Abstract
CYP106A2 (cytochrome P450meg) is a bacterial enzyme originally isolated from B. megaterium, and has been shown to hydroxylate a wide variety of substrates, including steroids. The regio- and stereochemistry of CYP106A2 hydroxylation has been shown to be dependent on a variety of factors, and hydroxylation often occurs at more than one site and/or with lack of stereospecificity for some substrates. Comprehensive backbone 15N, 1H and 13C resonance assignments based on multidimensional nuclear magnetic resonance (NMR) experiments performed with uniform and selective isotopically labeled CYP106A2 samples are reported herein, and broadening and splitting of resonances assigned to regions of the enzyme shown to be affected by substrate binding in other P450 enzymes indicate that substrate binding does not reduce structural heterogeneity as has been observed previously in P450 enzymes CYP101A1 and MycG. Paramagnetic relaxation enhancement (PRE) due to proximity between substrate protons and the heme iron were measured for three different substrates, and the relatively uniform nature of the PREs support the proposal that multiple substrate binding modes are occupied at saturating substrate concentrations.
Left) Two equal energy orientations of abietic acid in the CYP106A2 active site. Structure in gray sticks shows distance between hydroxylation site and heme. Structure in orange lines places the carboxylate close to the heme. Right) Structure of CYP106A2 with residues most affected by heterogeneity highlighted in red. [Display omitted]
•Comprehensive backbone NMR assignments for CYP106A2.•Multiple substrate binding modes give rise to heterogeneity in NMR spectra.•Most affected regions are those involved in substrate binding.•Paramagnetic relaxation patterns support multiple substrate binding orientations.