Methylene Oxidation of Alkyl Sulfates by Cytochrome P450_BM-3and a Role for Conformational Selection in Substrate Recognition

Abstract

Cytochrome P450 BM-3 (P450 BM-3 ) is a flavoprotein reductase− heme fusion protein from the bacterium Bacillus megaterium that has been well-characterized in many biophysical aspects. Although the enzyme is known to catalyze the hydroxylation of medium-and long-chain fatty acids at high rates, no definitive physiological function has been associated with this process in the organism other than a possible protective role. We found that P450 BM-3 rapidly hydroxylates alkyl sulfates, particularly those with 12−16 carbons (i.e., including dodecyl sulfate), in a similar manner to the fatty acids. The products were characterized as primarily ω-1 hydroxylated alkyl sulfates (plus some ω-2 and ω-3 hydroxylation products), and some further oxidation to dihydroxy and keto derivatives also occurred. Binding of the alkyl sulfates to P450 BM-3 converted the iron from the low-spin to high-spin form in a saturable manner, consistent with the catalytic results. Rates of binding decreased as a function of increasing concentration of dodecyl sulfate or the fatty acid myristate. This pattern is consistent with a binding model involving multiple events and with conformational selection (equilibrium of the unbound enzyme prior to binding) instead of an induced fit mechanism. Neither C−H bond-breaking nor product release was found to be rate-limiting in the oxidation of lauric acid. The conformational selection results rationalize some known crystal structures of P450 BM-3 and can help explain the flexibility of P450 BM-3 and engineered forms in accepting a great variety of substrates.

Kinetics of cytochrome P450 3A4 inhibition by heterocyclic drugs defines a general sequential multistep binding process

“…P450 cam (CYP101A1) binds its substrate camphor in a rapid reaction, leading to high-spin iron, in a kinetic mechanism that can be described by a two-state model with rapid binding ( 62 ). However, various biophysical approaches have been employed to demonstrate the existence of multiple conformational states of ligand-bound P450 cam ( 60 , 62 , 63 ). The binding of camphor can be understood in the context of a mechanism dominated by induced fit, although alternative substrates for the enzyme do not seem to fit this paradigm ( 66 ).…”

Stepwise binding of inhibitors to human cytochrome P450 17A1 and rapid kinetics of inhibition of androgen biosynthesis

“…Measurements of P450, b 5 , and POR were made with an OLIS-Aminco DW2 spectrophotometer (On-Line Instrument Systems). Pre–steady-state kinetic assays of binding of inhibitors were made using an OLIS RSM 1000 stopped-flow instrument equipped with a spinning band monochromator, at 23 °C (4 × 20 mm flow cell, 1.24-mm slits, 330–560 nm, 400 lines mm −1 /500 nm gratings), as described earlier ( 28 , 33 , 58 , 59 , 60 ). When indicated, traces were collected in the OLIS “pretrigger mode,” indicating some of the final absorbances from the end of the previous shot in order to judge whether a reaction has gone to completion.…”

“…Pre-steady-state kinetic assays of binding of inhibitors were made using an OLIS RSM 1000 stopped-flow instrument equipped with a spinning band monochromator, at 23 °C (4 mm  20 mm flow cell, 1.24 mm slits, 330-560 nm, 400 lines mm -1 /500 nm> gratings), as described earlier (28,33,(58)(59)(60). When indicated, traces were collected in the OLIS "pre-trigger mode,"…”

Latent Functions and Applications of Cytochrome P450 Monooxygenases from Thamnidium elegans: A Novel Biocatalyst for 14α-Hydroxylation of Testosterone

“… 29 Many researchers have explored the function and potential utility of fungal P450s. 31 − 33 However, most studies on fungal P450s have focused on basidiomycetous and ascomycetous fungi. This focus can be attributed to specific species in such fungal phyla being extensively studied for potential applications, even from the pregenomic era.…”