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Kanzo Sakata

Publications and source records attributed to Kanzo Sakata.

34 records · Page 2Linked to original sources
PubMed2005

Crystal structures of pyruvate phosphate dikinase from maize revealed an alternative conformation in the swiveling-domain motion.

Pyruvate phosphate dikinase (PPDK) reversibly catalyzes the conversion of ATP, phosphate, and pyruvate into AMP, pyrophosphate, and phosphoenolpyruvate (PEP), respectively. Since the nucleotide binding site (in the N-terminal domain) and the pyruvate/PEP binding site (in the C-terminal domain) are separated by approximately 45 A, it has been proposed that an intermediary domain, called the central domain, swivels between these remote domains to transfer the phosphate. However, no direct structural evidence for the swiveling central domain has been found. In this study, the crystal structures of maize PPDK with and without PEP have been determined at 2.3 A resolution. These structures revealed that the central domain is located near the pyruvate/PEP binding C-terminal domain, in contrast to the PPDK from Clostridium symbiosum, wherein the central domain is located near the nucleotide-binding N-terminal domain. Structural comparisons between the maize and C. symbiosum PPDKs demonstrated that the swiveling motion of the central domain consists of a rotation of at least 92 degrees and a translation of 0.5 A. By comparing the maize PPDK structures with and without PEP, we have elucidated the mode of binding of PEP to the C-terminal domain and the induced conformational changes in the central domain.

Amino Acid Sequence↗
PubMed2004

Arabidopsis CYP707As encode (+)-abscisic acid 8'-hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid.

Abscisic acid (ABA) is involved in a number of critical processes in normal growth and development as well as in adaptive responses to environmental stresses. For correct and accurate actions, a physiologically active ABA level is controlled through fine-tuning of de novo biosynthesis and catabolism. The hydroxylation at the 8'-position of ABA is known as the key step of ABA catabolism, and this reaction is catalyzed by ABA 8'-hydroxylase, a cytochrome P450. Here, we demonstrate CYP707As as the P450 responsible for the 8'-hydroxylation of (+)-ABA. First, all four CYP707A cDNAs were cloned from Arabidopsis and used for the production of the recombinant proteins in insect cells using a baculovirus system. The insect cells expressing CYP707A3 efficiently metabolized (+)-ABA to yield phaseic acid, the isomerized form of 8'-hydroxy-ABA. The microsomes from the insect cells exhibited very strong activity of 8'-hydroxylation of (+)-ABA (K(m) = 1.3 microm and k(cat) = 15 min(-1)). The solubilized CYP707A3 protein bound (+)-ABA with the binding constant K(s) = 3.5 microm, but did not bind (-)-ABA. Detailed analyses of the reaction products confirmed that CYP707A3 does not have the isomerization activity of 8'-hydroxy-ABA to phaseic acid. Further experiments revealed that Arabidopsis CYP707A1 and CYP707A4 also encode ABA 8'-hydroxylase. The transcripts of the CYP707A genes increased in response to salt, osmotic, and dehydration stresses as well as ABA. These results establish that the CYP707A family plays a key role in regulating the ABA level through the 8'-hydroxylation of (+)-ABA.

Abscisic Acid↗
PubMed2004

Furcatin hydrolase from Viburnum furcatum Blume is a novel disaccharide-specific acuminosidase in glycosyl hydrolase family 1.

Furcatin hydrolase (FH) is a unique disaccharide-specific acuminosidase, which hydrolyzes furcatin (p-allylphenyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside (acuminoside)) into p-allylphenol and the disaccharide acuminose. We have isolated a cDNA coding for FH from Viburnum furcatum leaves. The open reading frame in the cDNA encoded a 538-amino acid polypeptide including a putative chloroplast transit peptide. The deduced protein showed 64% identity with tea leaf beta-primeverosidase, which is another disaccharide glycosidase specific to beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides). The deduced FH also shared greater than 50% identity with various plant beta-glucosidases in glycosyl hydrolase family 1. The recombinant FH expressed in Escherichia coli exhibited the highest level of activity toward furcatin with a Km value of 2.2 mm and specifically hydrolyzed the beta-glycosidic bond between p-allylphenol and acuminose, confirming FH as a disaccharide glycosidase. The FH also hydrolyzed beta-primeverosides and beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside) but poorly hydrolyzed beta-gentiobiosides (6-O-beta-D-glucopyranosyl-beta-d-glucopyranosides), indicating high substrate specificity for the disaccharide glycone moiety. The FH exhibited activity toward p-allylphenyl beta-D-glucopyranoside containing the same aglycone as furcatin but little activity toward the other beta-D-glucopyranosides. Stereochemical analysis using 1H NMR spectroscopy revealed that FH is a retaining glycosidase. The subcellular localization of FH was analyzed using green fluorescent protein fused with the putative N-terminal signal peptide, indicating that FH is localized to the chloroplast. Phylogenetic analysis of plant beta-glucosidases revealed that FH clusters with beta-primeverosidase, and this suggests that the disaccharide glycosidases will form a new subfamily in glycosyl hydrolase family 1.

Amino Acid Sequence↗
PubMed2004

Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95).

A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the alpha-(1-->2) linkage of 2'-fucosyllactose, and a gene encoding 1,2-alpha-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2'-fucosyllactose was determined to be inversion by using (1)H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

Amino Acid Sequence↗
PubMed2003

Purification, crystallization and preliminary X-ray diffraction studies on pyruvate phosphate dikinase from maize.

Pyruvate phosphate dikinase (PPDK) from maize catalyzes the reversible conversion of ATP, orthophosphate and pyruvate to AMP, pyrophosphate and PEP. In higher plants, this enzyme is believed to be involved in the C(4) dicarboxylic acid pathway. PPDK was crystallized by the vapour-diffusion method using polyethylene glycol as a precipitant. The crystals belong to the orthorhombic space group C2, with unit-cell parameters a = 108.2, b = 100.2, c = 108.4 A, beta = 96.5 degrees, and diffract to 2.3 A using SPring-8 synchrotron radiation.

Chromatography, Agarose↗
PubMed2003

Beta-glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of beta-glycosidases.

An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K(i)) of 2.1 and 0.80 microM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.

Amidines↗
PubMed2003

Highly sensitive active-site titration of lipase in microscale culture media using fluorescent organophosphorus ester.

The fluorescent organophosphorus esters, diethyl 4-methylumbelliferyl phosphate (1), ethyl hexyl 4-methylumbelliferyl phosphate (2) and ethyl 4-methylumbelliferyl heptylphosphonate (3) have been synthesized and evaluated as a sensitive active-site titrant of lipase. The phosphorus esters 1, 2 and 3 inactivated the lipase from Pseudomonas aeruginosa (LPL-312) with a second-order rate constant for enzyme inactivation (k(on)) of 1.8, 32 and 5600 s(-1) M(-1), respectively. The long-chain phosphonate 3 turned out to be the most potent inactivator of the lipase to release a stoichiometric amount of highly fluorescent 4-methylumbelliferone (4MU) as a leaving group. By using the phosphate 3 as an active-site titrant, the low concentration (4.5 nM) of the active lipase was titrated successfully. The highly sensitive active-site titration with 3 enabled the direct determination of the concentration of the active lipase expressed in a microscale culture medium. Although the expression level differed significantly from one culture to another, the titrated concentration of the active lipase was proportional to the apparent activity for all the independent cultures. The molecular activity calculated for the expressed lipase was found to be the same as that of the purified lipase. The present active-site titration method is widely applicable to the biocatalytic engineering of lipases such as directed evolution, site-directed mutagenesis, chemical modification and immobilization.

Binding Sites↗
PubMed2003

Triadimefon, a fungicidal triazole-type P450 inhibitor, induces brassinosteroid deficiency-like phenotypes in plants and binds to DWF4 protein in the brassinosteroid biosynthesis pathway.

Triadimefon (Bayleton), a widely used triazole-type fungicide, affects gibberellin (GA) biosynthesis and 14 alpha-demethylase in sterol biosynthesis. The present study revealed that the phenotype of Arabidopsis treated with triadimefon resembled that of a brassinosteroid (BR)-biosynthesis mutant, and that the phenotype was rescued by brassinolide (BL), the most active BR, partly rescued by GA, and fully rescued by the co-application of BL and GA, suggesting that triadimefon affects both BR and GA biosynthesis. The target sites of triadimefon were investigated using a rescue experiment, feeding triadimefon-treated Arabidopsis BR-biosynthesis intermediates, and a binding assay to expressed DWF4 protein, which is reported to be involved in the BR-biosynthesis pathway. The binding assay indicated that the dissociation constant for triadimefon was in good agreement with the activity in an in planta assay. In the triadimefon-treated Arabidopsis cells, the CPD gene in the BR-biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR deficiency. These results strongly suggest that triadimefon inhibits the reaction catalysed by DWF4 protein and induces BR deficiency in plants. As triadimefon treatment has proved to be beneficial to plants, this result suggests that BR-biosynthesis inhibitors can be applied to crops.

Arabidopsis Proteins↗
PubMed2002

A direct continuous spectrophotometric assay for glycosidases with 3-nitro-2-pyridyl glycosides by tautomerization of 2-hydroxy-3-nitropyridine.

Two kinds of 3-nitro-2-pyridyl glycosides were synthesized and evaluated as substrates for continuous spectrophotometric assay for glycosidases. The liberated aglycon, 2-hydroxy-3-nitropyridine, immediately tautomerized to 3-nitro-2(1H)-pyridone, causing an absorption shift of ca. 60 nm even under acidic conditions (pH 3-6). Consequently, the enzymatic hydrolysis of these glycosides was monitored continuously in the acidic to neutral pH range (pH 4-7), the optimum pH for most glycosidases. The absorbance of liberated aglycon increased linearly at 390 nm until 10% consumption of the substrate to enable the initial rate to be determined at once without terminating the reaction. The kinetic parameters for the hydrolysis of 3-nitro-2-pyridyl glycosides were obtained from the slopes of the progress curves and were compared with those obtained from the conventional discontinuous assay using p- and o-nitrophenyl glycosides as substrates. The kinetic parameters indicated that 3-nitro-2-pyridyl glycosides were more activated and specific substrates, but with less affinity to the enzymes than the corresponding nitrophenyl glycosides. Moreover, the absorbance shift by tautomerization should promise further applications to continuous spectrophotometric assays for other enzymes acting under acidic conditions, such as acid proteases and acid phosphatases.

Aspergillus niger↗
PubMed2002

Cloning of beta-primeverosidase from tea leaves, a key enzyme in tea aroma formation.

A beta-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The beta-primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The beta-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The beta-primeverosidase protein sequence was 50% to 60% identical to beta-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the beta-primeverosidase expressed in Escherichia coli was able to hydrolyze beta-primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl beta-D-glucopyranoside. These results indicate that the beta-primeverosidase selectively recognizes the beta-primeverosides as substrates and specifically hydrolyzes the beta-glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl beta-primeveroside by the beta-primeverosidase was followed by (1)H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the beta-primeveroside by a retaining mechanism. The roles of the beta-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed.

Amino Acid Sequence↗
PubMed2002

Isolation and characterization of a beta-primeverosidase-like endo-manner beta-glycosidase from Aspergillus fumigatus AP-20.

A novel beta-glycosidase-producing microorganism was isolated from soil and identified as Aspergillus fumigatus AP-20 based on its taxonomical characteristics. The enzyme was found to be an extracellular protein in the culture of the isolated fungus and was purified 88-fold by fractionation with ammonium sulfate followed by successive column chromatographies on phenyl-Sepharose HP and Mono P HR. The molecular mass was estimated to be 47 kDa by SDS-PAGE and the isoelectric point to be pH 6.0 by isoelectric focusing. The purified enzyme was highly specific for a substrate, p-nitrophenyl beta-primeveroside (6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside), which was cleaved in an endo-manner into primeverose and p-nitrophenol.

Aspergillus fumigatus↗
PubMedDate not supplied

Morning glory systemically accumulates scopoletin and scopolin after interaction with Fusarium oxysporum.

An isolate of non-pathogenic Fusarium, Fusarium oxysporum 101-2 (NPF), induces resistance in the cuttings of morning glory against Fusarium wilt caused by F. oxysporum f. sp. batatas O-17 (PF). The effect of NPF on phenylpropanoid metabolism in morning glory cuttings was studied. It was found that morning glory tissues responded to treatment with NPF bud-cell suspension (108 bud-cells/ml) with the activation of phenylalanine ammonia-lyase (PAL). PAL activity was induced faster and greater in the NPF-treated cuttings compared to cuttings of a distilled water control. High performance liquid chromatography analysis of the extract from tissues of morning glory cuttings after NPF treatment showed a quicker induction of scopoletin and scopolin synthesis than that seen in the control cuttings. PF also the induced synthesis of these compounds at 10(5) bud-cells/ml, but inhibited it at 10(8) bud-cells/ml. Possibly PF produced constituent(s) that elicited the inhibitory effect on induction of the resistance reaction. These compounds could potentially be useful as markers to detect early beginning interactions between Fusarium and morning glory tissues cuttings.

Coumarins↗
PubMedDate not supplied

Phytotoxic components produced by pathogenic Fusarium against morning glory.

A pathogenic isolate of Fusarium, F. oxysporum f. sp. batatas O-17 (PF), causes wilt disease in leaf etiolation in sweet potato (Ipomoea batatas) and morning glory (Ipomoea tricolor). Extracts from PF cultures were screened for phytotoxic components using a growth inhibition assay with morning glory seedlings. The extracts were fractionated using differential solvent extraction and two active compounds, ergosterol and fusalanipyrone, were isolated from the less-polar fraction. Growth inhibition of morning glory seedlings showed a sigmoidal dose-response relationship, with fusalanipyrone exhibiting a two order of magnitude higher EC50 value than ergosterol (18 nM and 1.6 microM, respectively). Both compounds showed lower growth inhibition activity towards lettuce seedlings (Lactuca sativa). This study provides information on the phytotoxic components of PF and discusses the mechanism behind PFf-induced phytotoxicity.

Ergosterol↗