Towards a unified mechanism of biological methane oxidation

Abstract The biological oxidation of methane to methanol is catalysed by soluble and particulate forms of the enzyme methane monooxygenase. Little information is available regarding the structure and mechanism of the particulate enzyme whereas much is known about the soluble form of the enzyme. This review concentrates on current knowledge of the structure of the components of the soluble methane monooxygenase and draws together these results with those on the kinetics and substrate specificity of the enzyme in a possible chemical mechanism for enzymatic methane oxidation.     

Isolation and kinetic properties of 5′-nucleotidase from guinea-pig skeletal muscle

5′-Nucleotidase (EC 3.1.3.5) has been solubilized and purified 1200-fold from guinea-pig skeletal muscle, to a specific activity of 40 U/mg protein. The purified enzyme yields a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Guinea-pig skeletal muscle 5′-nucleotidase is extremely sensitive to inhibition by nucleoside di- and triphosphates. The inhibition is of the competitive type, and can be reversed only by strong excess of Mg²⁺. Nucleoside diphosphates are more powerful inhibitors than nucleoside triphosphates. The K_i values for ADP and ATP are 0.036 and 0.28 μM, respectively. The purified enzyme does not require exogenous cations for maximal activity and is inhibited by EDTA. This inhibition is reversed by divalent cations. This indicates that the enzyme contains a tightly bound metal cation.     

A high-resolution 1H-NMR investigation of the histidine-binding protein J of Salmonella Typhimurium: Substrate-induced conformational changes

High-resolution ¹ H-NMR spectroscopy at 600 MHz has been used to investigate the conformational transitions of the histidine-binding protein J of Salmonella Typhinmrium in solution as a function of pH and of l-histidine concentration. The dissociation constant for the binding of l-histidine to histidine-binding protein J increases from 6.0 × 10^?8 to 5.1 × 10^?7 M in going from pH 5.57 to 8.00. The conformation of this protein as observed by ¹H-NMR also changes over this range of pH. However, when l-histidine is bound, the changes in conformation with pH are much smaller. Also, the pk for the single histidyl residue in histidine-binding protein J changes from 6.75 in the absence of l-histidine to 6.52 when l-histidine is bound. Earlier work in this laboratory resulted in the identification of several proton resonances believed to be at or near the l-histidine-binding site. Two of these resonances have been assigned to a tyrosine and the single histidyl residue in the histidine-binding protein J molecule.     

Habitat correlates of the distribution and biomass of Seychelles' reef fishes

Synopsis Relationships between quantitative measures of habitat type and the biomass of Chaetodon, Scarus and Parupeneus species were investigated across 35 reef sites in the Inner Seychelles Group. Multiple regression was used to determine the proportion of variance in biomass between sites which could be explained by depth, exposure, vertical relief, topographic complexity, live coral cover, coral rubble cover, rock cover, sand cover, underlying carbonate substrate, underlying sand substrate, underlying rock substrate and an index of fishing intensity. A significant proportion of the variance in biomass was explained by habitat variables and the index of fishing intensity for 7 of 12 Chaetodon species (23–52% of variance explained), 3 of 6 Parupeneus species (33–40%), and 10 of 13 Scarus species (14–46%). Within genera, different groups of habitat variables explained the variance in biomass for different species and, of the variables studied, only the proportion of underlying sand substrate failed to explain a significant proportion of the variance in biomass for any species. Quantitative relationships between the biomass of Chaetodon and habitat were often in accordance with those suggested by previous studies of their ecology, life-history and distribution at other Indo-Pacific locations. However, the habitat associations of the Parupeneus and some Scarus species have not been studied at other locations and clearly warrant further investigation. It was concluded that habitat was an important determinant of the distribution of many Seychelles reef fishes, but that the habitat variables examined were rarely the most important determinant of biomass. However, the inclusion of a procedure to collect habitat data provided a useful means by which to reduce the unexplained variance associated with visual census biomass estimates and therefore improves the possibility of elucidating the effects of other factors on the biomass of Seychelles reef fishes.     

Site-directed mutagenesis of arginine-89 supports the role of its guanidino side-chain in substrate binding by Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase (IPNS) catalyses a key step in the penicillin and cephalosporin biosynthetic pathway which involves the oxidative cyclisation of the acyclic peptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N. Based on crystallographic evidence from the Aspergillus nidulans IPNS crystal structure complexed with the substrate ACV (Roach et al. (1997) Nature 387, 827-830), we were able to provide mutational evidence for the critical involvement of the conserved R-X-S motif in ACV binding in IPNS. The crystal structure further implicated arginine-87 in the binding of the aminoadipyl portion of ACV. Thus, in this study, the site-directed mutagenesis of the corresponding arginine-89 in Cephalosporium acremonium IPNS (cIPNS) was performed to ascertain its role in cIPNS. Alteration of arginine-89 to five amino acids from different amino acid groups, namely lysine, serine, alanine, aspartate and leucine, was performed and no activity was detected in all the mutants obtained when enzyme bioassays were performed. Furthermore, the solubility of the mutants was considerably lower than the wild-type cIPNS after expression at 37 degrees C, but could be recovered when the expression temperature was lowered to 25 degrees C. This suggests that arginine-89 could be critical for the activity of cIPNS due to its involvement in ACV binding and the solubility of wild-type enzyme.     

Some factors affecting successional change on uranium mine wastes: Insights for ecological restoration

Question: Are growth form and dispersal-mode replacement during vegetation succession in semi-arid Mediterranean conditions affected by the starting quality of the substrate and by site aspect? Location: Central-western Spain. Methods: We monitored successions on three waste materials left after uranium mining: unbroken waste, broken waste and wastes amended with a sandy material (Arkoses); both north and south aspects were also studied on each substrate. Results: The substrate starting quality had the greatest influence on spontaneous succession, separating the poorer quality substrates (broken and unbroken wastes) from the better ones (Arkoses) and two reference communities (Topsoil and Dehesa). The importance of aspect was confirmed then within each substrate type. Most species with a short life span (mostly annuals and a few biennials), together with some woody species on Arkoses, showed no response to age (years following the deposition of new soil). Others short-lived species declined over time on the poorer wastes but not on the better Arkoses. There was a tendency for life form replacement (from thero-phytes to hemicryptophytes) during succession only on the poorer-quality substrates. No dispersal-mode replacement sequence was found. Conclusion: Improving the abiotic conditions of the substrate had a great effect on vegetation succession, but this effect was modified by aspect. Aspect took longer to induce differences in floristic composition on the poorer substrates, where succession was slower. Some trends in species responses to successional change were found by considering species traits, particularly life-form.     

Steady-state kinetics of immobilized enzymes at very low substrate concentrations studied using the asarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

A method for determining initial velocities of enzymatic reactions at very low substrate concentrations is presented. It is based on teh continuous perfusiion of substrate-containing media through the enzyme, previously deposited as a thin layer on a solid support. An analytical rationalization of the dependence of the enzymatic activity upon the substrate supply and the flow rate was developed (substrate supply (μmol/min) = flow rate (ml/min) × inflowing substrate concentration (μmol/ml). This paper shows that a straight line should be expected from a double-reciprocal plot of the velocity of the enzymatic reaction and flow rate. The reciprocal of the ordinate at the origin is the strict initial velocity for a given, constant, and very low substrate concentration, since substrate consumption and product accumulation tend to zero. Results obtained with two different sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase preparations agree with the theoretical predictions. The method enabled the use of ATP concentrations in the range of 10^?8 M: it required neither an ATP-regerating system nor the dilution of the enzyme protein, and it presented no limitations for the reaction time. Both ATPase preparations showed two apparent K_m values for the substrate in the submicromolar and micromolar ranges: 0.25–12.0 μM for the purified ATPase, and 0.17–1.65 μM for the microsomal ATPase.     

Mechanism of the salt activation of laccase Lac15

Laccases (benzenediol: oxygen oxidoreductases, EC1.10.3.2) can oxidize various substrates, and those which are tolerant to and even activated by salts have attracted a lot of attention due to their application potential in certain industries. The mechanism of the salt activation of laccases is awaiting to be elucidated yet. Our previous study (Li, Xie et al. 2018) supposed that the salt activation of marine laccase Lac15 might be attributed to Cl^- ion specifically binding to some local sites to interfere substrate binding and/or electron transfer. In this study, we found two sites whose mutations resulted in elimination of the salt activation of Lac15’s activity towards catechol and dopamine respectively, and revealed that the mutations affected the activity by altering both E_m and k_cat, demonstrating the supposed mechanism. A model for the salt activation of laccases was accordingly proposed, albeit some details are to be elucidated.     

Identification of Meningeal Cell Released Neurite Promoting Activities for Embryonic Hippocampal Neurons

Primary cultures of meningeal cells from embryonic rat cerebra secrete neurite growth-inducing components into serum-free culture medium. This conditioned medium (CM) was analyzed by FPLC and immunochemical and enzymatic treatments and tested for neurite promoting activity (NPA) in a quantitative bioassay using hippocampal neurons from embryonic rat. By immunoprecipitation or specific adsorption we identified laminin (LN)-proteoglycan complexes and fibronectin (FN), respectively, as the major neurite promoting components within meningeal cell CM. The LN-proteoglycan complexes and their NPA were sensitive to chondroitinase (chondroitin ABC lyase, EC 4.2.2.4) and to a smaller extent to heparitinase (heparitin sulfate lyase, EC 4.2.2.8). Minor fractions of the total NPA in CM correlated with free LN and a putative but not yet characterized FN-proteoglycan complex.