Combined participation of hydroxylase active site residues and effector protein binding in a para to ortho modulation of toluene 4-monooxygenase regiospecificity

Toluene 4-monooxygenase (T4MO) is a diiron hydroxylase that exhibits high regiospecificity for para hydroxylation. This fidelity provides the basis for an assessment of the interplay between active site residues and protein complex formation in producing an essential biological outcome. The function of the T4MO catalytic complex (hydroxylase, T4moH, and effector protein T4moD) is evaluated with respect to effector protein concentration, the presence of T4MO electron-transfer components (Rieske ferredoxin, T4moC, and NADH oxidoreductase), and use of mutated T4moH isoforms with different hydroxylation regiospecificities. Steady-state kinetic analyses indicate that T4moC and T4moD form complexes of similar affinity with T4moH. At low T4moD concentrations, the steady-state hydroxylation rate is linearly dependent on T4moD-T4moH complex formation, whereas regiospecificity and the coupling efficiency between NADH consumption and hydroxylation are associated with intrinsic properties of the T4moD-T4moH complex. The optimized complex gives both efficient coupling and high regiospecificity with p-cresol representing >96% of total products from toluene. Similar coupling and regiospecificity for para hydroxylation are obtained with T3buV (an effector protein from a toluene 3-monooxygenase), demonstrating that effector protein binding does not uniquely determine or alter the regiospecificity of toluene hydroxylation. The omission of T4moD causes an approximately 20-fold decrease in hydroxylation rate, nearly complete uncoupling, and a decrease in regiospecificity so that p-cresol represents approximately 60% of total products. Similar shifts in regiospecificity are observed in oxidations of alternative substrates in the absence or upon the partial removal of either T4moD or T3buV from toluene oxidations. The mutated T4moH isoforms studied have apparent V(max)/K(M) specificities differing by approximately 2-4-fold and coupling efficiencies ranging from 88% to 95%, indicating comparable catalytic function, but also exhibit unique regiospecificity patterns for all substrates tested, suggesting unique substrate binding preferences within the active site. The G103L isoform has enhanced selectivity for ortho hydroxylation with all substrates tested except nitrobenzene, which gives only m-nitrophenol. The regiospecificity of the G103L isoform is comparable to that observed from naturally occurring variants of the toluene/benzene/o-xylene monooxygenase subfamily. Evolutionary and mechanistic implications of these findings are considered.     

Protected-Site Phosphorylation of Protein Kinase C in Hippocampal Long-Term Potentiation

Abstract: One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the α and βII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca²⁺, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a “protected site” model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.     

Effects of previous cropping and fungicide timing on the development of light leaf spot (Pyrenopeziza brassicae), seed yield and quality of winter oilseed rape (Brassica napus)

Light leaf spot, caused by Pyrenopeziza brassicae, was assessed regularly on double-low cultivars of winter oilseed rape during field experiments at Rothamsted in 1990-91 and 1991-92. Previous cropping and fungicide applications differed; seed yield and seed quality were measured at harvest. In each season, both the initial incidence of light leaf spot and the rate of disease increase were greater in oilseed rape crops sown after rape than those sown after cereals. The incidence of diseases caused by Phoma lingam or Alternaria spp. was also greater in second oilseed rape crops. In 1991-92 there was 42% less rainfall between September and March than in 1990-91, and much less light leaf spot developed. However, P. lingam and Alternaria spp. were more common. Only fungicide application schedules including an autumn spray decreased the incidence of light leaf spot on leaves, stems and pods, as indicated by decreased areas under the disease progress curves (AUDPC) and slower rates of disease increase. Summer sprays decreased incidence and severity of light leaf spot on pods only. In 1990-91, all fungicide treatments which included an autumn spray increased seed and oil yields of cv. Capricorn but only the treatment which included autumn, spring and summer sprays increased yields of cv. Falcon. No treatment increased the yields of cv. Capricorn or cv. Falcon in 1991-92. Fungicide applications decreased glucosinolate concentrations in the seed from a crop of cv. Cobra severely infected by P. brassicae in 1990-91, but did not increase yield.     

The Effect of Nitrate and Nitrite on the Microbial Flora of Wiltshire Bacon after Maturation and Vacuum-packed Storage

Unsmoked Wiltshire bacon was produced with and without nitrate and with different concentrations of nitrite. Micrococci, Moraxella spp. and Moraxella -like organisms were the most common bacteria on the bacon after maturation when there was little difference between the microflora of the various back bacon. Collar bacon cured without nitrate carried much higher numbers of Moraxella spp. than that cured with nitrate. Micrococci and lactic acid bacteria usually predominated after vacuum-packed storage. Inclusion of nitrate in the cure increased the numbers of micrococci but did not reduce stability on storage. The highest numbers of lactic acid bacteria were present on bacon containing the lowest initial concentration of nitrite (34 parts/10⁶) and it is thought that nitrite may delay the souring which these bacteria cause in vacuum-packed bacon.     

A model relating yield to individual plant area for predicting growth compensation in Central African field tobacco*

A model based on the concept of the circle of influence of a plant is used to estimate the relationship between yield and area/plant for tobacco crops with gaps in stand. The model predicts the average yield of plants in any configuration that includes missing plants and is used to predict the economic consequences of loss in stand from pest or other damage.