Impact of Phosphate on Iron Mineralization and Mobilization in Nonheme Bacterioferritin B from Mycobacterium tuberculosis.

Ferritins are supramolecular nanocage proteins, which synthesize hydrated ferric oxyhydroxide mineral via protein mediated rapid Fe2+ sequestration and ferroxidase reactions. Ferritin minerals are also associated with a significant amount of phosphate, which contribute toward their structure and reactivity. Like iron, phosphate also regulates the pathogenesis of Mycobacterium tuberculosis (Mtb), which expresses two types of ferritin: heme binding bacterioferritin A (BfrA) and nonheme binding bacterioferritin B (BfrB). Unlike Mtb BfrA, the rapid kinetics and mechanism of ferroxidase activity, and the mineral core formation/dissolution in Mtb BfrB are not well explored. Moreover, the effect of physiological levels of phosphate (0-10 mM) on the synthesis, structure, and reactivity of ferritin mineral core also require investigation in detail. Therefore, the stopped-flow rapid kinetics of ferroxidase activity (ΔA650/Δt) of Mtb BfrB was carried out, which detected a transient intermediate similar to diferric peroxo species as observed in frog and human ferritins. Increasing phosphate concentration increased the initial rate of iron mineralization (ΔA350/Δt) and dissolved O2 consumption (both ∼1.5-2-fold). Phosphate not only decreased the amount of iron loading and size of the BfrB mineral core (both up to 2-fold) but also decreased its crystallinity, resembling the variations observed in the core morphology of different native ferritins. In addition, phosphate inhibited the kinetics of reductive iron mobilization (∼6-8-fold) indicating its influence on the stability of the iron mineral core. Hence, the current work provides the kinetic/mechanistic insight toward the ferroxidase activity in Mtb BfrB, apart from demonstrating the role of phosphate toward the structure/reactivity of its iron mineral.

Enhanced Substrate Specificity of Thermostable Cytochrome P450 CYP175A1 by Site Saturation Mutation on Tyrosine 68.

Thermostable cytochrome P450 (CYP175A1) cloned from Thermus thermophilus shows mid-point unfolding temperature (Tm) of 88 °C (361 K) along with high thermodynamic stability making it a potential industrially viable biocatalyst. Molecular docking analyses, and structural superposition with steroidogenic and fatty acid metabolizing cytochrome P450 s suggested that the tyrosine 68 may have important role in binding as well as metabolism of substrates by the enzyme. Site-saturation mutation of the tyrosine 68 residue was carried out and several unique mutations were obtained that were properly folded and showed high thermostability. We investigated the effects of variation of the single residue, Tyr68 at the substrate binding pocket of the enzyme on the substrate specificity of CYP175A1. Screening of the mutant colonies of CYP175A1 obtained after saturation mutagenesis of Tyr68 using saturated fatty acid, myristic acid and poly unsaturated fatty acids showed that the Y68K had notable binding and catalytic activity for mono-oxygenation of the saturated fatty acid (myristic acid), which had no major detectable binding affinity towards the WT enzyme. The Y68R mutant of CYP175A1, on the other hand was found to selectively bind and catalyse reaction of cholesterol. The wild type as well as both the mutants of the enzyme however bind poly unsaturated fatty acids. The results thus show that saturation mutation of a single amino acid at the substrate binding pocket of the thermostable cytochrome P450 could induce sufficient changes in the substrate binding pocket of the enzyme that can efficiently change substrate specificity of the enzyme.