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I Urabe

Publications and source records attributed to I Urabe.

At least 37 records · Page 2Linked to original sources
PubMed1994

A frame-specific symmetry of complementary strands of DNA suggests the existence of genes on the antisense strand.

The bacterial DNA sequence in GenBank database were divided into coding and noncoding regions and examined for the base-trimer distribution in every triplet frame on the sense and antisense strands. The results revealed that for the noncoding region, both strands have very similar base-trimer distributions and have no frame specificity; that is, DNA is symmetric in the noncoding region. For the coding region, on the other hand, the symmetry is broken only in the triplet framework, and we found a special triplet-frame-specific symmetry which appears when the two complementary strands of the coding region are read from their 5' ends. In addition, the following frame specificity was also observed in the distribution of stop codons on the antisense strand of the coding region. When the antisense sequences of the open reading frames (ORFs) in the database are read in the three reading frames, the same reading frame as the corresponding ORF contains a significantly larger amount of long open frames without stop codons (i.e., nonstop frames [NSFs]) than expected, while the number of NSFs in the other two reading frames is similar to that of the expected one. That is, NSFs as well as ORFs are maintained in a frame-specific manner, and in this sense, DNA becomes symmetrical even in the coding region. These two kinds of frame-specific symmetries indicate that only an ORF and its complementary triplets are specifically recognized and maintained in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Antisense↗
PubMed1994

Insertion sequence IS6100 on plasmid pOAD2, which degrades nylon oligomers.

The nucleotide sequence of repeated sequence I, which appears in five regions on nylon oligomer-degrading plasmid pOAD2, harbored in Flavobacterium sp. strain K172, was determined. The five regions of repeated sequence I had 880 bp of identical sequence, and the sequence was identical to that of IS6100, an insertion sequence classified in the IS6 family, initially found in Mycobacterium fortuitum. Sequences homologous to that of IS6100 were found for another nylon oligomer-degrading plasmid, pNAD2, harbored in Pseudomonas sp. strain NK87, by Southern hybridization experiments.

Amino Acid Sequence↗
PubMed1993

Stabilization of xylanase by random mutagenesis.

Four heat-resistant mutants of xylanase (N56, N102, N104 and F1) were obtained by random mutagenesis. The mutant genes had the following amino acid changes: N56, Ser-26 to Trp, Gly-38 to Asp and Thr-126 to Ser; N102, Gly-38 to Asp; N104, Gly-38 to Ser and Arg-48 to Lys; F1, Ser-12 to Cys. Kinetic studies showed that N104 is stabilized by an increase in the activation enthalpy, while the other mutants are stabilized by a decrease in the activation entropy.

Endo-1,4-beta Xylanases↗
PubMed1993

Characterization of the 6-aminohexanoate-dimer hydrolase from Pseudomonas sp. NK87.

The DNA base sequence of the Pseudomonas sp. NK87 gene (P-nylB) for 6-aminohexanoate-dimer hydrolase (P-EII), a xenobiotic-compound-degrading enzyme, was determined. It has an open reading frame of 1188 bp, initiated by ATG and terminated by TAG, and coding for 396 amino acids. The base sequence of the open reading frame has 53% sequence similarity to that of the gene for the same enzyme of Flavobacterium sp. KI72 (F-nylB) and 35% sequence similarity with respect to the deduced amino acid sequence. The P-EII enzyme was purified from an Escherichia coli clone in which the P-EII gene was highly expressed. The P-EII enzyme was inhibited by a serine protease inhibitor, diisopropyl fluorophosphate, as was the F-EII enzyme. Double reciprocal plots obtained from various concentrations of 6-aminohexanoate-dimer indicated that the kcat value of the P-EII enzyme (9.2 s-1) was approximately half that of the F-EII enzyme (19 s-1), and the P-EII enzyme had higher affinity toward this substrate (Km for P-EII, 0.6 mM; Km for F-EII, 15 mM). The P-EII enzyme had a temperature optimum of 48 degrees C, and a pH optimum of 7.5. It is speculated that since the P-nylB and F-nylB genes are more diverged from each other than the corresponding nylA genes, the latter may have evolved more recently.

Amidohydrolases↗
PubMed1993

Nylon oligomer degradation gene, nylC, on plasmid pOAD2 from a Flavobacterium strain encodes endo-type 6-aminohexanoate oligomer hydrolase: purification and characterization of the nylC gene product.

A new type of nylon oligomer degradation enzyme (EIII) was purified from an Escherichia coli clone harboring the EIII gene (nylC). This enzyme hydrolyzed the linear trimer, tetramer, and pentamer of 6-aminohexanoate by an endo-type reaction, and this specificity is different from that of the EI (nylA gene product) and EII (nylB gene product). Amino acid sequencing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified EIII demonstrated that the enzyme is made of two polypeptide chains arising from an internal cleavage between amino acid residues 266 and 267.

Amidohydrolases↗
PubMed1992

Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus.

To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.

Aspartic Acid↗
PubMed1992

No stop codons in the antisense strands of the genes for nylon oligomer degradation.

Genes for nylon oligomer-degrading enzymes are unique in the sense that the enzymes encoded by them are found not to have any appropriate substrates during most of the period of their evolution. Furthermore, these nylB genes form a family not related to any other known gene families. The base sequences of these genes were examined and a common characteristic was found: a long stretch of sequence without chain-terminating base triplets, defined as a nonstop frame (NSF), is being maintained on the antisense strand. Moreover, a certain coding frame is open for both the sense and the antisense sequences, while the other frames have many stop codons. The probability of the presence of these NSFs on the antisense strand of a gene is very small (0.0001-0.0018). In addition, another gene for nylon oligomer degradation was found to have a NSF on its antisense strand, and this gene is phylogenically independent of the nylB genes. Therefore, the presence of these NSFs is very rare and improbable. Even if the common ancestral gene of the nylB family was originally endowed with a NSF on its antisense strand, the probability of this original NSF persisting in one of its descendants of today is only 0.007. Unless an unknown force was maintaining the NSF, it would have quickly disappeared by random emergences of chain terminators. Therefore, the presence of such rare NSFs on all three antisense strands of the nylB gene family suggests that there is some special mechanism for protecting these NSFs from mutations that generate the stop codons. Such a mechanism may enable NSFs to evolve into new functional genes and hence seems to be a basic mechanism for the birth of new enzymes.

Amidohydrolases↗
PubMed1992

Preparation and kinetic properties of 5-ethylphenazine-lactate-dehydrogenase-NAD+ conjugate, a semisynthetic lactate oxidase showing a hide-and-seek effect.

5-Ethylphenazine-lactate-dehydrogenase-NAD+ conjugate (EP(+)-LDH-NAD+) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD+ to lactate dehydrogenase. The average number of the ethylphenazine moieties bound per molecule of enzyme subunit was 0.46, and that of the NAD+ moieties was 0.32. This conjugate is a semisynthetic enzyme having lactate oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor; to make such conjugates seems to be a general method for artificially converting a dehydrogenase into an oxidase. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following kinetic constants: for the reaction system with oxygen, the turnover number per subunit is 2.3 min-1 and Km for oxygen is 1.91 mM; and for the system with MTT, the turnover number is 0.25 min-1 and Km for MTT is 0.076 mM. At the initial steady state of the oxidase reaction, only 2.1% of the NAD+ moieties of the conjugate are in the free state (i.e. not bound in the coenzyme-binding site of the lactate dehydrogenase moiety) and the rest are hidden in the coenzyme site; almost all the NAD+ moieties are in the reduced state. The apparent intramolecular rate constant for the reaction between a free NADH moiety and an oxidized ethylphenazine moiety is 2.3 s-1 and 2.1 s-1 for the systems with oxygen and with MTT, respectively. The apparent effective concentration of the free NADH moiety for the ethylphenazine moiety is 5.5 microM and is much smaller than that (0.34 mM) of the ethylphenazine moiety for the free NADH moiety; this difference is due to the effect of hiding the NADH moiety in the binding site, as the hidden NADH moiety cannot react with the ethylphenazine moiety.

Animals↗
PubMed1992

Principles for designing enzyme-like catalysts based on the rate-acceleration mechanisms of semisynthetic oxidases.

Combinations of substrate-binding sites and catalytic groups constitute various kinds of enzyme-like catalysts. The design of such catalysts can be evaluated by the enhancement of the overall catalytic activity by combining these parts into one catalyst. For a catalyst having one substrate-binding site and one catalytic group, an equation was obtained which shows the relationship between the rate-acceleration due to the combination, the affinity of the site (1/Kd), intrinsic effective concentration (kin/kex) and substrate concentration ([S]). The intrinsic effective concentration is the ratio of the first-order rate constant (kin) of the intramolecular reaction between the catalytic group and the bound substrate and the second-order rate constant (kex) of the intermolecular reaction between the catalytic group and the free substrate; the value depends on the method of linking the catalytic group and the binding site. This equation provides the following principles for designing catalysts of this type with a considerable grade of rate-acceleration: [S] less than or equal to kin/kex and (1/10)[S] less than or equal to Kd less than or equal to kin/kex. To increase kin/kex, the structure of the binding site is required not to reduce the reactivity of the bound substrate, and the linker connecting the binding site and the catalytic group is required to be flexible and to have an appropriate length. A subunit structure is also found to be effective to improve the catalytic activity: the activity of an n-mer is at most n2 times as high as that of the monomer. As for the substrate-binding sites, the sites of natural enzymes and antibodies are good candidates because various kinds of binding sites with high affinity and specificity to the corresponding substrates are available. In addition, the equation relating the rate-acceleration with Kd, kin/kex, and [S] is used for explaining the catalytic efficiency of enzymes energetically. The principle for designing a multifunctional catalyst having several kinds of binding sites for its substrates and intermediates and several kinds of catalytic groups was then investigated. In this case, the diffusion of the intermediates strongly affects the activity of the multifunctional catalyst, and such a diffusion process was also analyzed. On the basis of these analyses, the following principles were obtained.(ABSTRACT TRUNCATED AT 400 WORDS)

Catalysis↗
PubMed1992

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin-sensitive cells.

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.

Amino Acid Sequence↗
PubMed1992

Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030.

Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.

Amino Acid Sequence↗
PubMed1992

A new nylon oligomer degradation gene (nylC) on plasmid pOAD2 from a Flavobacterium sp.

Flavobacterium sp. strain KI725 harbors plasmid pOAD21, a derivative of nylon oligomer-degradative plasmid pOAD2, in which all of nylA (the gene for 6-aminohexanoate cyclic dimer hydrolase [EI]) was deleted but nylB (the gene for 6-aminohexanoate dimer hydrolase [EII]) was retained. KI725 showed no growth on unfractionated nylon oligomers (Nom1) obtained from a nylon factory as a sole carbon and nitrogen source (Nom1 minimum plate). Extracts of KI725 cells possessed hydrolytic activity for Nom1 (approximately 5% of the activity of KI72), but pOAD2-cured strains (KI722 and KI723) showed no activity. KI725R strains which grew on the Nom1 minimum plate were spontaneously isolated from KI725 at a frequency of 10(-7) per cell. Activity toward Nom1 was enhanced in KI725R strains (10 to 30% of the activity of KI72). This new Nom1 degrading enzyme (EIII, the nylC gene product) hydrolyzed not only Nom1 but also the N-carbobenzoxy-6-aminohexanoate trimer, a substrate which was not hydrolyzed by either EI or EII. Cloning and sequence analysis showed that the nylC gene is located close to nylB on pOAD21 and is a 1,065-bp open reading frame corresponding to 355 amino acid residues. The nucleotide sequence of the nylC gene and the deduced amino acid sequence of EIII had no detectable homology with the sequences of nylA (EI) and nylB (EII).

Amidohydrolases↗
PubMed1991

Sequence and properties of beta-xylosidase from Bacillus pumilus IPO. Contradiction of the previous nucleotide sequence.

The nucleotide sequence of the beta-xylosidase (xynB) gene from Bacillus pumilus has been reported previously [Moriyama, H., Fukusaki, E., Crespo, J.C., Shinmyo, A. & Okada, H. (1987) Eur. J. Biochem. 166, 539-545]. However, the sequence identified in the present study is quite different from the previously reported one. The total length of the PstI--EcoRI fragment of a plasmid pOXN295 containing the xynB gene is 2201 bp from our sequencing, while the length of the fragment in the previous data was 2466 bp. The sequences are similar in the N-terminal (500 bp) and C-terminal (260 bp) regions, but those in the central region are completely different. From the following observations, the previous sequence seems to have no reliable experimental basis. First, the restriction sites observed for pOXN295 are quite different from the sites deduced from the sequence. Second, the amino acid composition deduced from the sequence and the composition identified by amino acid analysis of the purified beta-xylosidase are very different. It is confirmed, on the other hand, that our new sequence agrees well with these experimental data. The enzyme was purified to homogeneity from Bacillus pumilus and Escherichia coli harboring a hybrid plasmid which highly expresses the xynB gene. The molecular mass of the enzyme was estimated to be 190 kDa by high performance gel filtration chromatography using TSK-G3000SW and 56 kDa by SDS/polyacrylamide gel electrophoresis. The pH optimum was 7.0, and the optimum temperature was 40 degrees C. The Vm value was estimated to be 1.23 +/- 0.14 mukat/mg (or p-nitrophenyl beta-D-xyloside) and 0.14 +/- 0.011 mukat/mg (for xylobiose), while Km was estimated to be 3.9 +/- 0.59 mM (for p-nitrophenyl beta-D-xyloside) and 8.9 +/- 1.19 mM (for xylobiose).

Amino Acid Sequence↗
PubMed1991

Preparation and kinetic properties of 5-ethylphenazine-glucose-dehydrogenase-NAD+ conjugate, a semisynthetic glucose oxidase.

5-Ethylphenazine-glucose-dehydrogenase-NAD+ conjugate (EP(+)-GlcDH-NAD+) was prepared by linking both poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD+ to glucose dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.8, and that of the NAD+ moieties was 1.2. This conjugate is a semisynthetic enzyme having glucose oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following values of the kinetic constants: for the system with oxygen, the turnover number per subunit is 0.40 s-1 and Km for oxygen is 1.57 mM; and for the system with MTT, the turnover number is 0.11 s-1 and Km for MTT is 0.072 mM. The catalytic cycle of the semisynthetic oxidase has two catalytic steps: reduction of the NAD+ moiety by the active site of the glucose dehydrogenase moiety and oxidation of the NADH moiety by another catalytic site of the ethylphenazine moiety. The apparent intramolecular rate constants of these steps were estimated, and the values are as follows: 0.39 s-1 for the reductions of the NAD+ moiety, 2.2 s-1 and 0.12 s-1 for the oxidation of the NADH moiety in the systems with oxygen and with MTT, respectively, and 3.2 s-1 and 0.18 s-1 for the reduction of the ethylphenazine moiety in the systems with oxygen and with MTT, respectively. On the bases of these results, the following three rate-acceleration mechanisms of the semisynthetic glucose oxidase are discussed: high effective concentration, intramolecular coupling of successive catalytic reactions, and multiple connection between the two kinds of the catalytic sites.

Binding Sites↗
PubMed1991

Amino acid alterations essential for increasing the catalytic activity of the nylon-oligomer-degradation enzyme of Flavobacterium sp.

The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase (EII; nylB) and its evolutionally related protein EII' (nylB') of Flavobacterium sp. KI72 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites. The specific activity of EII towards 6-aminohexanoate dimer is about 1000-fold that of EII'. Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII' enzyme, i.e. Gly181----Asp (EII type) and His266----Asn (EII type), enhanced the activity toward 6-aminohexanoate dimer 1000-fold.

Amidohydrolases↗
PubMed1991

Preparation and kinetic properties of 5-ethylphenazine-poly(ethylene-glycol)-glutamate-dehydrogenase conjugate. A semisynthetic NADH oxidase.

5-Ethylphenazine-poly(ethylene glycol)-glutamate dehydrogenase conjugate (EP(+)-PEG-GluDH) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine to glutamate dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.7. This conjugate is a semisynthetic enzyme having NADH oxidase activity; the ethylphenazine moiety works as a catalytic group, and the coenzyme-binding site of glutamate dehydrogenase works as a substrate-binding site. The effects of the presence of the substrate-binding site near the catalytic group were studied by using EP(+)-PEG-GluDH. Before the preparation of the conjugate, the reactivity of NADH bound in the coenzyme-binding site toward the ethylphenazine moiety was estimated for glutamate and lactate dehydrogenases. The results show that the NADH molecule bound in the site of glutamate dehydrogenase reacts with EP(+)-PEG at a rate of 43% of that of free NADH, but the NADH molecule bound in lactate dehydrogenase does not react with 1-(3-carboxypropyloxy)-5- ethylphenazine. Therefore, glutamate dehydrogenase was used as the substrate-binding site of the semisynthetic NADH oxidase. The results of the kinetic analysis of the activity of EP(+)-PEG-GluDH show that the apparent turnover number of the active site is 0.38 s-1, which corresponds to the apparent intramolecular rate constant of the oxidation of NADH bound in the active site. The apparent effective concentration of bound NADH for the catalytic group of the ethylphenazine moiety is 0.33 mM. This means that the presence of the substrate-binding site near the catalytic group increases the local NADH concentration by at most 0.33 mM, and this is the rate-accelerating effect of the binding site.

Binding Sites↗
PubMed1990

Electrostatic and redox potential effects on the rat of electron-transfer reaction of nicotinamide adenine dinucleotides with 1-substituted 5-ethylphenazines.

The effects of redox potential and electric charge on the rate of electron-transfer reaction by a two-electron process were investigated. For electron donors, beta-NADH, beta-NADPH and alpha-NADH were used; they have similar structures but different charges and different redox potentials. For electron acceptors, the following 5-ethylphenazine derivatives were used: 1-(3-carboxypropyloxy)-5-ethylphenazine, 1-(3-ethoxycarbonylpropyloxy)-5-ethylphenazine, and 1-[N-(2-aminoethyl)carbamoylpropyloxy]-5-ethylphenazine. They have similar structures and different charges. Using these donors and acceptors, the potential and the charge effects were estimated separately. In the potential effect, a linear free energy relationship was observed for the change in the redox potential of the donor with a Brønsted slope of about unity. On the other hand, the slope for the change in the potential of the acceptor was about 0.5. These results show that the potential effect due to electron donors is different from that due to electron acceptors. A linear relationship was also observed between activation free energy and electrostatic force (or potential). The redox potential effect and the electrostatic effect are independent and additive. New theory for the mechanism of electron-transfer reactions is needed to explain these results.

Electricity↗