Conformational heterogeneity suggests multiple substrate binding modes in CYP106A2.

CYP106A2 (cytochrome P450) 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 N, H and C 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.