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)

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

Archaeal Mo-Containing Glyceraldehyde Oxidoreductase Isozymes Exhibit Diverse Substrate Specificities through Unique Subunit Assemblies.

Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase (GAOR1, GAOR2 and GAOR3) from the thermoacidophilic archaeon Sulfolobus tokodaii. GAOR1-3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the STK17810/STK17830/STK17820 hetero-trimer, GAOR2 is a dimer of the STK23390/STK05620/STK05610 hetero-trimer, and GAOR3 is the STK24840/STK05620/STK05610 hetero-trimer. GAOR1-3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system

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)

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

Crystal Structures of Cytochrome P450 105P1 from Streptomyces avermitilis: Conformational Flexibility and Histidine Ligation State▿ †

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol in biological membranes. The filipin biosynthetic pathway of Streptomyces avermitilis contains two position-specific hydroxylases, C26-specific CYP105P1 and C1′-specific CYP105D6. In this study, we describe the three X-ray crystal structures of CYP105P1: the ligand-free wild-type (WT-free), 4-phenylimidazole-bound wild-type (WT-4PI), and ligand-free H72A mutant (H72A-free) forms. The BC loop region in the WT-free structure has a unique feature; the side chain of His72 within this region is ligated to the heme iron. On the other hand, this region is highly disordered and widely open in WT-4PI and H72A-free structures, respectively. Histidine ligation of wild-type CYP105P1 was not detectable in solution, and a type II spectral change was clearly observed when 4-phenylimidazole was titrated. The H72A mutant showed spectroscopic characteristics that were almost identical to those of the wild-type protein. In the H72A-free structure, there is a large pocket that is of the same size as the filipin molecule. The highly flexible feature of the BC loop region of CYP105P1 may be required to accept a large hydrophobic substrate