CYP261 enzymes from deep sea bacteria: A clue to conformational heterogeneity in cytochromes P450

Abstract

We have explored the adaptation of the cytochromes P450 (P450) of deep-sea bacteria to high hydrostatic pressures. Strict conservation of the protein fold and functional importance of proteinbound water make P450 a unique subject for the studies of high pressure adaptation. Earlier we expressed and purified a fatty-acid binding P450 from the deep-sea bacteria Photobacterium profundum SS9 (CYP261C1). Here we report purification and initial characterization of its mesophilic ortholog from the shallow-water P. profundum 3TCK (CYP261C2), as well as another piezophilic enzyme, CYP261D1 from deep-see Moritella sp. PE36. Comparison of the three enzymes revealed a striking peculiarity of the piezophilic enzymes. Both CYP261C1 and CYP261D1 possess an apparent pressure-induced conformational toggle actuated at the pressures commensurate with the physiological pressure of habitation of the host bacteria. Furthermore, in contrast to CYP261C2, the piezophilic CYP261 enzymes may be chromatographically separated into two fractions with different properties, and different thermodynamic parameters of spin equilibrium in particular. According to our concept, the changes in the energy landscape that evolved in pressure-tolerant enzymes must stabilize the less-hydrated, closed conformers, which may be transient in the catalytic mechanisms of non-piezophilic enzymes. The studies of enzymes of piezophiles should help unravel the mechanisms that control water access during the catalytic cycle.Keywords pressure-tolerant enzymes; piezophiles; conformational heterogeneity; cytochrome; P450; monooxygenases; molecular mechanism Cytochromes P450, the heme-thiolate enzymes that catalyze oxidation of a wide variety of endogenous and exogenous lipophilic compounds, play diverse physiological functions varying from the synthesis of steroid hormones, prostaglandins and second messengers to oxidation of exogenous compounds, such as drugs and other xenobiotics. Monooxygenation reactions require the input of two electrons (supplied by flavoprotein or ferredoxin partners) used to activate the dioxygen molecule. One oxygen atom is then inserted into the substrate, while the second atom is reduced and released in the form of a water molecule. However, an important fraction of the activated oxygen may be released without substrate oxidation through the "leaky" shortcuts in the catalytic cycle (see (1) for a review). This leakage, which results in a futile cycling and generation of reactive oxygen species, such as the superoxide anion-radical and hydrogen peroxide, is closely related to the degree of active site hydration. The mechanisms of conformational gating of the heme pocket that minimize futile cycling through regulation of its solvent accessibility, prevention of aberrant proton delivery and efficient expulsion of the water molecule formed in catalysis represents one of the most important challenges for the current mechanistic enzymology of cytochromes P450. In our search for new approaches to study the conformational dynamics during the...

In Silico Analysis of P450s and Their Role in Secondary Metabolism in the Bacterial Class Gammaproteobacteria

“…CYP226 family members are involved in the catabolism of dehydroabietic acid and abietic acid [ 69 , 70 ]. CYP261 family members were found to have a high affinity to fatty acids [ 71 ]. Based on the biosynthetic gene cluster analysis, in this study, we conclude that 49 P450s are involved in the synthesis of secondary metabolites ( Supplementary Dataset 1: Sheet 2 ).…”

Spectroscopic studies of the cytochrome P450 reaction mechanisms

“…High sensitivity and simple instrumentation, with no special requirements for experimental conditions, make UV-VIS spectroscopy a method of choice for thermodynamic and kinetic studies of cytochromes P450, as well as when employing cryogenic methods, [38,41,59,60,76,95] high pressure, [96,97,98,99] flash photolysis and photoacoustic methods in nanosecond range, [100,101,102,103,104] etc. Optical absorption spectra can be used for monitoring binding mode and for comparing substrate analogs, [105,106,107] or binding of the same substrate to single point mutants.…”

Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy

“…Along with the use of the naturally present C64, C98, and C468 SH groups, we also incorporated new points for R1 attachment by making T85C, A121C, T166C, A343C, T409C, and S495C replacements on the background of the CYP3A4 construct depleted of the native cysteine residues (except for the heme ligating C443). The selection of these sites was prompted by our recent study of high-pressure adaptation in cytochromes P450 from deep-sea bacteria, in which we demonstrated a pressure-actuated conformational switch in the high-pressure-adapted (piezophilic) cytochromes P450 261C1 and 261D1 (70). A comparison of the primary structures of these proteins with those of their mezophilic (not pressure-adapted) progenitors CYP261C2 and CYP261D2 allowed us to localize two regions affected by piezophilic adaptation: 1) the interface between the a-helices A, B 0 , K, and the meander loop; and 2) the interface between the C/D, J/J 0 , and the C-terminal loops of the protein (Fig.…”