homology modeling was performed on the Swiss-Model server (
Recombinant CYP158A3 protein was co-expressed with the molecular chaperones GroEL/ES in
Binding titration analysis of CYP158A3 was performed to identify putative substrates. Titration of purified CYP158A3 with myristic acid produced a decrease at 416 nm and an increase at 378 nm, indicative of type I binding (Fig. 3A). The binding affinity (
The catalytic activity of CYP158A3 towards 2-OH NQ was analyzed using LC-mass spectrometry. Purified CYP158A3 supported the catalytic turnover of 2-OH NQ, in the presence of
For further analysis of the molecular structure, we tried to obtain the three-dimensional structure of CYP158A3. However, we were unable to produce crystals of CYP158A3 that diffracted well. To improve protein crystallization, a clone of CYP158A3 was constructed in which the first seven amino acids were truncated from the N-terminus. The truncated clone was expressed in
To provide alternative structural information, a homology model of CYP158A3 was constructed using the crystal structure of CYP158A2 (PDB entry: 1T93) as a template (Fig. 5). Overall, the helical structure of the CYP158A2 homology model matched that of previously reported P450 enzymes (Fig. 5). When the model was superimposed on the crystal structure of CYP158A2, most residues from both structures aligned, implying that CYP158A3 may be a biflaviolin synthase in
The spectra for myristic acid and 2-HO NQ binding to purified CYP158A3 showed standard type I binding (Fig. 3). Previously, CYP158A2 displayed a similar type I spectral titration to 2-OH NQ with a
In conclusion, recombinant
This paper was supported by Konkuk University in 2014.