Methanol-dependent production of dihydroxyacetone and glycerol by mutants of the methylotrophic yeast Hansenula polymorpha blocked in dihydroxyacetone kinase and glycerol kinase

Summary Various factors controlling dihydroxyacetone (DHA) and glycerol production from methanol by resting cell suspensions of a mutant of Hansenula polymorpha, blocked in DHA kinase and glycerol kinase, were investigated. The presence of methanol (250mM) and an additional substrate (0.5%, w/v) to replenish the xylulose-5-phosphate required for the assimilation reaction (DHA synthase) was essential for significant triose production by this double mutant. A number of sugars were tested as additional substrates and C₅ sugars gave the highest triose accumulation (ca. 20mM after 45h). Glucose was the poorest additional substrate and triose production only started after its exhaustion, which occurred in the first few hours. Other sugars were metabolized at a much lower rate and accumulation of trioses began right at the start of the experiments and gradually increased with time. The production rate of total trioses increased, and the relative amount of glycerol diminished with higher oxygen supply rates. The data suggest that conversion of DHA into glycerol, catalysed by reduced nicotine adenine dinucleotide (NADH)-dependent DHA reductase, is partly regulated via intracellular NADH levels. Further support for this hypothesis was obtained in experiments with antimycin A, an inhibitor of the electron transport chain. Addition of higher amounts of methanol and xylose, either by increasing the initial concentrations or by repeated addition of these substrates, resulted in considerably enhanced productivity and a switch towards glycerol formation. After reaching a level of approximately 25mM the DHA concentration remained constant while the glycerol level gradually increased with time. After an incubation period of 350 h, a total of 3.9 M methanol and 0.62 M xylose had been converted, which resulted in accumulation of 0.76 M trioses, mostly glycerol.Offprint requests to: L. Dijkhuizen     

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.     

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.     

Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10

A bacterium that is able to utilize a number of halogenated short-chain hydrocarbons and halogenated carboxylic acids as sole carbon source for growth was identified as a strain of Xanthobacter autotrophicus. The organism constitutively produces two different dehalogenases. One enzyme is specific for halogenated alkanes, whereas the other, which is more heat stable and has a higher pH optimum, is specific for halogenated carboxylic acids. Haloalkanes were hydrolyzed in cell extracts to produce alcohols and halide ions, and a route for the metabolism of 1,2-dichlorethane is proposed. Both dehalogenases show a broad substrate specificity, allowing the degradation of bromine- and chlorine-substituted organic compounds. The results show that X. autotrophicus may play a role in the degradation of organochlorine compounds and that hydrolytic dehalogenases may be involved in the microbial metabolism of short-chain halogenated hydrocarbons in microorganisms.     

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+.     

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable α-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known -amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable -amylase. The half-time of inactivation of this -amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the -amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. -Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and -amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the -amylase was a growth-associated enzyme. The optimal temperature for -amylase production, however, was 40°C, with -amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal -amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O₂ concentration was found to be a critical factor in production of the -amylase.     

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).