Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

Chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure β-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-β-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a β-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A β-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both β- and α-amino acids.     

Preparation of a glycosynthase from the β-glycosidase of the Archaeon Pyrococcus horikoshii

The β-glycosidase from the hyperthermophilic Archaeon Pyrococcus horikoshii (Phoβ-gly) is a monomeric enzyme with wide substrate specificity belonging to family 1 of glycoside hydrolases classification. Inspection of the three-dimensional structure of the enzyme, recently resolved, showed that Phoβ-gly is membrane bound and that the residues putatively involved in the catalytic activity are Glu155 and Glu324 working as the general acid/base and the nucleophile of the reaction, respectively. We show here that mutation of the latter completely eliminated the activity of the enzyme and that it could be reactivated in the presence of sodium formate. Analysis of the products obtained in the presence of sodium formate buffer pH 4.0 at 75°C showed that the Glu324Gly mutant acts as a hyperthermophilic glycosynthase.     

Antigenic similarity between the β-subunits of the ATPases of a bacterium,a yeast and a higher plant

Antiserum raised against the β-subunit of wheat (Triticum aestivum) chloroplast ATPase cross-reacts with a 51000 protein located in the membrane fraction of Escherichia coli. The differential solubility of this polypeptide after chloroform treatment of unc⁺ and uncD409 strains indicates that this cross-reacting polypeptide is the bacterial β-subunit of ATPase. Thus a high degree of conservation of antigenic determinant sites exists between a bacterial β-subunit and the β-subunit of a monocot. This conservation also seems to extend to the β-subunit of mitochondrial ATPase of yeast (Saccharomyces cerevisiae).     

Effect of heterochiral inversions on the structure of a β-hairpin peptide

We study computationally a family of β-hairpin peptides with systematically introduced chiral inversions, in explicit water, and we investigate the extent to which the backbone structure is able to fold in the presence of heterochiral perturbations. In contrast to the recently investigated case of a helical peptide, we do not find a monotonic change in secondary structure content as a function of the number of L- to D-inversions. The effects of L- to D-inversions are instead found to be highly position-specific. Additionally, in contrast to the helical peptide, some inversions increase the stability of the folded peptide: in such cases, we compute an increase in β-sheet content in the aqueous solution equilibrium ensemble. However, the tertiary structures of the stable (folded) configurations for peptides for which inversions cause an increase in β-sheet content show differences from one another, as well as from the native fold of the nonchirally perturbed β-hairpin. Our results suggest that although some chiral perturbations can increase folding stability, chirally perturbed proteins may still underperform functionally, given the relationship between structure and function.     

Characterization of the Quenching of Chlorophyll a Fluorescence by β-Carotene Using the Non-Linear Analysis

Carotenoids (Car) regulate energy flow in photosynthesis by a specific Car-chlorophyll (Chl) interaction in the singlet-excited states, leading to a reduction in Chl fluorescence. We studied quenching of Chl a-fluorescence in benzene by trans--carotene. Non-linear analysis of the quenching process enables to explain the possible molecular mechanism leading to the de-excitation of Chl a. The fluorescence intensity was measured at 670 nm for excitation wavelengths of 380, 430, 640, and 650 nm. The -carotene concentrations ranged from 4×10^–5 M to 5×10^–3 M. When the samples were excited at 640 and 650 nm, the Stern-Volmer plots showed that the quenching process has high rate constants, hence -carotene is a very efficient quencher. Two different types of quenching process could take place.     

Studies on the rules of β-strand alignment in a protein β-sheet structure

To further disclose the underlying mechanisms of protein β-sheet formation, studies were made on the rules of β-strands alignment forming β-sheet structure using statistical and machine learning approaches. Firstly, statistical analysis was performed on the sum of β-strands between each β-strand pairs in protein sequences. The results showed a propensity of near-neighbor pairing (or called “first come first pair”) in the β-strand pairs. Secondly, based on the same dataset, the pairwise cross-combinations of real β-strand pairs and four pseudo-β-strand contained pairs were classified by support vector machine (SVM). A novel feature extracting approach was designed for classification using the average amino acid pairing encoding matrix (APEM). Analytical results of the classification indicated that a segment of β-strand had the ability to distinguish β-strands from segments of α-helix and coil. However, the result also showed that a β-strand was not strongly conserved to choose its real partner from all the alternative β-strand partners, which was corresponding with the ordination results of the statistical analysis each other. Thus, the rules of “first come first pair” propensity and the non-conservative ability to choose real partner, were possible important factors affecting the β-strands alignment forming β-sheet structures.     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

Lack of evidence for the involvement of a β-D-galactosyl-specific lectin in the fusion of chick myoblasts

The role of a β-D-galactosyl-specific lectin, first reported by Teichberg et al., in the fusion of myoblasts in vitro was investigated. The concentration of this lectin in embryonic chick skeletal muscle was found to reach maximal levels at the time of myoblast fusion in vivo. β-D-Galactosyl-β-thiogalactopyranoside and lactose are potent inhibitors of agglutination of trypsinized rabbit erythrocytes caused by the lectin. However, at concentrations of 50 mM these compounds had no effect on either nonsynchronous fusion of myoblasts or on the release of synchronized myoblast cultures from EGTA fusion block. The presence of the agglutinin in the external membranes of chick myoblasts and myotubes could not be demonstrated. It is, therefore, concluded that the involvement of the lectin in the fusion of chick myoblasts remains questionable.     

Molecular Analysis of the β-Globin Gene Cluster in the Niokholo Mandenka Population Reveals a Recent Origin of the βS Senegal Mutation

A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.     

Computational analysis of the interactions of a novel cephalosporin derivative with β-lactamases

Background

One of the main concerns of the modern medicine is the frightening spread of antimicrobial resistance caused mainly by the misuse of antibiotics. The researchers worldwide are actively involved in the search for new classes of antibiotics, and for the modification of known molecules in order to face this threatening problem. We have applied a computational approach to predict the interactions between a new cephalosporin derivative containing an additional β-lactam ring with different substituents, and several serine β-lactamases representative of the different classes of this family of enzymes.

Results

The results of the simulations, performed by using a covalent docking approach, has shown that this compound, although able to bind the selected β-lactamases, has a different predicted binding score for the two β-lactam rings, suggesting that one of them could be more resistant to the attack of these enzymes and stay available to perform its bactericidal activity.

Conclusions

The detailed analysis of the complexes obtained by these simulations suggests possible hints to modulate the affinity of this compound towards these enzymes, in order to develop new derivatives with improved features to escape to degradation.

     

Characterization of a β-glycosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius

In cell free extracts of the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius ATCC27009, we have identified β-gluco- and galactosidase activities showing a specific activity of 0.1 and 12 U/mg, respectively. The two enzymatic activities are associated with different polypeptides and we show here the functional cloning, the expression in Escherichia coli and the characterisation of the β-glucosidase (Aaβ-gly). The enzyme, which is optimally active and stable at temperatures above 65°C, belongs to glycoside hydrolase family 1 (GH1) and shows wide substrate specificity on different aryl-glycosides and cello-oligosaccharides with k _cat/K _M for 4-nitrophenyl-β-D-glucoside and cellobiose of 2,976 and 185 s⁻¹mM⁻¹, respectively. Interestingly, upstream to the β-glycosidase gene, we identified a second ORF homologous to the ATPase subunit of the bacterial ABC transporters (abc1) that is co-transcribed with the β-glycosidase gene glyB and that could be involved in the carbohydrate import. The activity of the enzyme on cello-oligosaccharides of up to five glucose units strongly indicates that the enzyme could be involved in vivo in the degradation of glucans together with endoglucanase enzymes previously described. This, together with the co-expression of the two genes, suggests a role for the glyB-abc1 cluster in A. acidocaldarius in the degradation of cellulose and hemicelluloses. Enzymes: EC 3.2.1.21; EC 3.2.1.23; EC 3.2.1.25; EC 3.2.1.38; EC 3.2.1.37 An erratum to this article can be found at     

Permeation through the cell membrane of a boron-based β-lactamase inhibitor

Bacteria express beta-lactamases to counteract the beneficial action of antibiotics. Benzo[b]-thiophene-2-boronic acid (BZB) derivatives are β-lactamase inhibitors and, as such, promising compounds to be associated with β-lactam antibacterial therapies. The uncharged form of BZB, in particular, is suggested to diffuse through the outer membrane of gram negative bacteria. In this study, through the combination of electrophysiological experiments across reconstituted PC/n-decane bilayers and metadynamics-based free energy calculations, we investigate the permeation mechanism of boronic compounds. Our experimental data establish that BZB passes through the membrane, while computer simulations provide hints for the existence of an aqueous, water-filled monomolecular channel. These findings provide new perspectives for the design of boronic acid derivatives with high membrane permeability.     

Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain

Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal β-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.     

Improvement of the lytic properties of a β-1,3-glucanase by directed evolution

BGLII is a bacterial endoglucanase that hydrolyzes the β-1,3-glucan present in yeast cell walls, resulting in lysis of Saccharomyces cerevisiae. As a result of this property, BGLII is considered a potential tool for downstream processing and recovery of biotechnological products produced in yeast. Here we describe the improvement of the yeast lytic activity of BGLII, achieved by a directed evolution approach involving random mutagenesis and screening for variants with improved catalytic activity, combined with site-directed mutagenesis. A BGLII variant having three times the wild-type hydrolytic activity on laminarin was identified. The purified enzyme also exhibited higher lytic activity on yeast cells. Mutations causing the improvements are located very close to each other in the amino acid sequence, suggesting that the region should be considered as a target for further improvements of the glucanase activity. These results demonstrate the feasibility of molecular evolution methods for the improvement of the BGLII hydrolytic activity, and open a window for further improvement of this or other properties in glycosyl hydrolases in general.