Crystallographic and cryogenic electron microscopic structures and enzymatic characterization of sulfur oxygenase reductase from Sulfurisphaera tokodaii.

Sulfur oxygenase reductases (SORs) are present in thermophilic and mesophilic archaea and bacteria, and catalyze oxygen-dependent oxygenation and disproportionation of elemental sulfur. SOR has a hollow, spherical homo-24-mer structure and reactions take place at active sites inside the chamber. The crystal structures of SORs from Acidianus species have been reported. However, the states of the active site components (mononuclear iron and cysteines) and the entry and exit paths of the substrate and products are still in dispute. Here, we report the biochemical and structural characterizations of SORs from the thermoacidophilic archaeon Sulfurisphaera tokodaii (StSOR) and present high-resolution structures determined by X-ray crystallography and cryogenic electron microscopy (cryo-EM). The crystal structure of StSOR was determined at 1.73 Å resolution. At the catalytic center, iron is ligated to His86, His90, Glu114, and two water molecules. Three conserved cysteines in the cavity are located 9.5-13 Å from the iron and were observed as free thiol forms. A mutational analysis indicated that the iron and one of the cysteines (Cys31) were essential for both activities. The cryo-EM structure was determined at 2.24 Å resolution using an instrument operating at 200 kV. The two structures determined by different methodologies showed similar main chain traces, but the maps exhibited different features at catalytically important components. A possible role of StSOR in the sulfur metabolism of S. tokodaii (an obligate aerobe) is discussed based on this study. Given the high resolution achieved in this study, StSOR was shown to be a good benchmark sample for cryo-EM.

Crystal structure of a feruloyl esterase belonging to the tannase family: a disulfide bond near a catalytic triad.

Feruloyl esterase (FAE) catalyzes the hydrolysis of the ferulic and diferulic acids present in plant cell wall polysaccharides, and tannase catalyzes the hydrolysis of tannins to release gallic acid. The fungal tannase family in the ESTHER database contains various enzymes, including FAEs and tannases. Despite the importance of FAEs and tannases in bioindustrial applications, three-dimensional structures of the fungal tannase family members have been unknown. Here, we determined the crystal structure of FAE B from Aspergillus oryzae (AoFaeB), which belongs to the fungal tannase family, at 1.5 Å resolution. AoFaeB consists of a catalytic α/ß-hydrolase fold domain and a large lid domain, and the latter has a novel fold. To estimate probable binding models of substrates in AoFaeB, an automated docking analysis was performed. In the active site pocket of AoFaeB, residues responsible for the substrate specificity of the FAE activity were identified. The catalytic triad of AoFaeB comprises Ser203, Asp417, and His457, and the serine and histidine residues are directly connected by a disulfide bond of the neighboring cysteine residues, Cys202 and Cys458. This structural feature, the "CS-D-HC motif," is unprecedented in serine hydrolases. A mutational analysis indicated that the novel structural motif plays essential roles in the function of the active site.

Crystal structure of glycoside hydrolase family 127 ß-l-arabinofuranosidase from Bifidobacterium longum.

Enzymes acting on ß-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of ß-l-arabinofuranosyl oligosaccharides in plant cells. Recently, a ß-l-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked ß-l-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and ß-l-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional ß-sandwich domains, with one ß-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn(2+) coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the ß-l-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.

Four Cys residues in heterodimeric 2-oxoacid:ferredoxin oxidoreductase are required for CoA-dependent oxidative decarboxylation but not for a non-oxidative decarboxylation.

Heterodimeric 2-oxoacid:ferredoxin oxidoreductase (OFOR) from Sulfolobus tokodaii (StOFOR) has only one [4Fe-4S]²âº cluster, ligated by 4 Cys residues, C12, C15, C46, and C197. The enzyme has no other Cys. To elucidate the role of these Cys residues in holding of the iron-sulfur cluster in the course of oxidative decarboxylation of a 2-oxoacid, one or two of these Cys residues was/were substituted with Ala to yield C12A, C15A, C46A, C197A and C12/15A mutants. All the mutants showed the loss of iron-sulfur cluster, except the C197A one which retained some unidentified type of iron-sulfur cluster. On addition of pyruvate to OFOR, the wild type enzyme exhibited a chromophore at 320nm and a stable large EPR signal corresponding to a hydroxyethyl-ThDP radical, while the mutant enzymes did not show formation of any radical intermediate or production of acetyl-CoA, suggesting that the intact [4Fe-4S] cluster is necessary for these processes. The stable radical intermediate in wild type OFOR was rapidly decomposed upon addition of CoA in the absence of an electron acceptor. Non-oxidative decarboxylation of pyruvate, yielding acetaldehyde, has been reported to require CoA for other OFORs, but StOFOR catalyzed acetaldehyde production from pyruvate independent of CoA, regardless of whether the iron-sulfur cluster is intact [4Fe-4S] type or not. A comprehensive reaction scheme for StOFOR with a single cluster was proposed.

Crystal structures of phosphoketolase: thiamine diphosphate-dependent dehydration mechanism.

Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,ß-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.

Characterization of phytochelatin synthase produced by the primitive red alga Cyanidioschyzon merolae.

Phytochelatins (PCs), non-protein peptides with the general structure [(γ-Glu-Cys)n-Gly (n≥ 2)], are involved in the detoxification of toxic heavy metals mainly in higher plants. The synthesis of the peptides is mediated by phytochelatin synthase (PCS), which is activated by a range of heavy metals. CmPCS, a PCS-like gene found in the genomic DNA of the primitive red alga Cyanidioschyzon merolae, was isolated and a recombinant protein (rCmPCS) fused with a hexahistidine tag at the N-terminus of CmPCS was produced. The finding that this protein mediated PC synthesis from glutathione in a metal-dependent way clearly establishes that rCmPCS is functional. The maximum activity was attained at a reaction temperature of 50 °C, considerably higher than the temperature required for the maximal activity of PCS isolated from the higher plant Silene cucubalus, probably due to the alga being a thermophile. CmPCS showed optimal pH in a slightly higher region than higher plant PCSs, probably due to the less effective charge relay network in the catalytic triad. In addition, the pattern of enzyme activation by metal ions was specific to rCmPCS, with Ag+, Cu2+, and Hg2+ showing only limited activation. In contrast to other eukaryotic PCSs, CmPCS has an extra domain in the N-terminal region from residues 1 to 109, and contains fewer cysteine residues in the C-terminal domain. These differences may be responsible for the metal specificity of the activation of CmPCS. Although the enzyme preparation lost PCS activity progressively when stored at 4 °C, the inclusion of Cd2+ in the preparation effectively prevented the reduction of activity. Furthermore, Cd2+ effectively restored the activity of the inactivated enzyme. These results indicate that Cd2+ ions bind the enzyme to maintain the structural integrity of the peptides.

Identification of the lysine residue responsible for coenzyme A binding in the heterodimeric 2-oxoacid:ferredoxin oxidoreductase from Sulfolobus tokodaii, a thermoacidophilic archaeon, using 4-fluoro-7-nitrobenzofurazan as an affinity label.

The heterodimeric 2-oxoacid:ferredoxin oxidoreductase (StOFOR) from Sulfolobus tokodaii, a thermoacidophilic archaeon, was inactivated by low concentrations of 4-fluoro-7-nitrobenzofurazan (NBD-F), with concomitant increase in fluorescence in subunit-b. The inactivation was prevented by CoA, suggesting that NBD-F covalently bound to the Lys which is responsible for CoA binding. The NBD-labeled subunit-b was isolated and digested with endoproteinase Lys-C. The resulting polypeptide mixture was separated by reverse phase HPLC and the fluorescent fraction was isolated. Amino acid sequencing of the fraction revealed that it comprised a mixture of two polypeptides containing Lys125 and Lys173, respectively. Two StOFOR mutants, K125A and K173A, were constructed, expressed and purified. K125A showed a large increase in the K(m) value for CoA and showed poor inactivation by NBD-F, compared with K173A and wild type StOFOR, indicating Lys125 in subunit-b is the critical residue that interacts with CoA.

Crystal structures of an ATP-dependent hexokinase with broad substrate specificity from the hyperthermophilic archaeon Sulfolobus tokodaii.

Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate by using ATP as a phosphoryl donor. Recently, we identified and characterized an ATP-dependent hexokinase (StHK) from the hyperthermophilic archaeon Sulfolobus tokodaii, which can phosphorylate a broad range of sugar substrates, including glucose, mannose, glucosamine, and N-acetylglucosamine. Here we present the crystal structures of StHK in four different forms: (i) apo-form, (ii) binary complex with glucose, (iii) binary complex with ADP, and (iv) quaternary complex with xylose, Mg(2+), and ADP. Forms i and iii are in the open state, and forms ii and iv are in the closed state, indicating that sugar binding induces a large conformational change, whereas ADP binding does not. The four different crystal structures of the same enzyme provide "snapshots" of the conformational changes during the catalytic cycle. StHK exhibits a core fold characteristic of the hexokinase family, but the structures of several loop regions responsible for substrate binding are significantly different from those of other known hexokinase family members. Structural comparison of StHK with human N-acetylglucosamine kinase and other hexokinases provides an explanation for the ability of StHK to phosphorylate both glucose and N-acetylglucosamine. A Mg(2+) ion and coordinating water molecules are well defined in the electron density of the quaternary complex structure. This structure represents the first direct visualization of the binding mode for magnesium to hexokinase and thus allows for a better understanding of the catalytic mechanism proposed for the entire hexokinase family.

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 structure of the ferredoxin component of carbazole 1,9a-dioxygenase of Pseudomonas resinovorans strain CA10, a novel Rieske non-heme iron oxygenase system.

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 catalyzes the dioxygenation of carbazole; the 9aC carbon bonds to a nitrogen atom and its adjacent 1C carbon as the initial reaction in the mineralization pathway. The CARDO system is composed of ferredoxin reductase (CarAd), ferredoxin (CarAc), and terminal oxygenase (CarAa). CarAc acts as a mediator in the electron transfer from CarAd to CarAa. To understand the structural basis of the protein-protein interactions during electron transport in the CARDO system, the crystal structure of CarAc was determined at 1.9 A resolution by molecular replacement using the structure of BphF, the biphenyl 2,3-dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model. CarAc is composed of three beta-sheets, and the structure can be divided into two domains, a cluster-binding domain and a basal domain. The Rieske [2Fe-2S] cluster is located at the tip of the cluster-binding domain, where it is exposed to solvent. While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other. The structure of the cluster-binding domain of CarAc is more compact and protruding than that of BphF, and the distribution of electric charge on its molecular surface is very different. Such differences are thought to explain why these ferredoxins can act as electron mediators in respective electron transport chains composed of different-featured components.

Crystal structure of a family 54 alpha-L-arabinofuranosidase reveals a novel carbohydrate-binding module that can bind arabinose.

As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 alpha-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a beta-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a beta-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu(221) and Asp(297), respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.

Molecular evolution of the ATPase subunit of three archaeal sugar ABC transporters.

The sequence of genes encoding extracellular amylopullulanase and putative maltodextrin ATP binding cassette (ABC) transporter of a hyperthermophilic archaeon, Thermococcus litoralis, was determined. The mdxK gene, which encodes an ATPase subunit of the putative maltodextrin ABC transporter, has extraordinarily high similarity with the malK gene, which encodes an ATPase subunit of trehalose/maltose ABC transporter of the same organism. DNA sequence comparison revealed that the malK gene was generated through the duplication of the mdxK gene before lateral gene transfer of the mal gene cluster from T. litoralis to Pyrococcus furiosus.

Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding.

Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K(m) and K(i) values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of betaTyr68 formed hydrogen bonds with both n-butyric acid and alphaSer112, which is located in the active center. The betaY68F mutant showed an elevated K(m) value and a significantly decreased k(cat) value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between alphaCys108 and alphaCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (alphaT109S and alphaY114T) were constructed, each residue being replaced with an iron-containing one. The alphaT109S mutant showed similar characteristics to the wild-type enzyme. However, the alphaY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of alphaCys111 and alphaCys113 residues were not observed. The alphaTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.

Crystal structure of an ADP-dependent glucokinase from Pyrococcus furiosus: implications for a sugar-induced conformational change in ADP-dependent kinase.

ADP-dependent kinases are used in the modified Embden-Meyerhoff pathway of certain archaea. Our previous study has revealed a mechanism for ADP-dependent phosphoryl transfer by Thermococcus litoralis glucokinase (tlGK), and its evolutionary relationship with ATP-dependent ribokinases and adenosine kinases (PFKB carbohydrate kinase family members). Here, we report the crystal structure of glucokinase from Pyrococcus furiosus (pfGK) in a closed conformation complexed with glucose and AMP at 1.9A resolution. In comparison with the tlGK structure, the pfGK structure shows significant conformational changes in the small domain and a region around the hinge, suggesting glucose-induced domain closing. A part of the large domain next to the hinge is also shifted accompanied with domain closing. In the pfGK structure, glucose binds in a groove between the large and small domains, and the electron density of O1 atoms for both the alpha and beta-anomer configurations was observed. The structural details of the sugar-binding site of ADP-dependent glucokinase were firstly clarified and then site-directed mutagenesis analysis clarified the catalytic residues for ADP-dependent kinase, such as Arg205 and Asp451 of tlGK. Homology search and multiple alignment of amino acid sequences using the information obtained from the structures reveals that eucaryotic hypothetical proteins homologous to ADP-dependent kinases retain the residues for the recognition of a glucose substrate.

Characterization of the cupin-type phosphoglucose isomerase from the hyperthermophilic archaeon Thermococcus litoralis.

The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli. The purified enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized. The inhibition constants for four competitive inhibitors were determined. The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer. The activity was enhanced by the addition of Fe(2+), but inhibited by Zn(2+) and EDTA. Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in k(cat). The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight differences.