Recognition of selected monosaccharides by Pseudomonas aeruginosa Lectin II analyzed by molecular dynamics and free energy calculations

In this study, interactions of selected monosaccharides with the Pseudomonas aeruginosa Lectin II (PA-IIL) are analyzed in detail. An interesting feature of the PA-IIL binding is that the monosaccharide is interacting via two calcium ions and the binding is unusually strong for protein-saccharide interaction. We have used Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) and normal mode analysis to calculate the free energy of binding. The impact of intramolecular hydrogen bond network for the lectin/monosaccharide interaction is also analyzed.     

The Fermentative Formation of CDP-Choline by Haploid Cells of Yeasts and the Change of the Temperature-sensitivity of a Mutant of a Yeast,Saccharomyces rouxii

Phosphorylation of CMP and formation of CDP-choline were tested with various haploid cells of yeasts. Most of them had more or less the ability, but a mutant (Lys–M7, alpha type) of Saccharomyces rouxii was found to lack the ability. Further study revealed the change of the temperature-sensitivity of the mutant, which could not produce CDP-choline when treated at 37°C, whereas it could at 16°C. The growth of the mutant was more sensitive to temperatures than that of the wild strain. The former did not grow at 36.3°C, while the latter grew.     

lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA

Most commonly used expression systems in bacteria are based on the Escherichia coli lac promoter. Furthermore, lac operon elements are used today in systems and synthetic biology. In the majority of the cases the gratuitous inducers IPTG or TMG are used. Here we report a systematic comparison of lac promoter induction by TMG and IPTG which focuses on the aspects inducer uptake, population heterogeneity and a potential influence of the transacetylase, LacA. We provide induction curves in E. coli LJ110 and in isogenic lacY and lacA mutant strains and we show that both inducers are substrates of the lactose permease at low inducer concentrations but can also enter cells independently of lactose permease if present at higher concentrations. Using a gfp reporter strain we compared TMG and IPTG induction at single cell level and showed that bimodal induction with IPTG occurred at approximately ten-fold lower concentrations than with TMG. Furthermore, we observed that lac operon induction is influenced by the transacetylase, LacA. By comparing two Plac-gfp reporter strains with and without a lacA deletion we could show that in the lacA⁺ strain the fluorescence level decreased after few hours while the fluorescence further increased in the lacA⁻ strain. The results indicate that through the activity of LacA the IPTG concentration can be reduced below an inducing threshold concentration—an influence that should be considered if low inducer amounts are used.     

Characterization of human D-amino acid oxidase

D-Amino acid oxidase (DAAO) has been proposed to be involved in the oxidation of D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.     

Site-directed alkylation of LacY: effect of the proton electrochemical gradient

Previous N-ethylmaleimide-labeling studies show that ligand binding increases the reactivity of single-Cys mutants located predominantly on the periplasmic side of LacY and decreases reactivity of mutants located for the most part of the cytoplasmic side. Thus, sugar binding appears to induce opening of a periplasmic pathway with closing of the cytoplasmic cavity resulting in alternative access of the sugar-binding site to either side of the membrane. Here we describe the use of a fluorescent alkylating reagent that reproduces the previous observations with respect to sugar binding. We then show that generation of an H⁺ electrochemical gradient (Δ_μ¯H+, interior negative) increases the reactivity of single-Cys mutants on the periplasmic side of the sugar-binding site and in the putative hydrophilic pathway. The results suggest that Δ_μ¯H+, like sugar, acts to increase the probability of opening on the periplasmic side of LacY.     

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.     

Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.     

The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc1 complexes

A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a_s-type with reduction potentials of + 200, + 400 and + 160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a_s, and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be + 320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane α-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc₁ complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc₁ complexes of bacteria and mitochondria, however with distinct subunits and heme types.     

Evaluation of carbohydrate molecular mechanical force fields by quantum mechanical calculations

A quantitative evaluation of 20 second-generation carbohydrate force fields was carried out using ab initio and density functional methods. Geometry-optimized structures (B3LYP/6-31G(d)) and relative energies using augmented correlation consistent basis sets were calculated in gas phase for monosaccharide carbohydrate benchmark systems. Selected results are: (i). The interaction energy of the alpha-d-glucopyranose.H(2)O heterodimer is estimated to be 4.9 kcal/mol, using a composite method including terms at highly correlated (CCSD(T)) level. Most molecular mechanics force fields are in error in this respect; (ii). The (3)E envelope (south) pseudorotational conformer of methyl 5-deoxy-beta-d-xylofuranoside is 0.66 kcal/mol more stable than the (3)E envelope (north) conformer and the alpha-anomer of methyl d-glucopyranoside is 0.82 kcal/mol more stable than the beta-anomer; (iii). The relative energies of the (gg, gt and tg) rotamers of methyl alpha-d-glucopyranoside and methyl alpha-d-galactopyranoside are (0.13, 0.00, 0.15) and (0.64, 0.00, 0.77) kcal/mol, respectively. The results of the quantum mechanical calculations are compared with the results of calculations using the 20 second-generation carbohydrate force fields. No single force field is consistently better than the others for all the test cases. A statistical assessment of the performance of the force fields indicates that CHEAT(95), CFF, certain versions of Amber and of MM3 have the best overall performance, for these gas phase monosaccharide systems.     

Purification and Characterization of β-D-Glucosidase (β-D-Fucosidase) from Bifidobacterium breve clb Acclimated to Cellobiose

The β-d-glucosidase (EC. 3.2.1.21) activity of Bifidobacterium breve 203 was increased by acclimation with cellobiose, and the enzyme was purified to homogeneity from cell-free extracts of an acclimatized strain of B. breve clb, by ammonium sulfate fractionation and column chromatographies of anion-exchange, gel filtration, Gigapaite, and hydrophobic interaction. This enzyme had not only β- d-glucosidase activity but also β- d-fucosidase activity, which is specific to Bifidobacteria in intestinal flora. The molecular weight of the purified enzyme was estimated to be 47,000–48,000 and the enzyme was assumed to be a monomeric protein. The optimum pH and temperature of the enzyme were around 5.5 and 45°C, respectively. The enzyme was stable up to 40°C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the K_m values for p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-fucoside were 1.3mm and 0.7 mm, respectively. This enzyme had also transferase activity for the β-d-fucosyl group but not for the β-d-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of β-d-glucosidase from other bacteria, actinomycetes, and plants.     

A selective Sc(OTf)3-catalyzed trialkylsilyl ether to acetyl ester exchange reaction with beta-l-idopyranoside and 3,4-O-isopropylidene-beta-D-galactopyranoside derivatives

The selective silylation of monosaccharide building blocks is useful for preparing complex oligosaccharides. We now report that the diol, methyl (dimethylthexylsilyl 3-O-pivaloyl-beta-L-idopyranosyl)uronate, can be selectively silylated at the O-2 position by trialkylsilyl triflates. After protection of O-4, the O-2 silyl group can be selectively replaced by acetate by taking advantage of a trialkylsilyl-acetate exchange reaction catalyzed by Sc(OTf)3 in the presence of acetic anhydride. The high O-2 selectivity is shown for triethylsilyl (TES), tert-butyldimethylsilyl (TBS), and triisopropylsilyl (TIPS). The selective cleavage reaction only worked well for TES and TBS derivatives. A selection of silyl triflates and silyl chlorides were used as silylating reagents with ethyl 3,4-O-isopropylidene-1-thio-beta-D-galactopyranoside. In most cases, silylation afforded 2,6-di-O-silylated products in high yields. Studies on the cleavage reaction showed that only the primary silylated protecting groups were replaced by acetyl groups. This reaction worked with a variety of silyl protecting groups but not the tert-butyldiphenylsilyl (TBDPS) protecting group. Unfortunately, the 1-thioethyl group was also sensitive to the Sc(OTf)3, leading in these conditions to alpha/beta mixtures of the 1-acetates, which compromised the synthetic utility of this reaction for these compounds. The sequence presented here is a useful synthetic route to differentially protected L-iduronic acid building blocks.     

Sugar binding in lactose permease: anomeric state of a disaccharide influences binding structure

Lactose permease in Escherichia coli (LacY) transports both anomeric states of disaccharides but has greater affinity for α-sugars. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions, binding structures, and global protein motions in response to sugar binding by investigating LacY (the experimental mutant and wild-type) embedded in a fully hydrated lipid bilayer. A total of 12 MD simulations of 20-25 ns each with β(α)-d-galactopyranosyl-(1,1)-β-d-galactopyranoside (ββ-(Galp)₂) and αβ-(Galp)₂ result in binding conformational families that depend on the anomeric state of the sugar. Both sugars strongly interact with Glu126 and αβ-(Galp)₂ has a greater affinity to this residue. Binding conformations are also seen that involve protein residues not observed in the crystal structure, as well as those involved in the proton translocation (Phe118, Asn119, Asn240, His322, Glu325, and Tyr350). Common to nearly all protein-sugar structures, water acts as a hydrogen bond bridge between the disaccharide and protein. The average binding energy is more attractive for αβ-(Galp)₂ than ββ-(Galp)₂, i.e. −10.7(±0.7) and −3.1(±1.0) kcal/mol, respectively. Of the 12 helices in LacY, helix-IV is the least stable with ββ-(Galp)₂ binding resulting in larger distortion than αβ-(Galp)₂.     

A short helix in the C-terminal region of LolA is important for the specific membrane localization of lipoproteins

The structures of a lipoprotein carrier, LolA, and a lipoprotein receptor, LolB, are similar except for an extra C-terminal loop containing a 3(10) helix and beta-strand 12 in LolA. Lipoprotein release was significantly reduced when beta-12 was deleted. Deletion of the 3(10) helix also inhibited the lipoprotein release. Furthermore, lipoproteins were non-specifically localized to membranes when LolA lacked the 3(10) helix. Thus, the membrane localization of lipoproteins with the LolA derivative lacking the 3(10) helix was independent of LolB whereas LolB was essential for the outer membrane localization of lipoproteins with the wild-type LolA.     

Characterization of a PA14 domain-containing galactofuranose-specific β-d-galactofuranosidase from Streptomyces sp.

As a constituent of polysaccharides and glycoconjugates, β-d-galactofuranose (Galf) exists in several pathogenic microorganisms. Although we recently identified a β-d-galactofuranosidase (Galf-ase) gene, ORF1110, in the Streptomyces strain JHA19, very little is known about the Galf-ase gene. Here, we characterized a strain, named JHA26, in the culture supernatant of which exhibited Galf-ase activity for 4-nitrophenyl β-d-galactofuranoside (pNP-β-d-Galf) as a substrate. Draft genome sequencing of the JHA26 strain revealed a putative gene, termed ORF0643, that encodes Galf-ase containing a PA14 domain, which is thought to function in substrate recognition. The recombinant protein expressed in Escherichia coli showed the Galf-specific Galf-ase activity and also released galactose residue of the polysaccharide galactomannan prepared from Aspergillus fumigatus, suggesting that this enzyme is an exo-type Galf-ase. BLAST searches using the amino acid sequences of ORF0643 and ORF1110 Galf-ases revealed two types of Galf-ases in Actinobacteria, suggesting that Galf-specific Galf-ases may exhibit discrete substrate specificities.     

Ab initio molecular dynamics simulations of beta-D-glucose and beta-D-xylose degradation mechanisms in acidic aqueous solution

Ab initio molecular dynamics simulations were employed to investigate, with explicit solvent water molecules, beta-D-glucose and beta-D-xylose degradation mechanisms in acidic media. The rate-limiting step in sugar degradation was found to be protonation of the hydroxyl groups on the sugar ring. We found that the structure of water molecules plays a significant role in the acidic sugar degradation pathways. Firstly, a water molecule competes with the hydroxyl group on the sugar ring for protons. Secondly, water forms hydrogen bonds with the hydroxyl groups on the sugar rings, thus weakening the C-C and C-O bonds (each to a different degree). Note that the reaction pathways could be altered due to the change of relative stability of the C-C and C-O bonds. Thirdly, water molecules that are hydrogen-bonded to sugar hydroxyls could easily extract a proton from the reaction intermediate, terminating the reaction. Indeed, the sugar degradation pathway is complex due to multiple protonation probabilities and the surrounding water structure. Our experimental data support multiple sugar acidic degradation pathways.     

Transglucosylation Catalyzed by Sucrose Phosphorylase from Leuconostoc mesenteroides and Production of Glucosyl-xylitol

The synthesis of glucooligosaccharides from α-D-glucose-1-phosphate by transglucosylation with sucrose phosphorylase from Leuconostoc mesenteroides was studied using the purified enzyme and high performance liquid chromatography. The enzyme had a rather broad acceptor specificity and transferred glucosyl residues to various acceptors such as sugars and sugar alcohols. Especially, 5-carbon sugar alcohols (pentitols), D- and L-arabitol were acceptors equal to D-fructose, which was known as a good acceptor. The transfer product of xylitol formed by the enzyme was investigated. The structure of the product was found to be 4-O-α-D-glucopyranosyl-xylitol (G-X) by acid hydrolysis and ¹³C-nuclear magnetic resonance analysis. G-X is a probable candidate for a preventive for dental caries because it reduced the synthesis of water-insoluble glucan by Streptococcus mutans and kept a neutral pH in the cell suspension.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

An octaheme c-type cytochrome from Shewanella oneidensis can reduce nitrite and hydroxylamine

A c-type cytochrome from Shewanella oneidensis MR-1, containing eight hemes, has been previously designated as an octaheme tetrathionate reductase (OTR). The structure of OTR revealed that the active site contains an unusual lysine-ligated heme, despite the presence of a CXXCH motif in the sequence that would predict histidine ligation. This lysine ligation has been previously observed only in the pentaheme nitrite reductases, suggesting that OTR may have a possible role in nitrite reduction. We have now shown that OTR is an efficient nitrite and hydroxylamine reductase and that ammonium ion is the product. These results indicate that OTR may have a role in the biological nitrogen cycle.