Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species

β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms.     

Tetrameric Structure of the GlfT2 Galactofuranosyltransferase Reveals a Scaffold for the Assembly of Mycobacterial Arabinogalactan

Biosynthesis of the mycobacterial cell wall relies on the activities of many enzymes, including several glycosyltransferases (GTs). The polymerizing galactofuranosyltransferase GlfT2 (Rv3808c) synthesizes the bulk of the galactan portion of the mycolyl-arabinogalactan complex, which is the largest component of the mycobacterial cell wall. We used x-ray crystallography to determine the 2.45-Å resolution crystal structure of GlfT2, revealing an unprecedented multidomain structure in which an N-terminal β-barrel domain and two primarily α-helical C-terminal domains flank a central GT-A domain. The kidney-shaped protomers assemble into a C₄-symmetric homotetramer with an open central core and a surface containing exposed hydrophobic and positively charged residues likely involved with membrane binding. The structure of a 3.1-Å resolution complex of GlfT2 with UDP reveals a distinctive mode of nucleotide recognition. In addition, models for the binding of UDP-galactofuranose and acceptor substrates in combination with site-directed mutagenesis and kinetic studies suggest a mechanism that explains the unique ability of GlfT2 to generate alternating β-(1→5) and β-(1→6) glycosidic linkages using a single active site. The topology imposed by docking a tetrameric assembly onto a membrane bilayer also provides novel insights into aspects of processivity and chain length regulation in this and possibly other polymerizing GTs.     

Plant O-Hydroxyproline Arabinogalactans Are Composed of Repeating Trigalactosyl Subunits with Short Bifurcated Side Chains

Classical arabinogalactan proteins partially defined by type II O-Hyp-linked arabinogalactans (Hyp-AGs) are structural components of the plant extracellular matrix. Recently we described the structure of a small Hyp-AG putatively based on repetitive trigalactosyl subunits and suggested that AGs are less complex and varied than generally supposed. Here we describe three additional AGs with similar subunits. The Hyp-AGs were isolated from two different arabinogalactan protein fusion glycoproteins expressed in tobacco cells; that is, a 22-residue Hyp-AG and a 20-residue Hyp-AG, both isolated from interferon α2b-(Ser-Hyp)₂₀, and a 14-residue Hyp-AG isolated from (Ala-Hyp)₅₁-green fluorescent protein. We used NMR spectroscopy to establish the molecular structure of these Hyp-AGs, which share common features: (i) a galactan main chain composed of two 1→3 β-linked trigalactosyl blocks linked by a β-1→6 bond; (ii) bifurcated side chains with Ara, Rha, GlcUA, and a Gal 6-linked to Gal-1 and Gal-2 of the main-chain trigalactosyl repeats; (iii) a common side chain structure composed of up to six residues, the largest consisting of an α-l-Araf-(1→5)-α-l-Araf-(1→3)-α-l-Araf-(1→3- unit and an α-l-Rhap-(1→4)-β-d-GlcUAp-(1→6)-unit, both linked to Gal. The conformational ensemble obtained by using nuclear Overhauser effect data in structure calculations revealed a galactan main chain with a reverse turn involving the β-1→6 link between the trigalactosyl blocks, yielding a moderately compact structure stabilized by H-bonds.     

Expression, purification and characterisation of soluble GlfT and the identification of a novel galactofuranosyltransferase Rv3782 involved in priming GlfT-mediated galactan polymerisation in Mycobacterium tuberculosis

The arabinogalactan (AG) component of the mycobacterial cell wall is an essential branched polysaccharide which tethers mycolic acids (m) to peptidoglycan (P), forming the mAGP complex. Much interest has been focused on the biosynthetic machinery involved in the production of this highly impermeable shield, which is the target for numerous anti-tuberculosis agents. The galactan domain of AG is synthesised via a bifunctional galactofuranosyltransferase (GlfT), which utilises UDP-Galf as its high-energy substrate. However, it has proven difficult to study the protein in its recombinant form due to difficulties in recovering pure soluble protein using standard expression systems. Herein, we describe the effects of glfT co-induction with a range of chaperone proteins, which resulted in an appreciable yield of soluble protein at 5 mg/L after a one-step purification procedure. We have shown that this purified enzyme transfers [¹⁴C]Galf to a range of both β(1 → 5) and β(1 → 6) linked digalactofuranosyl neoglycolipid acceptors with a distinct preference for the latter. Ligand binding studies using intrinsic tryptophan fluorescence have provided supporting evidence for the apparent preference of this enzyme to bind the β(1 → 6) disaccharide acceptor. However, we could not detect binding or galactofuranosyltransferase activity with an n-octyl β-d-Gal-(1 → 4)-α-l-Rha acceptor, which mimics the reducing terminus of galactan in the mycobacterial cell wall. Conversely, after an extensive bioinformatics analysis of the H37Rv genome, further cloning, expression and functional analysis of the Rv3792 open reading frame indicates that this protein affords galactofuranosyltransferase activity against such an acceptor and paves the way for a better understanding of galactan biosynthesis in Mycobacterium tuberculosis.     

STD-NMR studies of two acceptor substrates of GlfT2, a galactofuranosyltransferase from Mycobacterium tuberculosis: Epitope mapping studies

The major structural component of the mycobacterial cell wall, the mycolyl–arabinogalactan–peptidoglycan complex, possesses a galactan core composed of approximately 30 galactofuranosyl (Galf) resides attached via alternating β-(1→6) and β-(1→5) linkages. Recent studies have shown that the entire galactan is synthesized by two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. We report here saturation transfer difference (STD) NMR studies GlfT2 using two trisaccharide acceptor substrates, β-d-Galf-(1→6)-β-d-Galf-(1→5)-β-d-Galf-O(CH₂)₇CH₃ (2) and β-d-Galf-(1→5)-β-d-Galf-(1→6)-β-d-Galf-O(CH₂)₇CH₃ (3), as well as the donor substrate for the enzyme, UDP-Galf. Epitope mapping demonstrated a greater enhancement toward the ‘reducing’ ends of both trisaccharides, and that UDP-galactofuranose (UDP-Galf) made more intimate contacts through its nucleotide moiety. This observation is consistent with the greater flexibility required within the active site of the reaction between the growing polymer acceptor and the UDP-Galf donor. The addition of UDP-Galf to either 2 or 3 in the presence of GlfT2 generated a tetrasaccharide product, indicating that the enzyme was catalytically active.     

Structure and Molecular Characterization of Streptococcus pneumoniae Capsular Polysaccharide 10F by Carbohydrate Engineering in Streptococcus oralis

Although closely related at the molecular level, the capsular polysaccharide (CPS) of serotype 10F Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of Streptococcus oralis C104 have distinct ecological roles. CPS prevents phagocytosis of pathogenic S. pneumoniae, whereas RPS of commensal S. oralis functions as a receptor for lectin-like adhesins on other members of the dental plaque biofilm community. Results from high resolution NMR identified the recognition region of S. oralis RPS (i.e. Galfβ1–6GalNAcβ1–3Galα) in the hexasaccharide repeat of S. pneumoniae CPS10F. The failure of this polysaccharide to support fimbriae-mediated adhesion of Actinomyces naeslundii was explained by the position of Galf, which occurred as a branch in CPS10F rather than within the linear polysaccharide chain, as in RPS. Carbohydrate engineering of S. oralis RPS with wzy from S. pneumoniae attributed formation of the Galf branch in CPS10F to the linkage of adjacent repeating units through sub terminal GalNAc in Galfβ1–6GalNAcβ1–3Galα rather than through terminal Galf, as in RPS. A gene (wcrD) from serotype 10A S. pneumoniae was then used to engineer a linear surface polysaccharide in S. oralis that was identical to RPS except for the presence of a β1–3 linkage between Galf and GalNAcβ1–3Galα. This polysaccharide also failed to support adhesion of A. naeslundii, thereby establishing the essential role of β1–6-linked Galf in recognition of adjacent GalNAcβ1–3Galα in wild-type RPS. These findings, which illustrate a molecular approach for relating bacterial polysaccharide structure to function, provide insight into the possible evolution of S. oralis RPS from S. pneumoniae CPS.     

Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme: INSIGHTS OF N-TERMINAL β-SANDWICH IN SUBSTRATE SPECIFICITY AND ENZYMATIC ACTIVITY*

The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an α-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry {"type":"entrez-protein","attrs":{"text":"Q10625","term_id":"1707934","term_text":"Q10625"}}Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1→4 bond and making a new 1→6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-Å resolution. MtbGlgBWT contains four domains: N1 β-sandwich, N2 β-sandwich, a central (β/α)₈ domain that houses the catalytic site, and a C-terminal β-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) MtbΔ108GlgB protein. The N1 β-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 β-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and MtbΔ108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1→4 bond breakage) and isomerization (1→6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and MtbΔ108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (ECΔ112GlgB).     

Highly Hydrolytic Reuteransucrase from Probiotic Lactobacillus reuteri Strain ATCC 55730

Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the α-(1→4) glucosidic type (~70%). This reuteran also contains α-(1→6)- linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. The LB BIO glucansucrase gene (gtfO) was cloned and expressed in Escherichia coli, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing α-(1→4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of α-(1→4) linkages reported to date.     

Characterization of the oligosaccharides from lipid-linked oligosaccharides of mung bean seedlings

Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseolus aureus) seedlings in the presence of GDP-[¹⁴C] mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P-4 columns. Five different oligosaccharides were purified in this way. Based on their relative elution constants (K_d) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man₇GlcNAc₂, Man₅GlcNAc₂, Man₃GlcNAc₂, Man₂GlcNAc₂, and ManGlcNAc₂. These oligosaccharides were treated with endoglucosaminidase H and α- and β-mannosidase, and the products were examined on Biogel P-4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB³H₄ reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as follows: E, Manβ-GlcNAc-GlcNAc; D, Manα1→3Manβ-GlcNAc-GlcNAc; C, Manα1→2Manα1→3Manβ-GlcNAc-GlcNAc; B, Manα1→2Manα1→2Manα1→ 3(Manα1→6)Manβ-GlcNAc-GlcNAc; and A, Manα1→2Manα1→ 2Manα1→3(Manα1→ [Manα1→6]Manα1→6) Manβ-GlcNAc-GlcNAc. The synthesis of the Man₇GlcNAc₂ was greatly diminished when tunicamycin (10 μg/ml) was added to the incubation mixtures.     

Structure of the Type IX Group B Streptococcus Capsular Polysaccharide and Its Evolutionary Relationship with Types V and VII

The Group B Streptococcus capsular polysaccharide type IX was isolated and purified, and the structure of its repeating unit was determined. Type IX capsule →4)[NeupNAc-α-(2→3)-Galp-β-(1→4)-GlcpNAc-β-(1→6)]-β-GlcpNAc-(1→4)-β-Galp-(1→4)-β-Glcp-(1→ appears most similar to types VII and V, although it contains two GlcpNAc residues. Genetic analysis identified differences in cpsM, cpsO, and cpsI gene sequences as responsible for the differentiation between the three capsular polysaccharide types, leading us to hypothesize that type V emerged from a recombination event in a type IX background.     

The Formation of beta, 1 --> 4 Glucan from UDP-alpha-d-Glucose Catalyzed by a Phaseolus aureus Enzyme

Particulate enzyme preparations from Phaseolus aureus hypocotyls catalyze the formation of an alkali insoluble β, 1 → 4 linked [¹⁴C]-glucan using UDP-α-d [¹⁴C]-glucose as substrate. Particulate enzymes prepared from root tissue also catalyzed the production of β, 1 → 4 glucan. UDP-β-d-[¹⁴C]-glucose would not serve as a substrate for these enzymes. The presence or absence of β, 1 → 4 glucan synthetase activity was independent of tissue source, substrate concentration, or homogenization method.     

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2′:2,1′-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2′:2,1′-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).     

Domain Organization of the Polymerizing Mannosyltransferases Involved in Synthesis of the Escherichia coli O8 and O9a Lipopolysaccharide O-antigens

The Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional α-(1→2)-, α-(1→3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (α-(1→2), α-(1→3), and β-(1→2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdA^O9a contains two separable and functionally active domains, whereas WbdA^O8 possesses three. In WbdC^O9a and WbdB^O9a, substitution of the first Glu of the EX₇E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX₇E motif of the N-terminal WbdA^O9a domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdA^O9a domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only α-(1→2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdA^O9a possesses α-(1→2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases.     

Metabolism of the reserve polysaccharide of Streptococcus mitis. Properties of alpha-(1-->6)-glucosidase, its separation from transglucosylase, and the action of the two enzymes on branched oligosaccharides

1. An α-(1→6)-glucosidase has been separated from cell extracts of Streptococcus mitis. The enzyme was freed from transglucosylase by adsorption of the latter on retrograded amylose. 2. The enzyme was detected in five of the six strains of S. mitis that were studied; α-(1→6)-glucosidase was not found in strain RB1633, a strain that did not store polysaccharide. 3. The glucosidase could act on compounds in which α-glucose is joined through an α-(1→6)-bond to either a maltosaccharide or an isomaltosaccharide. 6²-α-Glucosylmaltose (panose) and 6³-α-glucosylmaltotriose were hydrolysed more rapidly and isomaltodextrins more slowly than isomaltose. 4. Transferring activity towards isomaltose and panose was appreciable when the concentration of substrate was 2% or higher. 5. The enzyme had no action on α-(1→4)-glucosidic linkages. 6-α-Maltodextrinylglucoses were hydrolysed only after transglucosylase action had attenuated them to isomaltose.     

Lysis of Yeast Cell Walls: Glucanases from Bacillus circulans WL-12

Endo-β-(1 → 3)- and endo-β-(1 → 6)-glucanases are produced in high concentration in the culture fluid of Bacillus circulans WL-12 when grown in a mineral medium with bakers' yeast cell walls as the sole carbon source. Much lower enzyme levels were found when laminarin, pustulan, or mannitol was the substrate. The two enzyme activities were well separated during Sephadex G-100 chromatography. The endo-β-(1 → 3)-glucanase was further purified by diethylaminoethyl-cellulose and hydroxyapatite chromatography, whereas the endo-β-(1 → 6)-glucanase could be purified further by diethylamino-ethyl-cellulose and carboxymethyl cellulose chromatography. The endo-β-(1 → 3)-glucanase was specific for the β-(1 → 3)-glucosidic bond, but it did not hydrolyze laminaribiose; laminaritriose was split very slowly. β-(1 → 4)-Bonds in oat glucan in which the glucosyl moiety is substituted in the 3-position were also cleaved. The kinetics of laminarin hydrolysis (optimum pH 5.0) were complex but appeared to follow Michaelis-Menten theory, especially at the lower substrate concentrations. Glucono-δ-lactone was a noncompetitive inhibitor and Hg²⁺ inhibited strongly. The enzyme has no metal ion requirements or essential sulfhydryl groups. The purified β-(1 → 6)-glucanase has an optimum pH of 5.5, and its properties were studied in less detail. In contrast to the crude culture fluid, the two purified β-glucanases have only a very limited hydrolytic action on cell wall of either bakers' yeast or of Schizosaccharomyces pombe. Although our previous work had assumed that the two glucanases studied here are responsible for cell wall lysis, it now appears that the culture fluid contains in addition a specific lytic enzyme which is eliminated during the extensive purification process.     

DEMONSTRATION OF A FIBRILLAR COMPONENT IN THE CELL WALL OF THE YEAST SACCHAROMYCES CEREVISIAE AND ITS CHEMICAL NATURE

The ultrastructure of isolated cell walls of Saccharomyces cerevisiae from the log and stationary phases of growth was studied after treatment with the following enzymes: purified endo-β-(1 → 3)-glucanase and endo-β-(1 → 6)-glucanase produced by Bacillus circulans; purified exo-β-glucanase and endo-β-(1 → 3)-glucanase produced by Schizosaccharomyces versatilis; commercial Pronase. While exo-β-glucanase from S. versatilis had no electron microscopically detectable effect on the walls, Pronase removed part of the external amorphous wall material disclosing an amorphous wall layer in which fibrils were indistinctly visible. Amorphous wall material was completely removed by the effect of either endo-β-(1 → 3)- or endo-β-(1 → 6)-glucanase of B. circulans or by a mixture of the two enzymes. As a result of these treatments a continuous fibrillar component appeared, composed of densely interwoven microfibrils resisting further action by both of the B. circulans enzymes. The fibrillar wall component was also demonstrated in untreated cell walls by electron microscopy after negative staining. Because of the complete disappearance of the fibrils following treatment with the S. versatilis endo-β-(1 → 3)-glucanase it can be concluded that this fibrillar component is composed of β-(1 → 3)-linked glucan. Bud scars were the only wall structures resistant to the effect of the latter enzyme.     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization

A (1→3)-β-glucan synthase has been isolated from petiole tissue of sugar beet (Beta vulgaris L.). Enzyme activity is associated with a membrane fraction with a density of 1.03 grams per cubic centimeter when subjected to isopycnic density gradient centrifugation in Percoll. The reaction product was determined to be a linear (1→3)-β-glucan by methylation analysis and by glucanase digestion. (1→3)-β-Glucan synthase activity is markedly stimulated by Ca²⁺; activation is half-maximal at about 50 micromolar Ca²⁺ and is nearly saturated at 100 micromolar. Other divalent cations tested, Mg²⁺, Mn²⁺, and Sr²⁺, also stimulate enzyme activity but are less effective. Enzyme activity was also stimulated up to 12-fold by β-glucosides. Sirofluor, the fluorochrome from aniline blue, inhibited enzyme activity 95% when included at 1 millimolar. The enzyme was solubilized in Zwittergent 3-14; 85% of total enzyme activity was solubilized in 0.03% detergent and the optimal detergent-to-protein ratio was 0.3 at 3 milligrams per milliliter protein.     

Characterization of the Corynebacterium glutamicum ΔpimB′ ΔmgtA Double Deletion Mutant and the Role of Mycobacterium tuberculosis Orthologues Rv2188c and Rv0557 in Glycolipid Biosynthesis

In this study, utilizing a Corynebacterium glutamicum ΔpimB′ ΔmgtA double deletion mutant, we unequivocally assign the in vivo functions of Rv2188c as an Ac₁PIM₁:mannosyltransferase (originally termed PimB′_Mt [Mycobacterium tuberculosis PimB′]) and Rv0557 as a GlcAGroAc₂:mannosyltransferase (originally termed PimB_Mt), which we have reassigned as PimB_Mt and MgtA_Mt, respectively, in Mycobacterium tuberculosis.The current model of mycobacterial phosphatidyl-myo-inositol mannoside (PIM) biosynthesis, supported by biochemical and genetic studies, follows a linear pathway from phosphatidylinositol (PI) → Ac₁PIM₂ → Ac₁PIM₄ → Ac₁PIM₆ (4, 17, 19) as shown in Fig. ​Fig.1.1. In this pathway, mycobacterial PI is glycosylated by an α-mannopyranosyl residue at the 2-OH position of inositol, followed by the acylation and mannosylation at the 6-OH position of PI to form Ac₁PIM₂ (3), which is further mannosylated to form Ac₁PIM₄ and Ac₁PIM₆, extending the 6-OH position of Ac₁PIM₂ (19).Open in a separate windowFIG. 1.Glycolipid biosynthetic pathways in Corynebacterineae. (A) PIM synthesis in M. tuberculosis; (B) PIMs; (C) ManGlcAGroAc₂ synthesis in C. glutamicum.In view of the identification of genes involved in PIM, lipomannan (LM), and lipoarabinomannan (LAM) biosynthesis, Schaeffer et al. (22) proposed Rv0557 as an α-d-mannose-α-(1→6)-phosphatidyl-myo-inositol-mannosyltransferase that transfers mannose from GDP-Man to Ac₁PIM₁ to form Ac₁PIM₂, a precursor of the immunomodulatory lipoglycans LM and LAM (4, 17). The study was based on a cell-free assay using GDP[¹⁴C]Man, Ac₁PIM_1, Mycobacterium smegmatis membranes, and/or partially purified recombinant Rv0557. On the basis of these in vitro studies, Rv0557 was assigned as PimB_Mt (Mycobacterium tuberculosis PimB) in the synthesis of Ac₁PIM₂. However, on the disruption of Rv0557 in Mycobacterium tuberculosis, PIM biosynthesis remains unaffected (G. S. Besra and L. S. Schlesinger, unpublished data), suggesting that either gene duplication or Rv0557 performed another function in M. tuberculosis. Interestingly, in a recent study, Rv0557 was also shown to be involved in the biosynthesis of 1,2-di-O-C₁₆/C_18:1-(α-d-mannopyranosyl)-(1→4)-(α-d-glucopyranosylu- ronic acid)-(1→3)-glycerol (ManGlcAGroAc₂) and an LM-like molecule in Corynebacterium glutamicum and was termed MgtA_Mt (M. tuberculosis MgtA) (25). More recently, Rv2188c was also proposed to be involved in the synthesis of Ac₁PIM₂ as the second α-d-mannose-α-(1→6)-phosphatidyl-myo-inositol-mannosyl transferase (termed PimB′_Mt) (13, 16), which has augmented ongoing confusion in the field. Due to the essentiality of M. tuberculosis PIM biosynthesis (3) in this study, we have generated C. glutamicum ΔpimB′ ΔmgtA, deficient in pimB′_Cg and mgtA_Cg (C. glutamicum pimB′ and mgtA) and subsequently overexpressed Rv2188c and Rv0557 individually to identify their true in vivo and in vitro biochemical activities.     

Studies on β-glucanases. Some properties of a bacterial endo-β-(1→3)-glucanase system

A commercial enzyme preparation, originally obtained from a Flavobacterium(Cytophaga), was fractionated by continuous electrophoresis, giving a protein fraction which hydrolysed laminarin, carboxymethylpachyman, barley β-glucan, lichenin and cellodextrin in random fashion. This enzymic activity was not very stable. Ion-exchange chromatography and molecular-sieve chromatography on Bio-Gel P-60 showed that this activity was due to two specific β-glucanases, an endo-β-(1→3)-glucanase and an endo-β-(1→4)-glucanase. The two enzymes occur in both high- and low-molecular-weight forms, the latter endo-β-(1→3)-glucanase having a molecular weight of about 16000.