Novel urushiols with human immunodeficiency virus type 1 reverse transcriptase inhibitory activity from the leaves of Rhus verniciflua.

Two novel urushiols, 1 and 2, and two known urushiols, 3 and 4, were isolated from the leaves of Rhus verniciflua and were examined for their human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitory activity. The novel urushiols were found to be 1,2-dihydroxyphenyl-3-[7'(E),9'(Z),11'(Z)-pentadecatrienyl]-14'-ol (1) and 1,2-dihydroxyphenyl-3-[8'(Z),10'(E),12'(E)-pentadecatrienyl]-14'-ol (2) by spectroscopic analyses. The absolute configuration at C-14' in 1 and 2 was determined to be a racemic mixture of (R) and (S) isomers by ozonolysis. Compound 2 (IC50: 12.6 µM) showed the highest HIV-1 RT inhibitory activity among the four urushiols, being 2.5-fold more potent than the positive control, adriamycin (IC50: 31.9 µM). Although the known urushiols were isolated from the sap and leaves of R. verniciflua, 1 was exclusively present in the leaves, and higher amounts of 2 were found in the leaves than in the sap. Present findings indicate that the leaves of R. verniciflua represent a new biological resource from which novel and known urushiols may be prepared, and the possible use of novel urushiols as bioactive products.

Design and synthesis of an ER-specific fluorescent probe based on carboxylesterase activity with quinone methide cleavage process.

CEs are important enzymes that catalyze the hydrolysis of prodrugs. In this Letter, we present a new mechanistic ER-specific fluorescent probe 1 based on CE activity. Permeation of 1 into cells and subsequent hydrolytic activation by CEs causes spontaneously quinone methide cleavage, resulting in bright red fluorescence in ER with high specificity. Probe 1 was developed for CE activity imaging and inhibitor screening at the cellular level.

Family M42 aminopeptidase from the syntrophic bacterium Symbiobacterium thermophilum: characterization using recombinant protein.

The chromosomal DNA of the syntrophic thermophile Symbiobacterium thermophilum contains open reading frames of the genes encoding family M42 aminopeptidases, Pep1079, Pep1080, and Pep1081. To characterize these peptidases, the genes were cloned into Escherichia coli and overexpressed. Our experiments using the recombinant proteins confirmed that Pep1079, Pep1080, and Pep1081 are components of arginyl or lysinyl aminopeptidases that require Co²+ for enzymatic activity. Coexistence of Pep1079 and Pep1080 is necessary for expressing high peptidase activity. Pep1081 enhances the activity of Pep1079 and Pep1080.

Expression, purification, physicochemical characterization and structural analysis of cytochrome c554 from Vibrio parahaemolyticus strain RIMD2210633.

The function of cytochrome c(554) of Vibrio parahaemolyticus has not yet been determined. We have determined the physicochemical properties and crystal structure of cytochrome c(554) at 1.8 A in order to help elucidate its function. The physicochemical properties and the tertiary structure of cytochrome c(554) resemble those of dimeric cytochrome c(552) from Pseudomonas nautica, but the Vibrio genus contains no gene for nitrite reductase, cytochrome cd(1), in its genome DNA. These results raise the possibility that both cytochromes denote an electron to an electron carrier and accept an electron from same electron carrier.

Heterodisaccharide 4-O-(N-acetyl-beta-D-glucosaminyl)-D-glucosamine is a specific inducer of chitinolytic enzyme production in Vibrios harboring chitin oligosaccharide deacetylase genes.

Vibrio parahaemolyticus KN1699 produces 4-O-(N-acetyl-beta-d-glucosaminyl)-d-glucosamine (GlcNAc-GlcN) as a major end product from chitin using two extracellular hydrolases: glycoside hydrolase family 18 chitinase, which produces (GlcNAc)(2) from chitin, and carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), which hydrolyzes the N-acetyl group at the reducing-end GlcNAc residue of (GlcNAc)(2). In this study, we clarified that this heterodisaccharide functions as an inducer of the production of the two above-mentioned chitinolytic enzymes, particularly chitinase. Similar results for chitinase production were obtained with other chitin-decomposing Vibrio strains harboring the CE family 4 COD gene; however, such an increase in chitinase production was not observed in chitinolytic Vibrio strains that did not harbor the COD gene. These results suggest that GlcNAc-GlcN is a unique inducer of chitinase production in Vibrio bacteria that have the COD-producing ability and that the COD involved in the synthesis of this signal compound is one of the key enzymes in the chitin catabolic cascade of these bacteria.

Design and screening strategies for alpha-glucosidase inhibitors based on enzymological information.

Alpha-glucosidase inhibitors are marketed as therapeutic drugs for diabetes that act through the inhibition of carbohydrate metabolism. Inhibitors of the alpha-glucosidases that are involved in the biosynthesis of N-linked oligosaccharide chains have been reported to have antitumor, antiviral, and apoptosis-inducing activities, and some have been used clinically. alpha-Glucosidase inhibitors have interesting biological activities, and their design, synthesis, and screening are being actively performed. In quite a few reports, however, alpha-glucosidases with different origins than the target alpha-glucosidases, have been used to evaluate inhibitory activities. There might be confusion regarding the naming of alpha-glucosidases. For example, the term alpha-glucosidase is sometimes used as a generic name for alpha-glucoside hydrolases. Moreover, IUBMB recommends the use of "alpha-glucosidase" (EC 3.2.1.20) for exo-alpha-1,4-glucosidases, which are further classified into four families based on amino acid sequence similarities. Accordingly, substrate specificity and susceptibility to inhibitors varies markedly among enzymes in the IUBMB alpha-glucosidases. The design and screening of inhibitors without consideration of these differences is not efficient. For the development of a practical inhibitor that is operational in cells, HTS using the target alpha-glucosidase and the computer-aided design of inhibitors based on enzymatic information concerning the same alpha-glucosidase are essential.

Crystallization and structural analysis of cytochrome c(6) from the diatom Phaeodactylum tricornutum at 1.5 A resolution.

We determined for the first time the crystal structure of diatom cytochrome c(6) from Phaeodactylum tricornutum at 1.5 A resolution. The overall structure of the protein was classified as a class I c-type cytochrome. The physicochemical properties of the protein were examined by denaturation with guanidine hydrochloride and urea, and compared with those of other algal cytochrome c(6).

Physicochemical properties of diheme cytochrome c4 of unknown function from Vibrio parahaemolyticus strain RIMD2210633.

To characterize a diheme cytochrome c4 of unknown functional of the Vibrio genus for the first time, the Vibrio parahaemolyticus cytochrome c4 was overexpressed in Escherichia coli periplasm using the endogenous signal sequence. The physicochemical properties of the purified recombinant protein, viz., molecular mass, UV/Vis, and CD spectra, and the redox potentials of the N- and C-terminal domain hemes were determined.

Cloning, expression and purification of cytochrome c(6) from the brown alga Hizikia fusiformis and complete X-ray diffraction analysis of the structure.

The primary sequence of cytochrome c(6) from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6 A resolution. The crystal belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 84.58, c = 232.91 A and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c(6) were most similar to those of red algal cytochrome c(6). These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host.

Production and secretion of a recombinant Vibrio parahaemolyticus chitinase by Escherichia coli and its purification from the culture medium.

An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined.

Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442.

Chitin binding proteins prepared from Vibrio proteolyticus were purified and the N-terminal amino-acid sequence of a protein from a 110-kDa band on SDS-PAGE was found to be 85-90% identical to the 22nd-41st residues of the N-termini of chitinase A precursor proteins from other vibrios. We cloned the corresponding gene, which encodes a putative protein of 850 amino acids containing a 26-residue signal sequence. The chitinase precursor from V. proteolyticus was 78-80% identical to those from Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio carchariae. However, the proteolytic cleavage site for C-terminal processing between R597 and K598 in the chitinase precursor of other vibrios was not observed in the amino acid sequence of V. proteolyticus, which instead had the sequence R600 and A601. Subsequently, full-length and truncated chitinases were generated in Escherichia coli. The specific activity of full-length chitinase expressed in E. coli was 17- and 20-folds higher for colloidal and alpha-chitins (insoluble substrate), respectively, than that of the C-terminal truncated enzyme. However, both recombinants showed similar hydrolysis patterns of hexa-N-acetyl-chitohexaose (soluble substrate), producing di-N-acetyl-chitobiose as major product on TLC analysis. We showed that the C-terminus of the V. proteolyticus chitinase A was important for expression of high specific activity against insoluble chitins.

Expression of the algal cytochrome c6 gene in Arabidopsis enhances photosynthesis and growth.

Photosynthetic plants convert light energy into ATP and NADPH in photosynthetic electron transfer and photophosphorylation, and synthesize mainly carbohydrates in the Calvin-Benson cycle. Here we report the enhancement of photosynthesis and growth of plants by introducing the gene of an algal cytochrome c6, which has been evolutionarily eliminated from higher plant chloroplasts, into the model plant Arabidopsis thaliana. At 60 d after planting, the plant height, leaf length and root length of the transformants were 1.3-, 1.1- and 1.3-fold those in the wild-type plants, respectively. At the same time, in the transgenic plants, the amounts of chlorophyll, protein, ATP, NADPH and starch were 1.2-, 1.1-, 1.9-, 1.4- and 1.2-fold those in the wild-type plants, respectively. The CO2 assimilation capacity of the transgenic plants was 1.3-fold that of the wild type. Moreover, in transgenic Arabidopsis expressing algal cytochrome c6, the 1-qP, which reflects the reduced state of the plastoquinone pool, is 30% decreased compared with the wild type. These results show that the electron transfer of photosynthesis of Arabidopsis would be accelerated by the expression of algal cytochrome c6. Our results demonstrate that the growth and photosynthesis of Arabidopsis plants could be enhanced by the expression of the algal cytochrome c6 gene.

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells.

An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.

Crystal structure of oxidized cytochrome c(6A) from Arabidopsis thaliana.

Compared with algal and cyanobacterial cytochrome c(6), cytochrome c(6A) from higher plants contains an additional loop of 12 amino acid residues. We have determined the first crystal structure of cytochrome c(6A) from Arabidopsis thaliana at 1.5 Angstrom resolution in order to help elucidate its function. The overall structure of cytochrome c(6A) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys16 and Cys19, and the iron has octahedral coordination with His20 and Met60 as the axial ligands. Two cysteine residues (Cys67 and Cys73) within the characteristic 12 amino acids loop form a disulfide bond, contributing to the structural stability of cytochrome c(6A). Our model provides a chemical basis for the known low redox potential of cytochrome c(6A) which makes it an unsuitable electron carrier between cytochrome b(6)f and PSI.

Planar catechin analogues with alkyl side chains: a potent antioxidant and an alpha-glucosidase inhibitor.

Planar catechin analogues having various alkyl side chain lengths were synthesized, and their remarkable antioxidative abilities and alpha-glucosidase inhibitory activities are shown.

Aglycon specificity profiling of alpha-glucosidases using synthetic probes.

We designed and synthesized hydrogen bond based probes 1-8 with the exception of known glycosidase inhibition mechanisms, and aglycon specificity of 11 different sources of alpha-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-D-ido-heptitol) showed a potent inhibition of S. cerevisiae alpha-glucosidase among all alpha-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae alpha-glucosidase with Ki 0.13 mM.

Design and synthesis of de novo cytochromes c.

Natural c-type cytochromes are characterized by the consensus Cys-X-X-Cys-His heme-binding motif (where X is any amino acid) by which the heme is covalently attached to protein by the addition of the sulfhydryl groups of two cysteine residues to the vinyl groups of the heme. In this work, the consensus sequence was used for the heme-binding site of a designed four-helix bundle, and the apoproteins with either a histidine residue or a methionine residue positioned at the sixth coordination site were synthesized and reacted with iron protoporphyrin IX (protoheme) under mild reducing conditions in vitro. These polypeptides bound one heme per helix-loop-helix monomer via a single thioether bond and formed four-helix bundle dimers in the holo forms as designed. They exhibited visible absorption spectra characteristic of c-type cytochromes, in which the absorption bands shifted to lower wavelengths in comparison with the b-type heme binding intermediates of the same proteins. Unexpectedly, the designed cytochromes c with bis-His-coordinated heme iron exhibited oxidation-reduction potentials similar to those of their b-type intermediates, which have no thioether bond. Furthermore, the cytochrome c with His and Met residues as the axial ligands exhibited redox potentials increased by only 15-30 mV in comparison with the cytochrome with the bis-His coordination. These results indicate that highly positive redox potentials of natural cytochromes c are not only due to the heme covalent structure, including the Met ligation, but also due to noncovalent and hydrophobic environments surrounding the heme. The covalent attachment of heme to the polypeptide in natural cytochromes c may contribute to their higher redox potentials by reducing the thermodynamic stability of the oxidized forms relatively against that of the reduced forms without the loss of heme.

alpha-Mannosidase-catalyzed synthesis of a (1-->2)-alpha-D-rhamnodisaccharide derivative.

Enzymatic transglycosylation using p-nitrophenyl alpha-D-rhamnopyranoside as the glycosyl donor and 6equiv of ethyl 1-thio-alpha-D-rhamnopyranoside as the glycosyl acceptor yielded a D-rhamnooligosaccharide derivative. The reaction was catalyzed by jack bean alpha-mannosidase in a 1:1 (v/v) mixture of 0.1 M sodium citrate buffer (pH4.5)-MeCN at 25 degrees C. The enzyme exhibited high catalytic activity for the reaction, to afford in 32.1% isolated yield (based on donor substrate) ethyl alpha-D-rhamnopyranosyl-(1-->2)-1-thio-alpha-D-rhamnopyranoside, which is a derivative of the common oligosaccharide unit of the antigenic lipopolysaccharides from Pseudomonas.

Development of a genetic system in chitinase-producing streptomyces and the application of an allosamidin-insensitive chitinase gene to homologous overexpression.

A transformation system for Streptomyces sp. AJ9463 strain (allosamidin producer) was successfully developed using protoplasts and a PEG-mediated method. To prepare protoplasts, the concentration of glycine and sucrose in YEME medium were optimized to 0.5% (w/v) and 34.0% (w/v), respectively. When the protoplasts of Streptomyces sp. AJ9463 were transformed with pUWL-KS, transformants could be obtained at a high efficiency of 7.0 x 10(4) transformants per microg DNA. To ensure that the transformation system worked properly, we then constructed a constitutive expression vector pYK1, in which the ermE* promoter drives transcription of the allosamidin-insensitive chitinase gene, chiIS. Although no transformant could be obtained by the genetic system using pYK1 isolated from Escherichia coli DH5alpha, pYK1 isolated from the methylase-deficient mutant E. coli SCS110, could be introduced into Streptomyces sp. AJ9463. This indicates that Streptomyces sp. AJ9463 has a methylation-specific restriction system, and that the chiIS and/or ermE* promoter region of pYK1 includes a restriction site of its endonuclease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that pYK1 in Streptomyces sp. AJ9463 started to obviously express ChiIS from 14-h. Moreover, the pYK1-introduced strain gave a five-fold higher chitinase activity than the wild-type, suggesting that this system can be widely applied for the overexpression and gene functional analysis.

A novel microperoxidase activity: methyl viologen-linked nitrite reducing activity of microperoxidase.

To investigate the nitrite reducing activity of microperoxidases (mps) in the presence of methyl viologen and dithionite, the fragments C14-K22 (mp9), V11-L32 (mp22), and G1-M65 (mp65) containing heme were prepared by enzymatic hydrolysis of commercially equine heart cytochrome c (Cyt c), in which His is axially coordinated to heme iron, and acts as its fifth ligand. The nitrite reducing activity of mps was measured under anaerobic condition, and the nitrite reducing activity of mps increased with the cutting of the peptide chain. The activity of the shortest nonapeptide mp9 was approximately 120-fold that of Cyt c (104 amino acid residues) and 3.2-fold that of nitrite reductase (EC 1.7.7.1) from Escherichia coli. In the nitrite reduction by mp, nitrite was completely reduced to ammonia. We presumed that ferrous mps reduced NO2- to NO by donating one electron, the NO was completely reduced to NH4+ under anaerobic condition via ferrous-NO complexes as a reaction intermediate using visible spectra and ESR spectra, and this overall reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized mp9 had a nitrite reducing activity similar to that of mp9 in solution, and the resin retained the activity after five uses and even 1-year storage. The mp will be able to use as a substitute for nitrite reductase.