The First Crystal Structure of the Novel Class of Fructose-1,6-Bisphosphatase Present in Thermophilic Archaea

AbstractAs the first structure of the novel class of fructose-1,6-bisphosphatase (FBPase) present in thermophilic archaea, we solved the crystal structure of the ST0318 gene product (St-Fbp) of Sulfolobus tokodaii strain 7. The St-Fbp structure comprises a homooctamer of the 422 point-group. The protein folds as a four-layer α-β-β-α sandwich with a novel topology, which is completely different from the sugar phosphatase fold. The structure contains an unhydrolyzed FBP molecule in the open-keto form, as well as four hexacoordinated magnesium ions around the 1-phosphoryl group of FBP. The arrangement of the catalytic side chains and metal ligands is consistent with the three-metal ion assisted catalysis proposed for conventional FBPases. The structure provides an insight into the structural basis of the strict substrate specificity of St-Fbp

Comparative analysis of two glyceraldehyde-3-phosphate dehydrogenases from a thermoacidophilic archaeon, Sulfolobus tokodaii

AbstractSulfolobus tokodaii, a thermoacidophilic archaeon, possesses two structurally and functionally different enzymes that catalyze the oxidation of glyceraldehyde-3-phosphate (GAP): non-phosphorylating GAP dehydrogenase (St-GAPN) and phosphorylating GAP dehydrogenase (St-GAPDH). In contrast to previously characterized GAPN from Sulfolobus solfataricus, which exhibits V-type allosterism, St-GAPN showed K-type allosterism in which the positive cooperativity was abolished with concomitant activation by glucose 1-phosphate (G1P). St-GAPDH catalyzed the reversible oxidation of GAP to 1,3-bisphosphoglycerate (1,3-BPG) with high gluconeogenic activity, which was specific for NADPH, while both NAD+ and NADP+ were utilized in the glycolytic direction.Structured summary of protein interactionsGAPDH and GAPDH bind by molecular sieving (View interaction) GAPN and GAPN bind by 2.2molecular sieving (View interaction)

Molecular analysis of cyclic α-maltosyl-(1→6)-maltose binding protein in the bacterial metabolic pathway

Cyclic α-maltosyl-(1→6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic α-nigerosyl-(1→6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM

Crystal structure of polysaccharide lyase family 20 endo-β-1,4-glucuronan lyase from the filamentous fungus Trichoderma reesei

AbstractThe crystal structure of endo-β-(1→4)-glucuronan lyase from Trichoderma reesei (TrGL) has been determined at 1.8Å resolution as the first three-dimensional structure of polysaccharide lyase (PL) family 20. TrGL has a typical β-jelly roll fold, which is similar to glycoside hydrolase family 16 and PL7 enzymes. A calcium ion is bound to the site far from the cleft and appears to contribute to the stability. There are several completely conserved residues in the cleft. Possible catalytic residues are predicted based on structural comparison with PL7 alginate lyase A1–II′

A Novel Enzymatic System against Oxidative Stress in the Thermophilic Hydrogen-Oxidizing Bacterium Hydrogenobacter thermophilus

Rubrerythrin (Rbr) is a non-heme iron protein composed of two distinctive domains and functions as a peroxidase in anaerobic organisms. A novel Rbr-like protein, ferriperoxin (Fpx), was identified in Hydrogenobacter thermophilus and was found not to possess the rubredoxin-like domain that is present in typical Rbrs. Although this protein is widely distributed among aerobic organisms, its function remains unknown. In this study, Fpx exhibited ferredoxin:NADPH oxidoreductase (FNR)-dependent peroxidase activity and reduced both hydrogen peroxide (H2O2) and organic hydroperoxide in the presence of NADPH and FNR as electron donors. The calculated Km and Vmax values of Fpx for organic hydroperoxides were comparable to that for H2O2, demonstrating a multiple reactivity of Fpx towards hydroperoxides. An fpx gene disruptant was unable to grow under aerobic conditions, whereas its growth profiles were comparable to those of the wild-type strain under anaerobic and microaerobic conditions, clearly indicating the indispensability of Fpx as an antioxidant of H. thermophilus in aerobic environments. Structural analysis suggested that domain-swapping occurs in Fpx, and this domain-swapped structure is well conserved among thermophiles, implying the importance of structural stability of domain-swapped conformation for thermal environments. In addition, Fpx was located on a deep branch of the phylogenetic tree of Rbr and Rbr-like proteins. This finding, taken together with the wide distribution of Fpx among Bacteria and Archaea, suggests that Fpx is an ancestral type of Rbr homolog that functions as an essential antioxidant and may be part of an ancestral peroxide-detoxification system

Cysteine Nucleophiles in Glycosidase Catalysis : Application of a Covalent β-L-Arabinofuranosidase Inhibitor

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)(3)(Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived beta-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This beta-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.Medical BiochemistryBio-organic Synthesi

Structure and evolution of the bifidobacterial carbohydrate metabolism proteins and enzymes

Bifidobacteria have attracted significant attention because they provide health-promoting effects in the human gut. In this review, we present a current overview of the three-dimensional structures of bifidobacterial proteins involved in carbohydrate uptake, degradation, and metabolism. As predominant early colonizers of the infant's gut, distinct bifidobacterial species are equipped with a panel of transporters and enzymes specific for human milk oligosaccharides (HMOs). Interestingly, Bifidobacterium bifidum and Bifidobacterium longum possess lacto-N-biosidases with unrelated structural folds to release the disaccharide lacto-N-biose from HMOs, suggesting the convergent evolution of this activity from different ancestral proteins. The crystal structures of enzymes that confer the degradation of glycans from the mucin glycoprotein layer provide a structural basis for the utilization of this sustainable nutrient in the gastrointestinal tract. The utilization of several plant dietary oligosaccharides has been studied in detail, and the prime importance of oligosaccharide-specific ATP-binding cassette (ABC) transporters in glycan utilisations by bifidobacteria has been revealed. The structural elements underpinning the high selectivity and roles of ABC transporter binding proteins in establishing competitive growth on preferred oligosaccharides are discussed. Distinct ABC transporters are conserved across several bifidobacterial species, e.g. those targeting arabinoxylooligosaccharide and α-1,6-galactosides/glucosides. Less prevalent transporters, e.g. targeting β-mannooligosaccharides, may contribute to the metabolic specialisation within Bifidobacterium. Some bifidobacterial species have established symbiotic relationships with humans. Structural studies of carbohydrate-utilizing systems in Bifidobacterium have revealed the interesting history of molecular coevolution with the host, as highlighted by the early selection of bifidobacteria by mucin and breast milk glycans

Identification and Characterization of an ATP-Dependent Hexokinase with Broad Substrate Specificity from the Hyperthermophilic Archaeon Sulfolobus tokodaii

As a new member of the glucose-phosphorylating enzymes, the ATP-dependent hexokinase from the hyperthermophilic crenarchaeon Sulfolobus tokodaii was purified, identified, and characterized. Our results revealed that the enzyme differs from other known enzymes in primary structure and its broad substrate specificity for both phosphoryl donors and acceptors