Anaerobic degradation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by a marine Desulfobacterium strain

Dimethylsulfoniopropionate, an osmolyte of marine algae, is thought to be the major precursor of dimethyl sulfide, which plays a dominant role in biogenic sulfur emission. The marine sulfate-reducing bacterium Desulfobacterium strain PM4 was found to degrade dimethylsulfoniopropionate to 3-S-methylmercaptopropionate. The oxidation of one of the methyl groups of dimethylsulfoniopropionate was coupled to the reduction of sulfate; this process is similar to the degradation betaine to dimethylglycine which was described earlier for the same strain. Desulfobacterium PM4 is the first example of an anaerobic marine bacterium that is able to demethylate dimethylsulfoniopropionate.Abbreviations DMSP dimethylsulfoniopropionate - DMS dimethyl sulfide - MMPA 3-S-methylmercaptopropionate     

Fructosebisphosphatase isoenzymes of the chemoautotroph Xanthobacter flavus.

Xanthobacter flavus employs two fructosebisphosphatase (FBPase)-sedoheptulosebisphosphatase (SBPase) enzymes. One of these is constitutively expressed and has a high FBPase-to-SBPase ratio. The alternative enzyme, which is encoded by cbbF, is induced during autotrophic growth. The cbbF gene was expressed in Escherichia coli, and the FBPase was purified to homogeneity. The purified enzyme has a specific FBPase activity of 114 mumol/min/mg of protein, a Michaelis constant for fructosebisphosphate of 3 microM, and a low FBPase-to-SBPase ratio. CbbF was activated by ATP and inhibited by Ca2+.     

Chorismate mutase and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the methylotrophic actinomycete Amycolatopsis methanolica.

Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII and CMII synthesis to L-Phe repression was isolated. DSII was partially purified (a 42-kDa protein); its activity was strongly inhibited by L-Tyr. CMII was purified to homogeneity (93.6 fold) and characterized as a homodimeric protein with 16-kDa subunits, completely insensitive to feedback inhibition by L-Phe and L-Tyr. The activity of CMII was activated by CMIb; the activity of CMII plus CMIb was again inhibited by L-Phe and L-Tyr. A tightly blocked L-Phe- plus L-Tyr-requiring derivative of mutant strain GH141, GH141-19, that had lost both CMIa and CMII activities was isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Current views on the regulation of autotrophic carbon dioxide fixation via the Calvin cycle in bacteria

The Calvin cycle of carbon dioxide fixation constitutes a biosynthetic pathway for the generation of (multi-carbon) intermediates of central metabolism from the one-carbon compound carbon dioxide. The product of this cycle can be used as a precursor for the synthesis of all components of cell material. Autotrophic carbon dioxide fixation is energetically expensive and it is therefore not surprising that in the various groups of autotrophic bacteria the operation of the cycle is under strict metabolic control. Synthesis of phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase, the two enzymes specifically involved in the Calvin cycle, is regulated via end-product repression. In this control phosphoenolpyruvate most likely has an alarmone function. Studies of the enzymes isolated from various sources have indicated that phosphoribulokinase is the target enzyme for the control of the rate of carbon dioxide fixation via the Calvin cycle through modulation of existing enzyme activity. In general, this enzyme is strongly activated by NADH, whereas AMP and phosphoenol-pyruvate are effective inhibitors. Recent studies of phosphoribulokinase inAlcaligenes eutrophus suggest that this enzyme may also be regulated via covalent modification.     

Metabolic regulation in the facultative methylotroph Arthrobacter P1. Growth on primary amines as carbon and energy sources

During growth of the facultative methylotroph Arthrobacter P1 on methylamine or ethylamine both substrates are metabolized initially in an identical fashion, via the respective aldehydes. The regulatory mechanisms governing the synthesis and activities of enzymes involved in amine and aldehyde utilization were studied in substrate transition experiments. Transfer of ethylamine-grown cells into a medium with methylamine resulted in immediate exeretion of low levels of formaldehyde (max. 0.5 mM) and formate. In the reverse experiment, transfer of methylaminegrown cells into a medium with ethylamine, excretion of much higher levels of acetaldehyde (max. 3.5 mM) occurred. These different levels of aldehyde accumulation were also observed in studies with mutants of Arthrobacter P1 blocked in the synthesis of hexulose phosphate synthase or acetaldehyde dehydrogenase. In wild type Arthrobacter P1, aldehyde production resulted in rapid induction of the synthesis of enzymes involved in their degradation but also in temporary inhibition of further amine utilization and growth. The latter aetivities only resumed at normal rates after the disappearance of the aldehydes from the cultures. Acetaldehyde utilization resulted in intermittent excretion of ethanol and acetate, whereas formaldehyde utilization resulted in further accumulation of formate.During growth of Arthrobacter P1 in the presence of methylamine accumulation of toxic levels of formaldehyde is prevented because of the rapid synthesis of hexulose phosphate synthase to high activities and, in transient state situations, by feedback inhibition of formaldehyde on the activities of the methylamine transport system and amine oxidase.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoate) - HPS hexulosephosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha.

Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95%, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k = 0.78 h-1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0.27 h-1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as D-amino acid oxidase from Candida boidinii.     

Engineering of Hydrolysis Reaction Specificity in the Transglycosylase Cyclodextrin Glycosyltransferase

Abstract

Cyclodextrin glycosyltransferase (CGTase) is a member of the α-amylase family, a large group of enzymes that act on α-glycosidic bonds in starch and related compounds. Over twenty different reaction and product specificities have been found in this family. Although three-dimensional structure elucidation and the biochemical characterization of site-directed mutants have yielded a detailed insight into the mechanism of bond cleavage, the variation in reaction and product specificity is far from understood. This article gives an overview of recent developments in the undersanding and engineering of transglycosylation and hydrolysis specificity in CGTase, which is one of the best-studied α-amylase family enzymes.     

Functional and Structural Neural Network Characterization of Serotonin Transporter Knockout Rats

Brain serotonin homeostasis is crucially maintained by the serotonin transporter (5-HTT), and its down-regulation has been linked to increased vulnerability for anxiety- and depression-related behavior. Studies in 5-HTT knockout (5-HTT^-/-) rodents have associated inherited reduced functional expression of 5-HTT with increased sensitivity to adverse as well as rewarding environmental stimuli, and in particular cocaine hyperresponsivity. 5-HTT down-regulation may affect normal neuronal wiring of implicated corticolimbic cerebral structures. To further our understanding of its contribution to potential alterations in basal functional and structural properties of neural network configurations, we applied resting-state functional MRI (fMRI), pharmacological MRI of cocaine-induced activation, and diffusion tensor imaging (DTI) in 5-HTT^-/- rats and wild-type controls (5-HTT^+/+). We found that baseline functional connectivity values and cocaine-induced neural activity within the corticolimbic network was not significantly altered in 5-HTT^-/- versus 5-HTT^+/+ rats. Similarly, DTI revealed mostly intact white matter structural integrity, except for a reduced fractional anisotropy in the genu of the corpus callosum of 5-HTT^-/- rats. At the macroscopic level, analyses of complex graphs constructed from either functional connectivity values or structural DTI-based tractography results revealed that key properties of brain network organization were essentially similar between 5-HTT^+/+ and 5-HTT^-/- rats. The individual tests for differences between 5-HTT^+/+ and 5-HTT^-/- rats were capable of detecting significant effects ranging from 5.8% (fractional anisotropy) to 26.1% (pharmacological MRI) and 29.3% (functional connectivity). Tentatively, lower fractional anisotropy in the genu of the corpus callosum could indicate a reduced capacity for information integration across hemispheres in 5-HTT^-/- rats. Overall, the comparison of 5-HTT^-/- and wild-type rats suggests mostly limited effects of 5-HTT genotype on MRI-based measures of brain morphology and function.     

Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase

Cyclodextrin-glycosyltransferases (CGTases) (EC ) preferably catalyze transglycosylation reactions with glucosyl residues as acceptor, whereas the homologous alpha-amylases catalyze hydrolysis reactions using water as acceptor. This difference in reaction specificity is most likely caused by the acceptor binding site. To investigate this in detail we altered the acceptor site residues Lys-232, Phe-183, Phe-259, and Glu-264 of Bacillus circulans strain 251 CGTase using site-directed mutagenesis. Lys-232 is of general importance for catalysis, which appears to result mainly from stabilization of the conformation of the loop containing the catalytic nucleophile Asp-229 and His-233, a residue that has been implied in transition state stabilization. Glu-264 contributes to the disproportionation reaction only, where it is involved in initial binding of the (maltose) acceptor. Phe-183 and Phe-259 play important and distinct roles in the transglycosylation reactions catalyzed by CGTase. Mutation of Phe-183 affects especially the cyclization and coupling reactions, whereas Phe-259 is most important for the cyclization and disproportionation reactions. Moreover, the hydrophobisity of Phe-183 and Phe-259 limits the hydrolyzing activity of the enzyme. Hydrolysis can be enhanced by making these residues more polar, which concomitantly results in a lower transglycosylation activity. A double mutant was constructed that yielded an enzyme preferring hydrolysis over cyclization (15:1), whereas the wild type favors cyclization over hydrolysis (90:1).