Asp-196-->Ala mutant of Leuconostoc mesenteroides sucrose phosphorylase exhibits altered stereochemical course and kinetic mechanism of glucosyl transfer to and from phosphate

Mutagenesis of Asp-196 into Ala yielded an inactive variant of Leuconostoc mesenteroides sucrose phosphorylase (D196A). External azide partly complemented the catalytic defect in D196A with a second-order rate constant of 0.031 M-1 s-1 (pH 5, 30 degrees C) while formate, acetate and halides could not restore activity. The mutant utilized azide to convert alpha-D-glucose 1-phosphate into beta-D-glucose 1-azide, reflecting a change in stereochemical course of glucosyl transfer from alpha-retaining in wild-type to inverting in D196A. Phosphorolysis of beta-D-glucose 1-azide by D196A occurred through a ternary complex kinetic mechanism, in marked contrast to the wild-type whose reactions feature a common glucosyl enzyme intermediate and Ping-Pong kinetics. Therefore, Asp-196 is identified unambiguously as the catalytic nucleophile of sucrose phosphorylase, and its substitution by Ala forces the reaction to proceed via single nucleophilic displacement. D196A is not detectably active as alpha-glucosynthase.     

The role of Asp-295 in the catalytic mechanism of Leuconostoc mesenteroides sucrose phosphorylase probed with site-directed mutagenesis

Replacements of Asp-295 by Asn (D295N) and Glu (D295E) decreased the catalytic center activity of Leuconostoc mesenteroides sucrose phosphorylase to about 0.01% of the wild-type level (k(cat)=200s(-1)). Glucosylation and deglucosylation steps of D295N were affected uniformly, approximately 10(4.3)-fold, and independently of leaving group ability and nucleophilic reactivity of the substrate, respectively. pH dependences of the catalytic steps were similar for D295N and wild-type. The 10(5)-fold preference of the wild-type for glucosyl transfer compared with mannosyl transfer from phosphate to fructose was lost in D295N and D295E. Selective disruption of catalysis to glucosyl but not mannosyl transfer in the two mutants suggests that the side chain of Asp-295, through a strong hydrogen bond with the equatorial sugar 2-hydroxyl, stabilizes the transition states flanking the beta-glucosyl enzyme intermediate by > or = 23kJ/mol.     

A novel family of glucosyl 1,5-anhydro-d-fructose derivatives synthesised by transglucosylation with dextransucrase from Leuconostoc mesenteroides NRRL B-512F

1,5-Anhydro-d-fructose (AF), a metabolite of starch/glycogen degradation, is a good antioxidant. With the prospect of increasing its applications and use as a food ingredient, AF glucosylation catalysed by the dextransucrase from Leuconostoc mesenteroides NRRL B-512F was performed in the presence of sucrose. This led to AF glucosylated derivatives containing alpha-(1-->6) linkages named 1,5-anhydro-d-fructo-glucooligosaccharides (AFGOS). LC-MS analyses showed that AFGOS with a degree of polymerisation (DP) of up to 7 were synthesised. The amount of AFGOS produced and the average DP increased by using a high sucrose/AF molar ratio and high total sugar concentration. AFGOS were proved to present antioxidant properties quite similar to AF.     

Regioselective O-glucosylation by sucrose phosphorylase: a promising route for functional diversification of a range of 1,2-propanediols

1,2-Propanediol and 3-aryloxy/alkyloxy derivatives thereof are bulk commodities produced directly from glycerol. Glycosylation is a promising route for their functional diversification into useful fine chemicals. Regioselective glucosylation of the secondary hydroxyl in different 1,2-propanediols was achieved by a sucrose phosphorylase-catalyzed transfer reaction where sucrose is the substrate and 2-O-α-d-glucopyranosyl products are exclusively obtained. Systematic investigation for optimization of the biocatalytic synthesis included prevention of sucrose hydrolysis, which occurs in the process as a side reaction of the phosphorylase. In addition to ‘nonproductive’ depletion of donor substrate, the hydrolysis also resulted in formation of maltose and kojibiose (up to 45%) due to secondary enzymatic glucosylation of the glucose thus produced. Using 3-ethoxy-1,2-propanediol as the acceptor substrate (1.0 M), the desired transfer product was obtained in about 65% yield when employing a moderate (1.5-fold) excess of sucrose donor. Loss of the glucosyl substrate to ‘glucobiose’ by-products was minimal (<7.5%) under these conditions. The reactivity of other acceptors decreased in the order, 3-methoxy-1,2-propanediol > 1,2-propanediol > 3-allyloxy-1,2-propanediol > 3-(o-methoxyphenoxy)-1,2-propanediol > 3-tert-butoxy-1,2-propanediol. Glucosylated 1,2-propanediols were not detectably hydrolyzed by sucrose phosphorylase so that their synthesis by transglucosylation occurred simply under quasi-equilibrium reaction conditions.     

Enzymatic alpha-glucosaminylation of maltooligosaccharides catalyzed by phosphorylase

This paper describes phosphorylase-catalyzed enzymatic alpha-glucosaminylation for the direct incorporation of a 2-amino-2-deoxy-alpha-d-glucopyranose unit into maltooligosaccharides. When the reaction of 2-amino-2-deoxy-alpha-d-glucopyranosyl 1-phosphate as the glycosyl donor with maltotetraose as a glycosyl acceptor was performed in the presence of phosphorylase, glucosaminylated oligosaccharides were produced, which were characterized by MALDI-TOF MS measurement after N-acetylation of the crude products. The N-acetylated derivative of the main product in this system was isolated by using HPLC, and its structure was confirmed by MS and (1)H NMR spectra. Furthermore, glucoamylase-catalyzed reaction of the isolated compound provided support that the alpha-glucosamine unit is positioned at the non-reducing end of the oligosaccharide.     

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.     

An efficient synthesis of methyl 1,3-O-isopropylidene-alpha-D-fructofuranoside and 2,3:5,6-di-O-isopropylidene-D-glucose dimethyl acetal derivatives from sucrose

Acetalation of sucrose with 2,2-dimethoxypropane in 1,4-dioxane in the presence of p-toluenesulfonic acid, followed by acetylation, afforded methyl 4,6-di-O-acetyl-1,3-O-isopropylidene-alpha-D-fructofuranoside and 4-O-acetyl-2,3:5,6-di-O-isopropylidene-D-glucose dimethyl acetal as major products, while tosylation of the intermediate acetals provided methyl 6-O-tosyl-1,3-O-isopropylidene-alpha-D-fructofuranose.     

Oxidative kinetic resolution of 1-phenylethanol catalyzed by sugar-based salen-Mn(III) complexes

Three new chiral salen-Mn(III) complexes with sugars at the C-5(5') positions of the salicylaldehyde moieties of the salen ligand were synthesized. Their structures were characterized by FTIR, MS, and elemental analysis. The complexes together with two previously reported ones were successfully used as chiral catalysts for the oxidative kinetic resolution (OKR) of 1-phenylethanol using PhI(OAc)2 as an oxidant and KBr as an additive. Excellent enantiomeric excess (up to 89%) of the product was achieved in 0.5h at 20 degrees C. The results showed that the sugars at C-5(5') of salicylaldehyde moieties in the ligand had influences on the catalytic performances of the complexes. It was concluded that the sugars with the same rotation direction of polarized light as the diimine bridge within the complex could enhance the chiral induction of the complex in the OKR of 1-phenylethanol, but the sugars with the opposite one would reduce that of the corresponding complex.     

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.     

Reaction on D-glucal by an inverting phosphorylase to synthesize derivatives of 2-deoxy-beta-D-arabino-hexopyranosyl-(1-->4)-D-glucose (2II-deoxycellobiose)

Four derivatives of 2(II)-deoxycellobiose were synthesized from d-glucal and acceptor sugars (d-glucose, d-xylose, d-mannose, and 2-deoxy-d-arabino-hexose) using a cellobiose phosphorylase from Cellvibrio gilvus. The enzyme was found to be an effective catalyst to synthesize the beta-(1-->4) linkage of 2-deoxy-d-arabino-hexopyranoside. The acceptor specificity for the d-glucal reaction was identical to that for the alpha-d-glucose 1-phosphate reaction, but the activity of d-glucal was approximately 500 times less than that of alpha-d-glucose 1-phosphate, using 10mM substrates.     

Efficient synthesis of 5-thio-D-arabinopyranose and 5-thio-D-xylopyranose from the corresponding D-pentono-1,4-lactones

5-Thio-D-arabinopyranose (5) and 5-thio-D-xylopyranose (10) were synthesized from the corresponding D-pentono-1,4-lactones. After regioselective bromination at C-5, transformation into 5-S-acetyl-5-thio derivatives, reduction into lactols and deprotection afforded the title compounds in 49 and 42% overall yield, respectively.     

Ring-opening mechanism revealed by crystal structures of NagB and its ES intermediate complex

Glucosamine 6-phosphate deaminase (NagB) catalyzes the conversion of d-glucosamine 6-phosphate (GlcN6P) to d-fructose 6-phosphate and ammonia. This reaction is the final step of N-acetylglucosamine utilization and decides its metabolic fate. The enzyme from Streptococcus mutans belongs to the monomeric subfamily of NagB. The crystal structure of the native SmuNagB (NagB from S. mutans) presented here, compared with the structures of its homologs BsuNagB (NagB from Bacillus subtilis) and EcoNagB (NagB from E. coli), implies a conformational change of the ‘lid’ motif in the activation of the monomeric NagB enzyme. We have also captured the enzyme-substrate intermediate complex of the NagB family at low pH, where a remarkable loss of the catalytic activity of SmuNagB was detected. The enzyme-substrate intermediate presents the initial step of the GlcN6P deaminase reaction. The structural evidence (1) supports the α-anomer of GlcN6P as the specific natural substrate of NagB; (2) displays the substrate-binding pocket at the active site; and (3) together with the site-directed mutagenesis studies, demonstrates the ring-opening mechanism of an Asn-His-Glu triad that performs the proton transfer from O1 to O5 to open the sugar ring.     

Predicted bacteriorhodopsin from Exiguobacterium sibiricum is a functional proton pump

The predicted Exigobacterium sibiricum bacterirhodopsin gene was amplified from an ancient Siberian permafrost sample. The protein bacteriorhodopsin from Exiguobacterium sibiricum (ESR) encoded by this gene was expressed in Escherichia coli membrane. ESR bound all-trans-retinal and displayed an absorbance maximum at 534 nm without dark adaptation. The ESR photocycle is characterized by fast formation of an M intermediate and the presence of a significant amount of an O intermediate. Proteoliposomes with ESR incorporated transport protons in an outward direction leading to medium acidification. Proton uptake at the cytoplasmic surface of these organelles precedes proton release and coincides with M decay/O rise of the ESR.     

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.     

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.     

Synthesis and intramolecular transesterifications of pivaloylated methyl alpha-D-galactopyranosides

Selective pivaloylations of methyl alpha-D-galactopyranoside have been studied under various reaction conditions. Partially pivaloylated products were submitted to additional acetylations. The structures were established by 1H and 13C NMR spectroscopies. Both, 2,6- and 3,6-dipivalates underwent intramolecular cyclization in neutral conditions (phosphate buffered saline, pH 7.2) to give a stable 2,3-orthoacid with a parallel 6-->4 migration of the pivaloyl group.     

Real time analysis of lipoprotein transfer from LolA to LolB by means of surface plasmon resonance

Lipoproteins of Escherichia coli are sorted to the outer membrane through a pathway composed of five Lol proteins. LolA transports lipoproteins released from the inner membrane by LolCDE to LolB on the outer membrane via the periplasm. Interaction between LolA and LolB was speculated to be strong when LolA binds lipoprotein. However, due to a lack of a sensitive method, the kinetics of this reaction have not been examined in detail. We report here the detection of lipoprotein transfer in real time by means of surface plasmon resonance. The kinetic parameters of lipoprotein transfer were determined with wild-type LolA and a mutant defective in it.

Structured summary

MINT-7259948: mlolB (uniprotkb:P61320) binds (MI:0407) to pal (uniprotkb:P0A912) by surface plasmon resonance (MI:0107)     

Enzymatic synthesis of D-glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) and NMR analysis of its isomeric forms

D-Glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) was prepared from D-glucosone (D-arabino-hexos-2-ulose) by enzymatic conversion with hexokinase. The isomeric composition of D-glucosone 6-phosphate in aqueous solution was quantitatively determined by NMR spectroscopy and compared to D-glucosone. The main isomers are the alpha-anomer (58%) and the beta-anomer (28%) of the hydrated pyranose form, and the beta-D-fructofuranose form (14%).     

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

Structured summary of protein interactions

dicPAH and dicPAHbind by molecular sieving (View Interaction: 1, 2, 3, 4)     

Synthesis of 2,3,4,5-tetra-O-methyl-D-glucono-1,6-lactone as a monomer for the preparation of copolyesters

2,3,4,5-tetra-O-methyl-D-glucono-1,6-lactone has been prepared as a crystalline compound in acceptable yield by two different routes. An initial assay of copolymerization with L-lactide by ring-opening polymerization was carried out. The incorporation of the carbohydrate monomer into the polymer chain was about 2%.