Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation of O-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR

Abstract

Cysteine is a building block for many biomolecules that are crucial for living organisms. O-Acetylserine sulfhydrylase (OASS), present in bacteria and plants but absent in mammals, catalyzes the last step of cysteine biosynthesis. This enzyme has been deeply investigated because, beside the biosynthesis of cysteine, it exerts a series of “moonlighting” activities in bacteria. We have previously reported a series of molecules capable of inhibiting Salmonella typhimurium (S. typhymurium) OASS isoforms at nanomolar concentrations, using a combination of computational and spectroscopic approaches. The cyclopropane-1,2-dicarboxylic acids presented herein provide further insights into the binding mode of small molecules to OASS enzymes. Saturation transfer difference NMR (STD-NMR) was used to characterize the molecule/enzyme interactions for both OASS-A and B. Most of the compounds induce a several fold increase in fluorescence emission of the pyridoxal 5′-phosphate (PLP) coenzyme upon binding to either OASS-A or OASS-B, making these compounds excellent tools for the development of competition-binding experiments.     

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53 269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80° C; ammonia-dependent reaction, 90° C). Received: 30 October 1996 / Accepted: 13 January 1997     

Further improvement of phosphite dehydrogenase thermostability by saturation mutagenesis

Phosphite dehydrogenase represents a new enzymatic system for regenerating reduced nicotinamide cofactors for industrial biocatalysis. We previously engineered a variant of phosphite dehydrogenase with relaxed cofactor specificity and significantly increased activity and stability. Here we performed one round of random mutagenesis followed by comprehensive saturation mutagenesis to further improve the enzyme thermostability while maintaining its activity. Two new thermostabilizing mutations were identified. These, along with the 12 mutations previously identified, were subjected to saturation mutagenesis using the parent enzyme or the engineered thermostable variant 12x as a template, followed by screening of variants with increased thermostability. Of the 12 previously identified sites, 6 yielded new variants with improved stability over the parent enzyme. Several mutations were found to be context-dependent. On the basis of molecular modeling and biochemical analysis, various mechanisms of thermostabilization were identified. Combining the most thermostabilizing mutation at each site resulted in a variant that showed a 100-fold increase in half-life at 62 degrees C over the 12x mutant. The final mutant has improved the half-life of thermal inactivation at 45 degrees C by 23,000-fold over the parent enzyme. The engineered phosphite dehydrogenase will be useful in NAD(P)H regeneration.     

Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Summary Pea (Pisum sativum) root nodule cells infected by the diazotrophRhizobium leguminosarum have been well characterized by chemical fixation techniques. Propane-jet freezing and high pressure freezing were used in this study to compare rapidly frozen and chemically fixed pea root nodule cells. Cells that had been incubated in 2-(N-morpholino)ethanesulfonic acid buffer and frozen with the propane-jet freezer were better preserved than cells that had been chemically fixed or frozen with the high-pressure freezer. Rapidly frozen infected nodule cells showed that the rough endoplasmic reticulum had a high frequency of associations with the peribacteroid membrane and the infection thread. The peribacteroid space also varied in size depending on the method of preservation; however, it was most reduced in size and devoid of inclusions in the propane-jet frozen tissue. The biological significance of these observations is discussed.Abbreviations HPF high-pressure freezing - MES 2-(N-morpholino)ethanesulfonic acid - PBM peribacteroid membrane - PBS peribacteroid space - PJF propane-jet freezing - RER rough endoplasmic reticulum     

Photosynthetic Responses to Light Intensity in Intact Leaves of Some Coastal Desert and Tropical Rain Forest Plant Species in Atmospheres with Different CO2 Concentrations

The photosynthetic responses to light (A/light response) at different CO2 concentration levels were studied in five coastal desert plant species (two C4 species and three C3 species) and five tropical rain forest plant species (all C3' s) using LI-6400 Portable Photosynthesis System. The measurements were carried out in the Coastal Desert and Tropical Rain Forest mesocosms of Biosphere 2 located in Oracle, Arizona, USA. All the species measured had been growing in Biosphere 2 under very high CO2 concentrations (1500～4000 μmol · mol-1) for about four and half years. The authors measured A/light response curves in intact leaves of different species under a series of CO2 concentrations, 350 to 1500 μmol ~ mol-1. Except for the C4 grass, Panicurn maximum, the initial slopes and convexity (0) of A/light curves of all the species increased with the elevation of CO2 concentration. For most C3 species the light compensation points (LCP) and light saturation points (LSP) had increased when CO2 concentrations were elevated, especialy the LSPs. But for the C4 species, changes in LCPs and LSPs were small. In all the C3 species and the C4 shrub, Atriplesc canescens, the apparent maximum quantum yield of CO2 fixation (Φm, app) and maximum assimilation rate (Amax) increased under elevated CO2. But both Φmapp and Amax in Panicum maximum (C4) decreased. The Amax in Biosphere 2 increased by 127 % when CO2 concentration was doubled (from 375 to 750 μmol · mol-1), and C4 species by 39%. Such great enhancement could possibly be due to the fact that the plants had developed under very high CO2 concentration and large growth rooms in Biosphere 2.     

Excretion of ammonia and urea during development in the oyster toadfish,Opsanus tau

The oyster toadfish is one of several teleosts that has been found to produce and excrete large amounts of nitrogenous waste as urea. To clarify the role of urea in the oyster toadfish, urea and ammonia excretion rates were examined in developing fish. Ammonia and urea excretion rates were measured for groups of developing toadfish for three days a week over eight weeks of development. A distinct and significant increase in the excretion rate of urea occurred between the first three weeks (mean = 0.38 mg N/kg‐h) and the fourth through sixth and eighth week of development (mean = 4.68 mg N/kg‐h). This increase of urea excretion occurs at the time of hatching and may be important during development. Preliminary analysis (temperature, pH and salinity) was conducted on water at one toadfish nesting site to provide insight into conditions to which toadfish are exposed.     

Spatial variation of permittivity of an electrolyte solution in contact with a charged metal surface: a mini review

Contact between a charged metal surface and an electrolyte implies a particular ion distribution near the charged surface, i.e. the electrical double layer. In this mini review, different mean-field models of relative (effective) permittivity are described within a simple lattice model, where the orientational ordering of water dipoles in the saturation regime is taken into account. The Langevin–Poisson–Boltzmann (LPB) model of spatial variation of the relative permittivity for point-like ions is described and compared to a more general Langevin–Bikerman (LB) model of spatial variation of permittivity for finite-sized ions. The Bikerman model and the Poisson–Boltzmann model are derived as limiting cases. It is shown that near the charged surface, the relative permittivity decreases due to depletion of water molecules (volume-excluded effect) and orientational ordering of water dipoles (saturation effect). At the end, the LPB and LB models are generalised by also taking into account the cavity field.     

Sensitivity of coral calcification to ocean acidification: a meta‐analysis

To date, meta‐analyses of effects of acidification have focused on the overall strength of evidence for statistically significant responses; however, to anticipate likely consequences of ocean acidification, quantitative estimates of the magnitude of likely responses are also needed. Herein, we use random effects meta‐analysis to produce a systematically integrated measure of the distribution of magnitudes of the response of coral calcification to decreasing Ω_Arag. We also tested whether methodological and biological factors that have been hypothesized to drive variation in response magnitude explain a significant proportion of the among‐study variation. We found that the overall mean response of coral calcification is ~15% per unit decrease in Ω_Arag over the range 2 < Ω_Arag < 4. Among‐study variation is large (standard deviation of 8% per unit decrease in Ω_Arag). Neither differences in carbonate chemistry manipulation method, study duration, irradiance level, nor study species growth rate explained a significant proportion of the among‐study variation. However, studies employing buoyant weighting found significantly smaller decreases in calcification per unit Ω_Arag (~10%), compared with studies using the alkalinity anomaly technique (~25%). These differences may be due to the greater tendency for the former to integrate over light and dark calcification. If the existing body of experimental work is indeed representative of likely responses of corals in nature, our results imply that, under business as usual conditions, declines in coral calcification by end‐of‐century will be ~22%, on average, or ~15% if only studies integrating light and dark calcification are considered. These values are near the low end of published projections, but support the emerging view that variability due to local environmental conditions and species composition is likely to be substantial.     

Untersuchungen zum Einsatz von Pferdebohnen-Molkepräparat in den Milchaustauschfuttermitteln für frühabgesetzte Ferkel

Nach N‐Bilanzversuchen an Leghornhennen 5 unterschiedlicher genetischer Kombinationen in der Legespitze, bei fortgeschrittener Legeperiode sowie am Ende der Legepriode und Fütterung eines Sojaextraktionsschrot/Fischmehl‐Proteins erfolgte eine regressive Ermittlung des NEB/LM_kg ^0,67 durch Approximation der N‐Bilanzwerte aus N‐Steigerungsversuchen an eine Exponentialfunktion.

Als Eiweißverwertungsgrößen ließen sich aus den N‐Retentionskurven (e‐Funktion) die PNu‐Kurven (Physiologischer Nutzwert) und aus den N‐Bilanz‐Funktionen die PEW‐Funktionen (Produktiver Eiweißwert) beschreiben.

Weiterhin wurden die Beziehungen zwischen N‐Aufhahme und Ei‐N‐Abgabe, bezogen auf metabolische LM, regressiv errechnet und dargestellt. Eine Subtraktion der Ei‐N‐Abgabe von der N‐Bilanz ermöglichte jeweils die Abschätzung des Körper‐N‐Ansatzes. Abschließend wurde das Leistungsvermögen der 5 Herkünfte bei gleicher Eiweiß‐ bzw. schwefelhaltiger (‐ limitierende AS)‐Aufnahme verglichen.