An artificially constructed dimer through deformation of a short zinc-binding loop

We have analyzed the crystal structure of the dimeric form of d-glycero-d-manno-heptose-1,7-bisphosphate phosphatase from Burkholderia thailandensis (BtGmhB), catalyzing the removal of the phosphate at the 7 position of d-glycero-d-manno-heptose-1,7-bisphosphate. The crystal structure of BtGmhB revealed a dimeric form caused by a disruption of a short zinc-binding loop. The dimeric BtGmhB structure was induced by triggering the loss of Zn^2 + via the protonation of cysteine residues at pH 4.8 of the crystallization condition. Similarly, the addition of EDTA also causes the dimerization of BtGmhB. It appears there are two dimeric forms in solution with and without the disulfide bridge mediated by Cys95. The disulfide-free dimer produced by the loss of Zn^2 + in the short zinc-binding loop is further converted to a stable disulfide-bonded dimer in vitro. Though the two dimeric forms are reversible, both of them are inactive due to a deformation of the active site. Single and triple mutant experiments confirmed the presence of two dimeric forms in vitro. Phosphatase assay results showed that only a zinc-bound monomeric form contains catalytic activity in contrast to the inactive zinc-free dimeric forms. The monomer-to-dimer transition caused by the loss of Zn^2 + observed in this study is an example of reversal phenomenon caused by artificial proteins containing protein engineered zinc-finger motifs where the monomer-to-dimer transitions occurred in the presence of Zn^2 +. Therefore, this unusual dimerization process may be applicable to designing proteins possessing a short zinc-binding loop with a novel regulatory role.     

General assay for enzymes in the heptose biosynthesis pathways using electrospray ionization mass spectrometry

The ADP-l-glycero-β-d-manno-heptose and the GDP-6-deoxy-α-d-manno-heptose biosynthesis pathways play important roles in constructing lipopolysaccharide of Gram-negative bacteria. Blocking the pathways is lethal or increases antibiotic susceptibility to pathogens. Therefore, the enzymes involved in the pathways are novel antibiotic drug targets. Here, we designed an efficient method to assay the whole enzymes in the pathways using mass spectrometry and screened 148 compounds. One promising lead is (?)-nyasol targeting d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC) included in the GDP-6-deoxy-α-d-manno-heptose biosynthesis pathway from Burkholderia pseudomallei. The inhibitory activity of the lead compound against HddC has been confirmed by blocking the system transferring the guanosine monophosphate (GMP) moiety to α-d-glucose-1-phosphate. (?)-Nyasol exhibits the half maximal inhibitory concentration (IC50) value of 17.6 μM. A further study is going on using (?)-nyasol derivatives to find better leads with high affinity.     

A short synthesis of d-glycero-d-manno-heptose 7-phosphate

d-glycero-d-manno-Heptopyranose 7-phosphate—an intermediate in the biosynthesis of nucleotide-activated heptoses—has been prepared in good overall yield from benzyl 5,6-dideoxy-2,3-O-isopropylidene-α-d-lyxo-(Z)-hept-5-enofuranoside by a short-step synthesis. Phosphitylation using the phosphoramidite procedure followed by in situ oxidation afforded the corresponding 7-O-phosphotriester derivative in high yield. Subsequent osmylation proceeded in good diastereoselectivity (4:1) to furnish the d-glycero-d-manno-configured derivative, which was separated from the l-glycero-l-gulo-isomer by chromatography. Hydrogenolysis led to simultaneous removal of the benzyl and isopropylidene groups and afforded the target compound in high yield, which serves as a substrate of bacterial heptose 7-phosphate kinases.     

Crystal structure of d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase from Yersinia pseudotuberculosis

The Gram-negative bacterium Yersinia pseudotuberculosis is the causative agent of yersiniosis. d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC) is the fourth enzyme of the GDP-d-glycero-α-d-manno-heptose biosynthesis pathway which is important for the virulence of the microorganism. Therefore, HddC is a potential target of antibiotics against yersiniosis. In this study, HddC from the synthesized HddC gene of Y. pseudotuberculosis has been expressed, purified, crystallized. Synchrotron X-ray data from a selenomethionine-substituted HddC crystal were also collected and its structure was determined at 2.0 Å resolution. Structure analyses revealed that it belongs to the glycosyltransferase A type superfamily members with the signature motif GXGXR for nucleotide binding. Despite of remarkable structural similarity, HddC uses GTP for catalysis instead of CTP and UTP which are used for other major family members, cytidylyltransferase and uridylyltransferase, respectively. We suggest that EXXPLGTGGA and L(S/A/G)X(S/G) motifs are probably essential to bind with GTP and a FSFE motif with substrate.     

The synthesis and degradation of rat liver and kidney fructose bisphosphatase in vivo.

Sedoheptulose 1,7-bisphosphate has been shown to be present in extracts of normal rat liver. Its concentration in this tissue, estimated by colorimetric and enzymatic assays, is in the range of 5–7 nmol/g tissue. The concentration of sedoheptulose 7-phosphate in these extracts was 110 nmol/g tissue. Also present were mono- and bisphosphate esters of d-glycero-d-ido-octulose and d-glycero-d-altro-octulose, in concentrations ranging from 1–10 nmol/g tissue. Sedoheptulose 1,7-bisphosphate may function as a reservoir for erythrose 4-phosphate. The possible origin of the eight-carbon sugars and their function are discussed.     

Isomerization ofd-glycero-d-galacto-heptose tod-manno-heptulose by selected yeasts

Selected eight yeast strains isomerized-glycero-d-galacto-heptose tod-manno-heptulose. The conversion is 7–10%. Under identical conditions, the reverse isomerization ofd-manno-heptulose tod-glycero-d-galacto-heptose ord-glycero-d-talo-heptose does not take place.     

The crystal structure of the Y140F mutant of ADP-l-glycero-d-manno-heptose 6-epimerase bound to ADP-��-d-mannose suggests a one base mechanism

Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate L ‐glycero‐D ‐manno‐heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6″ position converting ADP‐D ‐glycero‐D ‐manno‐heptose into ADP‐L ‐glycero‐D ‐manno‐heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP‐L ‐glycero‐D ‐manno‐heptose 6‐epimerase (AGME). The structure of the enzyme was known but the arrangement of the catalytic site with respect to the substrate is unclear. We now report the structure of AGME bound to a substrate mimic, ADP‐β‐D ‐mannose, which has the same stereochemical configuration as the substrate. The complex identifies the key residues and allows mechanistic insight into this novel enzyme.     

A study of inhibitors of d-glycero-β-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei as a potential antibiotic target

d-Glycero-β-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) is the fourth enzyme in the ADP‐l‐glycero‐β‐d‐manno‐heptose biosynthesis pathway producing a lipopolysaccharide core. Therefore, BpHldC is an anti-melioidosis target. Three ChemBridge compounds purchased from ChemBridge Corporation (San Diego, CA) were found to have an effective inhibitory activity on BpHldC. Interestingly, ChemBridge 7929959 was the most effective compound due to the presence of the terminal benzyl group. The enzyme kinetic study revealed that most of them show mixed type inhibitory modes against ATP and βG1P. The induced-fit docking indicated that the medium affinity of ChemBridge 7929959 is originated from its benzyl group occupying the substrate-binding pocket of BpHldC. The inhibitory role of terminal aromatic groups was proven with ChemBridge 7570508. Combined with the previous study, ChemBridge 7929959 is found to work as a dual inhibitor against both HldC and HddC. Therefore, three ChemBridge compounds can be developed as a potent anti-melioidosis agent with a novel inhibitory concept.     

Spontaneous reactions of the irreversible β-D-galactosidase inhibitor 2,6-anhydro-1-deoxy-1-diazo-D-glycero-L-manno-heptitol

2,6-Anhydro-1-deoxy-1-diazo-D-glycero-L-manno-heptitol (2) decomposes in 0.01M methanolic sodium methoxide with a half-life of approx. 18 min. Decomposition in aqueous solution is too rapid for spectrophotometric measurement. Seven products could be identified in methanolic and aqueous reaction mixtures. 2,6-Anhydro-1-deoxy-D-galacto-hept-1-enitol (6), 2,7-anhydro-1-deoxy-β-D-galacto-heptulopyranose (10), and 4-O-vinyl-D-lyxose (12) are products of rapid intramolecular reactions. The major portion consists of the direct solvolysis products 2,6-anhydro-1-O-methyl-D-glycero-L-manno-heptitol (3) and 2,6-anhydro-D-glycero-L-manno-heptitol (5).     

Isolation and phylogenetic analysis of cultivable manganese bacteria in sediments from the Arctic Ocean

Isolation, molecular identification and phylogenetic analysis were carried out to investigate the biodiversity of manganese bacteria in sediments which were collected from the Arctic Ocean during the 2nd Chinese Arctic Scientific Expedition. 21 and 19 species of cultivable strains were isolated from sediments at Stations P11 and S11, respectively, according to their distinct morphological character on the screening plate of manganese medium. Molecular identification and phylogenetic analysis showed that the cultivable manganese bacteria from Station P11 were basically composed of γ-Proteobacteria (γ subgroup of the Proteobacteria branch of the domain Bacteria) and Actinobacteria, which accounted for 86% and 14%, respectively. The isolates of γ-Proteobacteria mainly included Psychrobacter, Shewanella, Acinetobacter and Marinobacter, of which Psychrobacter was the major genus, which accounted for 67% of the γ-Proteobacteria. The cultivable manganese bacteria from Station S11 included α-Proteobacteria, γ-Proteobacteria and Flavobacteria of Bacteroides. The γ-Proteobacteria mainly included Shewanella, Marinomonas and Alteromonas. The majority of α-Proteobacteria was Sphingomonas. The phylogenetic analysis indicated that bacteria from sediments at Stations P11 and S11 had different cultivable manganese microbial communities. All tested strains had higher resistance to Mn2+, of which Marinomonas sp. S11-S-4 had the highest resistant ability.     

Reaction of methyl β-d-glycero-l-manno-heptopyranoside with cyclohexanone: Synthesis of the 4- and 6-methyl ethers of d-glycero-l-manno-heptitol

Acid-catalysed condensation of methyl β-d-glycero-l-manno-heptopyranoside with cyclohexanone yielded an approximately 3:1 mixture of the 2,3:6,7- and 2,3:4,7-di-O-cyclohexylideneheptosides (1 and 2), which could be separated either as their benzoates (3 and 4) or as their methyl ethers (5 and 6). The latter compounds afforded the 4- and 6-methyl ethers (7 and 8) of d-glycero-l-manno-heptitol.     

Synthesis of phosphorylated 3,4-branched trisaccharides corresponding to LPS inner core structures of Neisseria meningitidis and Haemophilus influenzae

2-(N-Benzyloxycarbonyl)aminoethyl 7-O-acetyl-6-O-allyl-2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-l-glycero-α-d-manno-heptopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl-(1→4)]-6,7-di-O-acetyl-2-O-benzyl-l-glycero-α-d-manno-heptopyranoside, a spacer-equipped protected derivative of the common 3,4-branched diheptoside trisaccharide structure of the lipopolysaccharide core of Neisseria meningitidis and Haemophilus influenzae has been synthesized. The protecting group pattern installed allows regioselective introduction of phosphoethanolamine residues in the 3- and 6-position of the second heptose unit in accordance with native structures. From this intermediate the 3-and 6-monophosphoethanolamine as well as the non-phosphorylated deprotected trisaccharides have been synthesized to be used in evaluation of antibody binding specificity and in investigation of the substrate specificity of glycosyl transferases involved in the biosynthesis of LPS core structures.     

Crystal structure of D‐glycero‐Β‐D‐manno‐heptose‐1‐phosphate adenylyltransferase from Burkholderia pseudomallei

The crystal structure of HldC from B. pseudomallei (BpHldC), the fourth enzyme of the heptose biosynthesis pathway, has been determined. BpHldC converts ATP and d ‐glycero‐β‐d ‐manno‐heptose‐1‐phosphate into ADP‐d ‐glycero‐β‐d ‐manno‐heptose and pyrophosphate. The crystal structure of BpHldC belongs to the nucleotidyltransferase α/β phosphodiesterase superfamily sharing a common Rossmann‐like α/β fold with a conserved T/HXGH sequence motif. The invariant catalytic key residues of BpHldC indicate that the core catalytic mechanism of BpHldC may be similar to that of other closest homologues. Intriguingly, a reorientation of the C‐terminal helix seems to guide open and close states of the active site for the catalytic reaction.     

Acid-catalyzed isopropylidenation of some heptoses

Acid-catalyzed acetonation of d-glycero-d-galacto-heptose yields solely the 1,2:3,4:6,7-tri-O-isopropylidene pyranoid derivative, whereas d-glycero-l-gluco- and d-glycero-l-manno-heptose react in the furanose form to give 1,2:5,6-(major) and 1,2:6,7-di-O-isopropylidene-d-glycero-l-gluco-heptose (minor), and 2,3:5,6-(major) and 2,3:6,7-di-O-isopropylidene-d-glycero-l-manno-heptose (minor), respectively.     

Lipid A with 2,3-diamino-2,3-dideoxy-glucose in lipopolysaccharides from slow-growing members of Rhizobiaceae and from “Pseudomonas carboxydovorans”

Lipid A's from two Bradyrhizobium species and from the phylogenetically closely related species Pseudomonas carboxydovorans were found to contain 2,3-diamino-2,3-dideoxy-glucose as lipid A backbone sugar. In contrast, three representatives of the genus Rhizobium, as well as the phylogenetically related species Agrobacterium tumefaciens, contain solely glucosamine as lipid A backbone sugar. These findings suppor independent studies on the phylogenetical relatedness based on 16S rRNA-data of the genus Bradyrhizobium with Pseudomonas carboxydovorans and Rhodopseudomonas palustris, which form a tight phylogenetical cluster and which all contain the 2,3-diamino-2,3-dideoxy-glucose-containing lipid A. The relatedness of these species to the glucosamine-containing species of the genus Rhizobium and to Agrobacterium tumefaciens is rather distant as documented by 16S rRNA studies.Abbreviations LPS lipopolysaccharide - KDO 2-keto-3-deoxyoctonic acid - GalA galacturonic acid - ld-heptose l-glycero-d-manno-heptose - dd-heptose d-glycero-d-manno-heptose - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - DAG 2,3-diamino-2,3-dideoxy-glucose     

Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells

Lipopolysaccharide (LPS) is considered as an important virulence factor of Helicobacter pylori, and contributes to infection persistence and disease severity. ADP-l-glycero-d-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia colirfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP⁺, instead of NAD⁺, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.     

A conservative distribution of tridomain NDP-heptose synthetases in actinobacteria

Heptoses are important structural components of Gram-negative bacterium cell wall and participate in bacterial colonization, infection, and immune recognition. Current knowledge of NDP-heptose originating from d-sedoheptulose 7-phosphate in Grampositive bacterium remains limited. Here, in silico analysis suggested that the special tridomain NDP-heptose synthetases with isomerase, kinase, and nucleotidyltransferase activities are conservatively distributed in Actinobacteria class of Gram-positive bacterium. Enzymatical characterization of the tridomain proteins from different strains showed that they are involved in ADP-d-glycero-β-d-manno-heptose biosynthesis despite the unexpected discovery of kinase activities deficient in some proteins. The presence of three types of NDP-heptose synthetases in Gram-positive bacterium suggests that it is also a rich source of heptoses and the heptose moieties may play important roles in vivo. Our work updates the understanding of NDP-heptose biosynthesis in Gram-positive bacterium and lays a solid foundation for further physiological function explorations.

     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Lipopolysaccharides of Escherichia coli K12 Strains That Express Cloned Genes for the Ogawa and Inaba Antigens of Vibrio cholerae O1: Identification of O-Antigenic Factors

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O-polysaccharide chain composed of an α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine (4-amino-4,6-dideoxy-D -manno-pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N-(3-deoxy-L -glycero-tetronyl)-2-O-methyl-D -perosamine at the non-reducing terminus of the O-polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co-existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O-antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O-forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2-O-methyl group at the non-reducing terminus of the α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine homopolymer that forms the O-polysaccharide chain of LPS of V. cholerae O1 (Ogawa).     

Lipopolysaccharides of Thiobacillus species containing lipid A with 2,3-diamino-2,3-dideoxyglucose

Lipopolysaccharides were isolated from two strains of Thiobacillus ferrooxidans and one strain each of Thiobacillus thiooxidans, Thiobacillus novellus and Thiobacillus sp. IFO 14570. Neutral sugars, 2-keto-3-deoxyoctonate, fatty acids and the rare 2,3-diamino-2,3-dideoxyglucose were detected in all lipopolysaccharides. Lipopolysaccharides of both T. ferrooxidans strains contained l-glycero-d-manno-heptose, whereas that of T. thiooxidans contained both l-glycero-d-manno-heptose and d-glycero-d-manno-heptose. On the other hand, heptoses were absent in lipopolysaccharides of T. novellus and Thiobacillus sp. IFO 14570. Lipid A of T. ferrooxidans and T. thiooxidans contained both glucosamine and 2,3-diamino-2,3-dideoxyglucose, in contrast, lipid A of T. novellus and the Thiobacillus sp. IFO 14570 most likely contain only 2,3-diamino-2,3-dideoxyglucose as backbone sugar. Deoxycholate polyacrylamide gel electrophoresis revealed S-type character for all lipopolysaccharides studied. The significance of the lipopolysaccharide composition for taxonomic and phylogenetic questions with regard to thiobacilli is discussed.Abbreviations DAG 2,3-diamino-2,3-dideoxyglucose - DOC sodium deoxycholate - GC gas-liquid chromatography - GC/MS gas-liquid chromatography/mass spectrometry - d,d-Heptose d-glycero-d-manno-heptose - l,d-Heptose l-glycero-d-manno-heptose - KDO 2-keto-3-deoxyoctonate - LPS lipopolysaccharide - 3-OH-14:0 3-hydroxy-tetradecanoic acid - PAGE polyacrylamide gel electrophoresis - PCP phenol-chloroform-petroleum ether