Structural Insights into the Specificity of Xyn10B from Paenibacillus barcinonensis and Its Improved Stability by Forced Protein Evolution

Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (β/α)₈ barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the β/α motifs. One of these loops -L7- located at the β7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His²⁴⁹–Glu²⁵⁰, which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme.     

Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis

Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade β-propeller fold linked to a C-terminal β-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (−1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the −1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.     

Crystal Structures of β-Primeverosidase in Complex with Disaccharide Amidine Inhibitors

β-Primeverosidase (PD) is a disaccharide-specific β-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. PD hydrolyzes β-primeveroside (6-O-β-d-xylopyranosyl-β-d-glucopyranoside) at the β-glycosidic bond of primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones. PD only accepts primeverose as the glycone substrate, but broadly accepts various aglycones, including 2-phenylethanol, benzyl alcohol, linalool, and geraniol. We determined the crystal structure of PD complexes using highly specific disaccharide amidine inhibitors, N-β-primeverosylamidines, and revealed the architecture of the active site responsible for substrate specificity. We identified three subsites in the active site: subsite −2 specific for 6-O-β-d-xylopyranosyl, subsite −1 well conserved among β-glucosidases and specific for β-d-glucopyranosyl, and wide subsite +1 for hydrophobic aglycone. Glu-470, Ser-473, and Gln-477 act as the specific hydrogen bond donors for 6-O-β-d-xylopyranosyl in subsite −2. On the other hand, subsite +1 was a large hydrophobic cavity that accommodates various aromatic aglycones. Compared with aglycone-specific β-glucosidases of the glycoside hydrolase family 1, PD lacks the Trp crucial for aglycone recognition, and the resultant large cavity accepts aglycone and 6-O-β-d-xylopyranosyl together. PD recognizes the β-primeverosides in subsites −1 and −2 by hydrogen bonds, whereas the large subsite +1 loosely accommodates various aglycones. The glycone-specific activity of PD for broad aglycone substrates results in selective and multiple release of temporally stored alcoholic volatile aglycones of β-primeveroside.     

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase

ΔN₁₂₃-glucan-binding domain-catalytic domain 2 (ΔN₁₂₃-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN₁₂₃-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite −1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN₁₂₃-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN₁₂₃-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN₁₂₃-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.     

Increasing pCO2 correlates with low concentrations of intracellular dimethylsulfoniopropionate in the sea anemone Anemonia viridis

Marine anthozoans maintain a mutualistic symbiosis with dinoflagellates that are prolific producers of the algal secondary metabolite dimethylsulfoniopropionate (DMSP), the precursor of the climate-cooling trace gas dimethyl sulfide (DMS). Surprisingly, little is known about the physiological role of DMSP in anthozoans and the environmental factors that regulate its production. Here, we assessed the potential functional role of DMSP as an antioxidant and determined how future increases in seawater pCO₂ may affect DMSP concentrations in the anemone Anemonia viridis along a natural pCO₂ gradient at the island of Vulcano, Italy. There was no significant difference in zooxanthellae genotype and characteristics (density of zooxanthellae, and chlorophyll a) as well as protein concentrations between anemones from three stations along the gradient, V1 (3232 μatm CO₂), V2 (682 μatm) and control (463 μatm), which indicated that A. viridis can acclimate to various seawater pCO₂. In contrast, DMSP concentrations in anemones from stations V1 (33.23 ± 8.30 fmol cell⁻¹) and V2 (34.78 ± 8.69 fmol cell⁻¹) were about 35% lower than concentrations in tentacles from the control station (51.85 ± 12.96 fmol cell⁻¹). Furthermore, low tissue concentrations of DMSP coincided with low activities of the antioxidant enzyme superoxide dismutase (SOD). Superoxide dismutase activity for both host (7.84 ± 1.37 U·mg⁻¹ protein) and zooxanthellae (2.84 ± 0.41 U·mg⁻¹ protein) at V1 was 40% lower than at the control station (host: 13.19 ± 1.42; zooxanthellae: 4.72 ± 0.57 U·mg⁻¹ protein). Our results provide insight into coastal DMSP production under predicted environmental change and support the function of DMSP as an antioxidant in symbiotic anthozoans.     

Crystal Structure and Mutational Analysis of Isomalto-dextranase,a Member of Glycoside Hydrolase Family 27

Arthrobacter globiformis T6 isomalto-dextranase (AgIMD) is an enzyme that liberates isomaltose from the non-reducing end of a polymer of glucose, dextran. AgIMD is classified as a member of the glycoside hydrolase family (GH) 27, which comprises mainly α-galactosidases and α-N-acetylgalactosaminidases, whereas AgIMD does not show α-galactosidase or α-N-acetylgalactosaminidase activities. Here, we determined the crystal structure of AgIMD. AgIMD consists of the following three domains: A, C, and D. Domains A and C are identified as a (β/α)₈-barrel catalytic domain and an antiparallel β-structure, respectively, both of which are commonly found in GH27 enzymes. However, domain A of AgIMD has subdomain B, loop-1, and loop-2, all of which are not found in GH27 human α-galactosidase. AgIMD in a complex with trisaccharide panose shows that Asp-207, a residue in loop-1, is involved in subsite +1. Kinetic parameters of the wild-type and mutant enzymes for the small synthetic saccharide p-nitrophenyl α-isomaltoside and the polysaccharide dextran were compared, showing that Asp-207 is important for the catalysis of dextran. Domain D is classified as carbohydrate-binding module (CBM) 35, and an isomaltose molecule is seen in this domain in the AgIMD-isomaltose complex. Domain D is highly homologous to CBM35 domains found in GH31 and GH66 enzymes. The results here indicate that some features found in GH13, -31, and -66 enzymes, such as subdomain B, residues at the subsite +1, and the CBM35 domain, are also observed in the GH27 enzyme AgIMD and thus provide insights into the evolutionary relationships among GH13, -27, -31, -36, and -66 enzymes.     

Depletion of Vitamin E Increases Amyloid �� Accumulation by Decreasing Its Clearances from Brain and Blood in a Mouse Model of Alzheimer Disease

Increased oxidative damage is a prominent and early feature in Alzheimer disease. We previously crossed Alzheimer disease transgenic (APPsw) model mice with α-tocopherol transfer protein knock-out (Ttpa^−/−) mice in which lipid peroxidation in the brain was significantly increased. The resulting double-mutant (Ttpa^−/−APPsw) mice showed increased amyloid β (Aβ) deposits in the brain, which was ameliorated with α-tocopherol supplementation. To investigate the mechanism of the increased Aβ accumulation, we here studied generation, degradation, aggregation, and efflux of Aβ in the mice. The clearance of intracerebral-microinjected ¹²⁵I-Aβ_1–40 from brain was decreased in Ttpa^−/− mice to be compared with wild-type mice, whereas the generation of Aβ was not increased in Ttpa^−/−APPsw mice. The activity of an Aβ-degrading enzyme, neprilysin, did not decrease, but the expression level of insulin-degrading enzyme was markedly decreased in Ttpa^−/− mouse brain. In contrast, Aβ aggregation was accelerated in Ttpa^−/− mouse brains compared with wild-type brains, and well known molecules involved in Aβ transport from brain to blood, low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein, were up-regulated in the small vascular fraction of Ttpa^−/− mouse brains. Moreover, the disappearance of intravenously administered ¹²⁵I-Aβ_1–40 was decreased in Ttpa^−/− mice with reduced translocation of LRP-1 in the hepatocytes. These results suggest that lipid peroxidation due to depletion of α-tocopherol impairs Aβ clearances from the brain and from the blood, possibly causing increased Aβ accumulation in Ttpa^−/−APPsw mouse brain and plasma.     

Novel reaction of succinyl coenzyme A (Succinyl-CoA) synthetase: activation of 3-sulfinopropionate to 3-sulfinopropionyl-CoA in Advenella mimigardefordensis strain DPN7T during degradation of 3,3'-dithiodipropionic acid

The sucCD gene of Advenella mimigardefordensis strain DPN7^T encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3′-dithiodipropionic acid (DTDP) degradation was observed in Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7^T disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ΔsucCD. These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7^T activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCD_Am) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCD_Am is Mg²⁺ or Mn²⁺ dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate (V_max = 9.85 ± 0.14 μmol min⁻¹ mg⁻¹, K_m = 0.143 ± 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% (V_max = 0.12 ± 0.01 μmol min⁻¹ mg⁻¹) and the affinity was 6-fold lower (K_m = 0.818 ± 0.046 mM). Based on the present results, we conclude that SucCD_Am is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP.     

Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β-d-glucosyl, β-d-xylosyl, β-d-mannosyl and β-d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β-d-N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β-d-N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity. Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209. Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β-d-N-acetylglucosaminyl residue at +1 subsite. The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors.     

Atherosclerosis in ApoE-deficient mice progresses independently of the NLRP3 inflammasome

The interleukin-1 (IL-1) family of cytokines has been implicated in the pathogenesis of atherosclerosis in previous studies. The NLRP3 inflammasome has recently emerged as a pivotal regulator of IL-1β maturation and secretion by macrophages. Little is currently known about a possible role for the NLRP3 inflammasome in atherosclerosis progression in vivo. We generated ApoE−/− Nlrp3−/−, ApoE−/− Asc−/− and ApoE−/− caspase-1−/− double-deficient mice, fed them a high-fat diet for 11 weeks and subsequently assessed atherosclerosis progression and plaque phenotype. No differences in atherosclerosis progression, infiltration of plaques by macrophages, nor plaque stability and phenotype across the genotypes studied were found. Our results demonstrate that the NLRP3 inflammasome is not critically implicated in atherosclerosis progression in the ApoE mouse model.     

Stability of thioester intermediates in ubiquitin‐like modifications

Ubiquitin-like modifications are important mechanisms in cellular regulation, and are carried out through several steps with reaction intermediates being thioester conjugates of ubiquitin-like proteins with E1, E2, and sometimes E3. Despite their importance, a thorough characterization of the intrinsic stability of these thioester intermediates has been lacking. In this study, we investigated the intrinsic stability by using a model compound and the Ubc9∼SUMO-1 thioester conjugate. The Ubc9∼SUMO-1 thioester intermediate has a half life of approximately 3.6 h (hydrolysis rate k = 5.33 ± 2.8 ×10⁻⁵ s⁻¹), and the stability decreased slightly under denaturing conditions (k = 12.5 ± 1.8 × 10⁻⁵ s⁻¹), indicating a moderate effect of the three-dimensional structural context on the stability of these intermediates. Binding to active and inactive E3, (RanBP2) also has only a moderate effect on the hydrolysis rate (13.8 ± 0.8 × 10⁻⁵ s⁻¹ for active E3 versus 7.38 ± 0.7 × 10⁻⁵ s⁻¹ for inactive E3). The intrinsically high stability of these intermediates suggests that unwanted thioester intermediates may be eliminated enzymatically, such as by thioesterases, to regulate their intracellular abundance and trafficking in the control of ubiquitin-like modifications.     

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase

We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10–13 nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (ΔG° approximately −6.7 kcal/mol). The relative contribution of the oxoG unit of DNA (ΔG° approximately −3.3 kcal/mol) together with other specific interactions (ΔG° approximately −0.7 kcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the k_cat term instead; the rate increases by 6–7 orders of magnitude for cognate DNA as compared with non-cognate one. The k_cat values for substrates of different sequences correlate with the DNA twist, while the K_M values correlate with ΔG° of the DNA fragments surrounding the lesion (position from −6 to +6). The functions for predicting the K_M and k_cat values for different sequences containing oxoG were found.     

Structural elucidation of dextran degradation mechanism by streptococcus mutans dextranase belonging to glycoside hydrolase family 66

Dextranase is an enzyme that hydrolyzes dextran α-1,6 linkages. Streptococcus mutans dextranase belongs to glycoside hydrolase family 66, producing isomaltooligosaccharides of various sizes and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln¹⁰⁰ to Ile⁷³² of S. mutans dextranase, devoid of its N- and C-terminal variable regions, was determined at 1.6 Å resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like β-sandwich fold; domain A contained the enzyme''s catalytic module, comprising a (β/α)₈-barrel; and domain C formed a β-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined, and, in the enzyme-isomaltotriose complex structure, the bound isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites, −4 to −1, in the catalytic cleft. The complexed structure with 4′,5′-epoxypentyl-α-d-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp³⁸⁵ side chain. These structures collectively indicated that Asp³⁸⁵ was the catalytic nucleophile and that Glu⁴⁵³ was the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzyme''s catalytic machinery.     

HP1α mediates defective heterochromatin repair and accelerates senescence in Zmpste24-deficient cells

Heterochromatin protein 1 (HP1) interacts with various proteins, including lamins, to play versatile functions within nuclei, such as chromatin remodeling and DNA repair. Accumulation of prelamin A leads to misshapen nuclei, heterochromatin disorganization, genomic instability, and premature aging in Zmpste24-null mice. Here, we investigated the effects of prelamin A on HP1α homeostasis, subcellular distribution, phosphorylation, and their contribution to accelerated senescence in mouse embryonic fibroblasts (MEFs) derived from Zmpste24^−/− mice. The results showed that the level of HP1α was significantly increased in Zmpste24^−/− cells. Although prelamin A interacted with HP1α in a manner similar to lamin A, HP1α associated with the nuclease-resistant nuclear matrix fraction was remarkably increased in Zmpste24^−/− MEFs compared with that in wild-type littermate controls. In wild-type cells, HP1α was phosphorylated at Thr50, and the phosphorylation was maximized around 30 min, gradually dispersed 2 h after DNA damage induced by camptothecin. However, the peak of HP1α phosphorylation was significantly compromised and appeared until 2 h, which is correlated with the delayed maximal formation of γ-H2AX foci in Zmpste24^−/− MEFs. Furthermore, knocking down HP1α by siRNA alleviated the delayed DNA damage response and accelerated senescence in Zmpste24^−/− MEFs, evidenced by the rescue of the delayed γ-H2AX foci formation, downregulation of p16, and reduction of senescence-associated β-galactosidase activity. Taken together, these findings establish a functional link between prelamin A, HP1α, chromatin remodeling, DNA repair, and early senescence in Zmpste24-deficient mice, suggesting a potential therapeutic strategy for laminopathy-based premature aging via the intervention of HP1α.     

Characterization of the Functional Roles of Amino Acid Residues in Acceptor-binding Subsite +1 in the Active Site of the Glucansucrase GTF180 from Lactobacillus reuteri 180

α-Glucans produced by glucansucrase enzymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure of glucansucrase GTF180-ΔN from Lactobacillus reuteri 180 in complex with the acceptor substrate maltose, we identified several residues (Asp-1028 and Asn-1029 from domain A, as well as Leu-938, Ala-978, and Leu-981 from domain B) near subsite +1 that may be critical for linkage specificity determination, and we investigated these by random site-directed mutagenesis. First, mutants of Ala-978 (to Leu, Pro, Phe, or Tyr) and Asp-1028 (to Tyr or Trp) with larger side chains showed reduced degrees of branching, likely due to the steric hindrance by these bulky residues. Second, Leu-938 mutants (except L938F) and Asp-1028 mutants showed altered linkage specificity, mostly with increased (α1→6) linkage synthesis. Third, mutation of Leu-981 and Asn-1029 significantly affected the transglycosylation reaction, indicating their essential roles in acceptor substrate binding. In conclusion, glucansucrase product specificity is determined by an interplay of domain A and B residues surrounding the acceptor substrate binding groove. Residues surrounding the +1 subsite thus are critical for activity and specificity of the GTF180 enzyme and play different roles in the enzyme functions. This study provides novel insights into the structure-function relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailor-made α-glucans.     

4,6-α-glucanotransferase, a novel enzyme that structurally and functionally provides an evolutionary link between glycoside hydrolase enzyme families 13 and 70

Lactobacillus reuteri 121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the α-d-glucan reuteran, an exopolysaccharide. Upstream of gtfA lies another putative glucansucrase gene, designated gtfB. Previously, we have shown that the purified recombinant GTFB protein/enzyme is inactive with sucrose. Various homologs of gtfB are present in other Lactobacillus strains, including the L. reuteri type strain, DSM 20016, the genome sequence of which is available. Here we report that GTFB is a novel α-glucanotransferase enzyme with disproportionating (cleaving α1→4 and synthesizing α1→6 and α1→4 glycosidic linkages) and α1→6 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-α-glucanotransferase). Characterization of the types of compounds synthesized from maltoheptaose by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), methylation analysis, and 1-dimensional ¹H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more α1→6 glycosidic linkages were introduced into the final products, ranging from 18% in the incubation mixture to 33% in an enriched fraction. In view of its primary structure, GTFB clearly is a member of the glycoside hydrolase 70 (GH70) family, comprising enzymes with a permuted (β/α)₈ barrel that use sucrose to synthesize α-d-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 α-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of α1→6 glycosidic linkages into linear oligosaccharide products. We conclude that GTFB represents a novel evolutionary intermediate between the GH13 and GH70 enzyme families, and we speculate about its origin.     

Loss of protein kinase C delta alters mammary gland development and apoptosis

As apoptotic pathways are commonly deregulated in breast cancer, exploring how mammary gland cell death is regulated is critical for understanding human disease. We show that primary mammary epithelial cells from protein kinase C delta (PKCδ) −/− mice have a suppressed response to apoptotic agents in vitro. In the mammary gland in vivo, apoptosis is critical for ductal morphogenesis during puberty and involution following lactation. We have explored mammary gland development in the PKCδ −/− mouse during these two critical windows. Branching morphogenesis was altered in 4- to 6-week-old PKCδ −/− mice as indicated by reduced ductal branching; however, apoptosis and proliferation in the terminal end buds was unaltered. Conversely, activation of caspase-3 during involution was delayed in PKCδ −/− mice, but involution proceeded normally. The thymus also undergoes apoptosis in response to physiological signals. A dramatic suppression of caspase-3 activation was observed in the thymus of PKCδ −/− mice treated with irradiation, but not mice treated with dexamethasone, suggesting that there are both target- and tissue-dependent differences in the execution of apoptotic pathways in vivo. These findings highlight a role for PKCδ in both apoptotic and nonapoptotic processes in the mammary gland and underscore the redundancy of apoptotic pathways in vivo.     

Murine and Bovine �æ� T Cells Enhance Innate Immunity against Brucella abortus Infections

γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ^−/− mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα^−/−, and GMCSF^−/− mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ^−/− mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections.     

β-Carotene conversion products and their effects on adipose tissue

Recent epidemiological data suggest that β-carotene may be protective against metabolic diseases in which adipose tissue plays a key role. Adipose tissue constitutes the major β-carotene storage tissue and its functions have been shown to be modulated in response to β-carotene breakdown products, especially retinal produced after cleavage by β-carotene 15,15′-monooxygenase (BCMO1), and retinoic acid arising from oxidation of retinal. However, the possibility exists that β-carotene in its intact form can also affect adipocyte function. Development of a knock out model and identification of a loss-of-function mutation have pointed out BCMO1 as being probably the sole enzyme responsible for provitamin A conversion into retinal in mammals. The utilisation of BCMO1^−/−mice should provide insights on β-carotene effect on its own in the future. In humans, intervention studies have highlighted the huge interindividual variation of β-carotene conversion efficiency, possibly due to genetic polymorphisms, which might impact on response to β-carotene. This brief review discusses the processes involved in β-carotene conversion and the effect of cleavage products on body fat and adipose tissue function.