Protein engineering of cytochromes P-450

The cytochromes P-450 are an immensely important superfamily of heme-containing enzymes. They catalyze the monooxygenation of an enormous range of substrates. In bacteria, cytochromes P-450 are known to catalyze the hydroxylation of environmentally significant substrates such as camphor, phenolic compounds and many herbicides. In eukaryotes, these enzymes perform key roles in the synthesis and interconversion of steroids, while in mammals hepatic cytochromes P-450 are vital for the detoxification of many drugs. As such, the cytochromes P-450 are of considerable interest in medicine and biotechnology and are obvious targets for protein engineering. The purpose of this article is to illustrate the ways in which protein engineering has been used to investigate and modify the properties of cytochromes P-450. Illustrative examples include: the manipulation of substrate selectivity and regiospecificity, the alteration of membrane binding properties, and probing the route of electron transfer.     

The Tortoise and the Hare. Small-Game Use, the Broad-Spectrum Revolution, and Paleolithic Demography

This study illustrates the potential of small-game data for identifying and dating Paleolithic demographic pulses such as those associated with modern human origins and the later evolution of food-producing economies. Archaeofaunal series from Israel and Italy serve as our examples. Three important implications of this study are that (1) early Middle Paleolithic populations were exceptionally small and highly dispersed, (2) the first major population growth pulse in the eastern Mediterranean probably occurred before the end of the Middle Paleolithic, and (3) subsequent demographic pulses in the Upper and Epi-Paleolithic greatly reshaped the conditions of selection that operated on human subsistence ecology, technology, and society. The findings of this study are consistent with the main premise of Flannery's broad-spectrum-revolution hypothesis. However, ranking small prey in terms of work of capture (in the absence of special harvesting tools) proved far more effective in this investigation of human diet breadth than have the taxonomic-diversity analyses published previously.     

Consequences of the non-specific binding of a protein to a linear polymer: reconciliation of stoichiometric and equilibrium titration data for the thrombin-heparin interaction

Theoretical aspects of the thermodynamic characterization of cooperative protein interactions with non-specific segments of a linear polymer lattice have been re-examined. This reconsideration has not only provided an alternative derivation of recursive expressions for the stoichiometry of random ligand binding prior to elimination of the parking problem but also extended that treatment to include binding with overlap of additional lattice units. The major obstacle to thermodynamic characterization of non-specific protein-polymer interactions is determination of the lattice capacity for ligand, which in turn defines the length of the polymer segment to which the protein binds. Although these parameters are most readily obtained from studies under conditions that ensure essentially stoichiometric interaction, the endpoint of such a titration is likely to reflect the irreversible rather than the equilibrium binding capacity of the lattice for ligand. Consideration of published results for spectrofluorometric titrations of the thrombin-heparin system under stoichiometric conditions in such terms has permitted their reconciliation with results of a later publication on the interaction under equilibrium conditions.     

Stereostructure and anti-inflammatory activity of three diastereomers of ocobullenone from Ocotea bullata

A novel diastereomer of ocobullenone. designated as sibyllenone, was isolated from the stem bark of mature Ocotea bullata in the course of a search for anti-inflammatory compounds from this plant. The stereostructure was established by X-ray crystallography and corroborated by NOESY analysis. Ocobullenone, obtained previously, was re-isolated and crystallised successfully for X-ray analysis, thus making possible an accurate spatial comparison of ocobullenone, iso-ocobullenone and the new stereoisomer. Tested pharmacologically for cyclooxygenase-1 and 2, and 5-lipoxygenase inhibition, sibyllenone was the only compound from O. bullata which showed good inhibitory activity towards 5-lipoxygenase.     

Unusual Cytochrome P450 Enzymes and Reactions

Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO³⁺), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function.     

The palatability of flavoured novel floating pellets made with brewer's spent grain to captive carp

Abstract

The palatability to common carp, Cyprinus carpio L. of three newly developed differently flavoured floating pellets made from a high proportion (40%) of brewer's spent grain (BSG) was tested using a multiple-offer feeding experiment. The addition of ‘bold’ flavours, such as vanilla or strawberry essence, may help mask the unpleasant taste of some piscicides; however, their inclusion must not compromise uptake by carp. There were no significant differences between the consumption rates of the three varieties, and all flavours were readily consumed. Therefore, it is suggested that highly flavoured pellets made with BSG have a strong potential to mask the flavour of an unpalatable toxin, and further research is now needed to test this hypothesis.     

Blebs produced by actin–myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs

Apoptosis is a fundamental homeostatic mechanism essential for the normal growth, development and maintenance of every tissue and organ. Dying cells have been defined as apoptotic by distinguishing features, including cell contraction, nuclear fragmentation, blebbing, apoptotic body formation and maintenance of intact cellular membranes to prevent massive protein release and consequent inflammation. We now show that during early apoptosis limited membrane permeabilization occurs in blebs and apoptotic bodies, which allows release of proteins that may affect the proximal microenvironment before the catastrophic loss of membrane integrity during secondary necrosis. Blebbing, apoptotic body formation and protein release during early apoptosis are dependent on ROCK and myosin ATPase activity to drive actomyosin contraction. We identified 231 proteins released from actomyosin contraction-dependent blebs and apoptotic bodies by adapted SILAC (stable isotope labeling with amino acids in cell culture) combined with mass spectrometry analysis. The most enriched proteins released were the nucleosomal histones, which have previously been identified as damage-associated molecular pattern proteins (DAMPs) that can initiate sterile inflammatory responses. These results indicate that limited membrane permeabilization occurs in blebs and apoptotic bodies before secondary necrosis, leading to acute and localized release of immunomodulatory proteins during the early phase of active apoptotic membrane blebbing. Therefore, the shift from apoptosis to secondary necrosis is more graded than a simple binary switch, with the membrane permeabilization of apoptotic bodies and consequent limited release of DAMPs contributing to the transition between these states.     

Selective κ Opioid Antagonists nor-BNI,GNTI and JDTic Have Low Affinities for Non-Opioid Receptors and Transporters

Background

Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets.

Results

In binding assays, the three antagonists showed no detectable affinity (K _i≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold). Nor-BNI bound weakly to the α_2C-adrenoceptor (K _i = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α_1A-adrenoceptor (EC₅₀ = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M₁ receptor antagonist (K _B = 3.7 µM). JDTic bound to the noradrenaline transporter (K _i = 54 nM), but only weakly inhibited transport (IC₅₀ = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (K _i = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers.

Conclusions

Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α_1A-adrenoceptors). This may contribute to GNTI''s severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.     

Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.     

Bacillus megaterium Has Both a Functional BluB Protein Required for DMB Synthesis and a Related Flavoprotein That Forms a Stable Radical Species

Despite the extensive study of the biosynthesis of the complex molecule B₁₂ (cobalamin), the mechanism by which the lower ligand 5,6-dimethylbenzimidazole (DMB) is formed has remained something of a mystery. However, recent work has identified and characterized a DMB-synthase (BluB) responsible for the oxygen-dependent, single enzyme conversion of FMN to DMB. In this work, we have identified BluB homologs from the aerobic purple, nonsulfur, photosynthetic bacterium Rhodobacter capsulatus and the aerobic soil bacterium Bacillus megaterium and have demonstrated DMB synthesis by the use of a novel complementation assay in which a B₁₂ deficient strain, substituted with the precursor cobinamide is recovered either by the addition of DMB or by the recombinant expression of a bluB gene. The DMB-synthetic activity of the purified recombinant BluB enzymes was further confirmed in vitro by providing the enzyme with FMNH₂ and oxygen and observing the formation of DMB by HPLC. The formation of a 4a-peroxyflavin intermediate, the first step in the oxygen dependent mechanism of DMB biosynthesis, is reported here and is the first intermediate in the enzyme catalysed reaction to be demonstrated experimentally to date. The identification and characterization of an FMN-binding protein found on the cobI operon of B. megaterium, CbiY, is also detailed, revealing an FMN-containing enzyme which is able to stabilize a blue flavin semiquinone upon reduction with a 1-electron donor.