Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: Implications for the mechanism of electron transfer in the P450 BM3 dimer

Bacillus megaterium P450 BM3 (BM3) is a P450/P450 reductase fusion enzyme, where the dimer is considered the active form in NADPH-dependent fatty acid hydroxylation. The BM3 W1046A mutant was generated, removing an aromatic “shield” from its FAD isoalloxazine ring. W1046A BM3 is a catalytically active NADH-dependent lauric acid hydroxylase, with product formation slightly superior to the NADPH-driven enzyme. The W1046A BM3 K_m for NADH is 20-fold lower than wild-type BM3, and catalytic efficiency of W1046A BM3 with NADH and NADPH are similar in lauric acid oxidation. Wild-type BM3 also catalyzes NADH-dependent lauric acid hydroxylation, but less efficiently than W1046A BM3. A hypothesis that W1046A BM3 is inactive [15] helped underpin a model of electron transfer from FAD in one BM3 monomer to FMN in the other in order to drive fatty acid hydroxylation in native BM3. Our data showing W1046A BM3 is a functional fatty acid hydroxylase are consistent instead with a BM3 catalytic model involving electron transfer within a reductase monomer, and from FMN of one monomer to heme of the other [12]. W1046A BM3 is an efficient NADH-utilizing fatty acid hydroxylase with potential biotechnological applications.     

On the evolution of diet and landscape during the Upper Paleolithic through Mesolithic at Franchthi Cave (Peloponnese, Greece)

Franchthi Cave in southern Greece preserves one of the most remarkable records of socioeconomic change of the Late Pleistocene through early Holocene. Located on the southern end of the Argolid Peninsula, the area around the site was greatly affected by climate variation and marine transgression. This study examines the complex interplay of site formation processes (material deposition rates), climate-driven landscape change, and human hunting systems during the Upper Paleolithic through Mesolithic at Franchthi Cave based on the H1B faunal series. Building on earlier work, we establish the full spectrum of the meat diet using taphonomic evidence, and we analyze these data for trends in socioeconomic reorganization. Foraging patterns during the Aurignacian and “Gravettoid” occupations at Franchthi were terrestrial and already rather diversified in comparison to Middle Paleolithic diets in southern Greece. Hunting shifted abruptly to a mixed marine-terrestrial pattern during the Final Paleolithic, and fishing activities intensified though the Mesolithic. The zooarchaeological data indicate two consecutive trends of increasing dietary breadth, the first within an exclusively terrestrial context, and the second as marine habitats came into use through the end of the Mesolithic. The intensity of the human occupations at this site increased in tandem with intensified use of animal and plants. Comparison to the inland site of Klissoura Cave 1 indicates that the trend toward broader diets was regional as well as local.     

The Golgin Coiled-Coil Proteins of the Golgi Apparatus

A number of long coiled-coil proteins are present on the Golgi. Often referred to as “golgins,” they are well conserved in evolution and at least five are likely to have been present in the last common ancestor of all eukaryotes. Individual golgins are found in different parts of the Golgi stack, and they are typically anchored to the membrane at their carboxyl termini by a transmembrane domain or by binding a small GTPase. They appear to have roles in membrane traffic and Golgi structure, but their precise function is in most cases unclear. Many have binding sites for Rab family GTPases along their length, and this has led to the suggestion that the golgins act collectively to form a tentacular matrix that surrounds the Golgi to capture Rab-coated membranes in the vicinity of the stack. Such a collective role might explain the lack of cell lethality seen following loss of some of the genes in human familial conditions or mouse models.Coiled-coils are widely occurring protein structural motifs in which two or more α-helices wind around each other to form an extended rod-like structure. Proteins containing such structures are found in many parts of the cell, and play diverse roles including organizing centrosomes, chromatin, and synapses, or serving as molecular motors. As such there may seem little reason to consider them collectively beyond an interest in the structural and biophysical properties of the coiled-coil itself. However, the Golgi is unique amongst the cellular compartments in that several different large coiled-coil proteins are present on its cytoplasmic surface (Gillingham and Munro 2003; Lupashin and Sztul 2005; Short et al. 2005; Ramirez and Lowe 2009). A number of these share a similar organization in that most of the protein is predicted to form a coiled-coil, and that their carboxyl termini mediate attachment to Golgi membranes. They are generally ubiquitously expressed and well conserved in evolution, but their coiled-coil regions are relatively poorly conserved suggesting that much of their length serves as spacer. Given that 500 residues of coiled-coil is ∼75 nm in length then the proteins could extend for ∼100–400 nm. Some of the proteins have regions which appear likely to be unstructured and hence could serve as extensions or hinges to increase the proteins’ reach and flexibility (Oas and Endow 1994; Yamakawa et al. 1996). These shared features suggest that the proteins serve related functions on the Golgi. The term “golgin” is often applied to these proteins having been coined in early studies when several were found as human autoantigens (Fritzler et al. 1993), but the term lacks a clear definition. To provide a focus to this article, I will concentrate on “golgins” as defined by being a protein that is found primarily, if not exclusively, on the Golgi and is predicted to form a homodimeric parallel coiled-coil over most of its length. Proteins with shorter regions of coiled-coil are more likely to have roles distinct to the golgins, especially if further domains are present.Golgin coiled-coil proteins are found on the cis-face of the Golgi, around the rims of the stack and on the trans-face of the Golgi (Fig. 1). The human golgins are summarized in Open in a separate windowFigure 1.The golgin coiled-coil proteins of humans.Schematic representations of known human golgins. Regions predicted to form coiled-coils are shown in gray, and known domains involved in protein function or subcellular targeting are indicated.

Table 1.

The canonical golgins of the human Golgi and their orthologs.

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.     

Between-session reliability of four hop tests and the agility T-test

The purposes of this study were firstly to investigate whether learning affects were present in the administration of 4 hop tests and the Agility T-test and secondly to assess the between-session reliability of these tests. Twenty-two recreational athletes (11 women: age 22.3 ± 3.7 years, height 167.7 ± 6.2 cm, weight 59.2 ± 6.9 kg and 11 men: age 22.8 ± 3.1 years, height 179.8 ± 4 cm, weight 79.6 ± 10 kg) took part in the study. The subjects performed 6 repetitions of each hop test and 4 repetitions of the Agility T-test once a week over a period of 3 weeks. Distances were normalized to leg length and presented as a percentage value for the single, triple and crossover hop. Results showed that there were significant differences in scores between genders and that learning affects were present in all tests. Intraclass correlation coefficients ranged from 0.76 to 0.92 for the hop tests and 0.82 to 0.96 for the Agility T-test. The results indicated that the hop and Agility T-tests are reliable tests for use with subjects in a clinical or team sport environment. The error measurement statistics presented could be of help to practitioners to determine whether changes in individuals' scores in the hop and Agility T-tests are because of a true change in performance or measurement error. Of most importance was the fact that all subjects achieved at least 90% limb symmetry index on all 4 hop tests. Therefore, we recommend that a minimum limb symmetry value of 90%, rather than previously recommended 85%, should be adopted during rehabilitation and conditioning.     

Transplantation of organic surface horizons of boreal soils into warmer regions alters microbiology but not the temperature sensitivity of decomposition

Changes in soil carbon, the largest terrestrial carbon pool, are critical for the global carbon cycle, atmospheric CO₂ levels and climate. Climate warming is predicted to be most pronounced in the northern regions and therefore the large soil carbon pool residing in boreal forests will be subject to larger global warming impact than soil carbon pools in the temperate or the tropical forest. A major uncertainty in current estimates of the terrestrial carbon balance is related to decomposition of soil organic matter (SOM). We hypothesized that when soils are exposed to warmer climate the structure of the ground vegetation will change much more rapidly than the dominant tree species. This change will alter the quality and amount of litter input to the soil and induce changes in microbial communities, thus possibly altering the temperature sensitivity of SOM decomposition. We transferred organic surface soil sections from the northern borders of the boreal forest zone to corresponding forest sites in the southern borders of the boreal forest zone and studied the effects of warmer climate after an adaptation period of 2 years. The results showed that initially ground vegetation and soil microbial community structure and community functions were different in northern and southern forest sites and that 2 years of exposure to warmer climate was long enough to cause changes in these ecological indicators. The rate of SOM decomposition was approximately equally sensitive to temperature irrespective of changes in vegetation or microbial communities in the studied forest sites. However, as temperature sensitivity of the decomposition increases with decreasing temperature regime, the proportional increase in the decomposition rate in northern latitudes could lead to significant carbon losses from the soils.     

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.