Cytochrome P450 102A2 Catalyzes Efficient Oxidation of Sodium Dodecyl Sulphate: A Molecular Tool for Remediation

Abstract

Bacterial cytochrome P450s (CYPs) constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. In the present work we report the characterization of CYP102A2 from B. subtilis with a focus on its substrate specificity. CYP102A2 is more active in oxidation of sodium dodecyl sulphate (SDS) than any other characterized CYP. The effect of SDS and NADPH concentration on reaction rate showed nonhyperbolic and hyperbolic dependence, respectively. The enzyme was found to exhibit a bell-shaped curve for plots of activity versus pH, over pH values 5.9-8.5. The rate of SDS oxidation reached the maximum value approximately at pH 7.2 and the pH transition observed controlled by two pK(a)s in the acidic (pK(a) = 6.7 ± 0.08) and basic (pK(a) = 7.3 ± 0.06) pH range. The results are discussed in relation to the future biotechnology applications of CYPs.

Figure 1

The effect of pH on V _max of the CYP102A2 SDS/NADPH reaction. Steady-state kinetic measurements were performed in 0.1 M potassium phosphate buffers adjusted to different pH values (5.9–8.5).

Figure 2

Kinetic analysis of CYP102A2 in 0.1 M potassium phosphate buffer, pH 7.2. (a) Initial velocity analysis with NADPH as variable substrate (6.6–100 μM) and SDS at saturation concentration. (b) Initial velocity analysis with SDS as variable substrate (0.34–1.73 mM) and NADPH at saturation concentration. The plot of rate versus SDS concentration is nonhyperbolic and is fitted to the Hill function.

Figure 3

Amino acid sequence alignments. Sequence alignments of heme domain of CYP102A1 (residues 1–472) of B. megaterium flavocytochrome P450 BM3 (NCBI accession number A34286) with the respective domain of CYP102A2 from B. subtilis (NCBI accession number O08394). The alignments were produced using Clustal W [25] and visualied using ESPript [26]. The secondary structure of CYP102A1 (pdb 1FAG) and numbering are shown above the alignment. Alpha helices and beta strands are represented as helices and arrows, respectively, and beta turns are marked with TT. Conserved areas are shown shaded. A column is framed, if more than 70% of its residues are similar according to physicochemical properties.

Figure 4

Kinetic analysis of CYP102A2. Initial velocity analysis of CYP102A2 with NADPH as the variable substrate for several fixed concentrations of SDS (mM): 0.7, (○); 1 mM, (•); 2 mM, (□).

Figure 5

SDS binding to CYP102A2. (a) The spectral changes induced on titration of CYP102A2 with SDS are shown. Arrows indicate directions of change of spectra induced by successive additions of SDS. Difference spectral titrations of CYP102A2 (curve a) and the CYP102A2-SDS 0.8653 mM (curve b) and CYP102A2-SDS 1.726 mM (curve c) and CYP102A2-SDS 2.1553 mM (curve d) complexes. (b) The difference absorbance at 420 nm as a function of the total SDS concentration.