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H Kagamiyama

Publications and source records attributed to H Kagamiyama.

At least 37 records · Page 2Linked to original sources
PubMed1999

Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues.

Directed evolution was used to change the substrate specificity of aspartate aminotransferase. A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 x 10(6)-fold increase in the catalytic efficiency (kcat/Km) for a non-native substrate, valine. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, is also changed significantly by the mutations. Interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-A resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding. The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.

Amino Acid Sequence↗
PubMed1999

A genetic approach to visualization of multisynaptic neural pathways using plant lectin transgene.

The wiring patterns among various types of neurons via specific synaptic connections are the basis of functional logic employed by the brain for information processing. This study introduces a powerful method of analyzing the neuronal connectivity patterns by delivering a tracer selectively to specific types of neurons while simultaneously transsynaptically labeling their target neurons. We developed a novel genetic approach introducing cDNA for a plant lectin, wheat germ agglutinin (WGA), as a transgene under the control of specific promoter elements. Using this method, we demonstrate three examples of visualization of specific transsynaptic neural pathways: the mouse cerebellar efferent pathways, the mouse olfactory pathways, and the Drosophila visual pathways. This strategy should greatly facilitate studies on the anatomical and functional organization of the developing and mature nervous system.

Animals↗
PubMed1999

Directed evolution of thermostable kanamycin-resistance gene: a convenient selection marker for Thermus thermophilus.

The whole-genome sequencing of an extreme thermophile, Thermus thermophilus, is now in progress. Like other genome projects, major concern is shifting from the sequence itself to post-sequencing research such as functional or structural genomics. Under such circumstances, the demand for convenient genetic-engineering tools is increasing. In this study we have increased the thermostability of a kanamycin-resistance gene product using strategies based on directed evolution in T. thermophilus to the upper limit of its growth temperature. The most thermostable mutant has 19 amino-acid substitutions, whereby the thermostability is increased by 20 degrees C, but the enzymatic activity is not significantly changed. Most of the mutated residues are located on the surface of the protein molecule, and, interestingly, five of the 19 substitutions are those to proline residues. The evolved kanamycin-resistance gene products could be used as selection markers at the optimum growth temperature of T. thermophilus. The development of such a convenient genetic-engineering tool would facilitate post-sequencing research on T. thermophilus.

Amino Acid Substitution↗
PubMed1998

The imine-pyridine torsion of the pyridoxal 5'-phosphate Schiff base of aspartate aminotransferase lowers its pKa in the unliganded enzyme and is crucial for the successive increase in the pKa during catalysis.

In aspartate aminotransferase, pyridoxal 5'-phosphate (PLP) forms a Schiff base with the epsilon-amino group of Lys258 (internal aldimine). The internal aldimine has a pKa value of 6.8. Binding of a substrate amino acid to the enzyme yields the Michaelis complex, in which PLP still forms the internal aldimine with Lys258. This is followed by a transaldimination process to form a Schiff base of PLP with the alpha-amino group of substrates (external aldimine). Kinetic analysis of the spectral changes during the reaction of the enzyme with a substrate analogue 2-methylaspartate showed that the aldimine is 6.4-8.6% protonated in the Michaelis complex and 32-43% in the external aldimine. The bases that accept protons from the aldimines are considered to be the substrate alpha-amino group in the Michaelis complex and the epsilon-amino group of Lys258 in the external aldimine. Therefore, the intrinsic pKa value of the aldimine is expected to increase over a range of 3 during transformation from the unliganded enzyme (pKa = 6.8) to the Michaelis complex (pKa = 8.8) and the external aldimine (pKa > 10). When the Lys258 side chain of the internal aldimine was "cleaved" by the construction of an enzyme in which Lys258 was replaced by Ala and the aldimine was reconstituted with methylamine, the pKa of the internal aldimine was increased to 9.6. This indicates that the low pKa value of the internal aldimine of the unliganded enzyme is provided by the side chain of Lys258 which destabilizes the planar conformation of the aldimine suitable for protonation. This strained conformation is partially relaxed in the Michaelis complex, and the pKa is moderately increased. On formation of the external aldimine, Lys258 is released and the aldimine is fixed to a near planar conformation and has a high pKa value. Thus, the aldimine pKa is modulated by a mechanism that exploits the conformational differences between the intermediate structures. The strain of the protonated internal aldimine is interpreted to enhance the catalytic ability of the enzyme by increasing the energy level of the free enzyme plus substrate at neutral pH relative to the transition state.

Alanine↗
PubMed1998

cDNA cloning and characterization of mouse nifS-like protein, m-Nfs1: mitochondrial localization of eukaryotic NifS-like proteins.

We have isolated a mouse cDNA which shows significant sequence similarity to the yeast nifS-like gene (y-NFS1), and termed it m-Nfs1. The deduced protein sequence (459 amino acids long) has several characteristic features common to those of bacterial NifS proteins, but distinct from them by its amino-terminal extension which contains a typical mitochondrial targeting presequence. m-Nfs1 was found to be a soluble 47-kDa protein in the matrix fraction of mouse liver mitochondria. The m-Nfs1 gene was ubiquitously expressed in most tissues, suggesting its housekeeping function in vivo. We also found that the gamma-NFS1 protein was localized in the mitochondrial matrix in yeast cells. These results suggest that both eukaryotic NifS-like proteins may play some roles in mitochondrial functions.

Amino Acid Sequence↗
PubMed1998

Crystal structures of Paracoccus denitrificans aromatic amino acid aminotransferase: a substrate recognition site constructed by rearrangement of hydrogen bond network.

Aminotransferase reversibly catalyzes the transamination reaction by a ping-pong bi-bi mechanism with pyridoxal 5'-phosphate (PLP) as a cofactor. Various kinds of aminotransferases developing into catalysts for particular substrates have been reported. Among the aminotransferases, aromatic amino acid aminotransferase (EC 2.6.1. 57) catalyzes the transamination reaction with both acidic substrates and aromatic substrates. To elucidate the multiple substrate recognition mechanism, we determined the crystal structures of aromatic amino acid aminotransferase from Paracoccus denitrificans (pdAroAT): unliganded pdAroAT, pdAroAT in a complex with maleate as an acidic substrate analog, and pdAroAT in a complex with 3-phenylpropionate as an aromatic substrate analog at 2.33 A, 2. 50 A and 2.30 A resolution, respectively. The pdAroAT molecule is a homo-dimer. Each subunit has 394 amino acids and one PLP and is divided into small and large domains. The overall structure of pdAroAT is essentially identical to that of aspartate aminotransferase (AspAT) which catalyzes the transamination reaction with only an acidic amino acid. On binding the acidic substrate analog, arginine 292 and 386 form end-on salt bridges with carboxylates of the analog. Furthermore, binding of the substrate induces the domain movement to close the active site. The recognition mechanism for the acidic substrate analog in pdAroAT is identical to that observed in AspAT. Binding of the aromatic substrate analog causes reorientation of the side-chain of the residues, lysine 16, asparagine 142, arginine 292* and serine 296*, and changes in the position of water molecules in the active site to form a new hydrogen bond network in contrast to the active site structure of pdAroAT in the complex with an acidic substrate analog. Consequently, the rearrangement of the hydrogen bond network can form recognition sites for both acidic and aromatic side-chains of the substrate without a conformational change in the backbone structure in pdAroAT.

Bacterial Proteins↗
PubMed1998

Activation of protein phosphatase 2A by cAMP-dependent protein kinase-catalyzed phosphorylation of the 74-kDa B'' (delta) regulatory subunit in vitro and identification of the phosphorylation sites.

Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B'' (delta) subunit, was phosphorylated at serine residues of B'' in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 microM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B'', respectively. The Km value of A-kinase for CAB'' was 0.17 +/- 0.01 microM in the presence of OA. The major in vitro phosphorylation sites of B'' were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B'' did not dissociate B'' from CA, and stimulated the molecular activity of CAB'' toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a.

Animals↗
PubMed1998

Directed evolution of an aspartate aminotransferase with new substrate specificities.

The substrate specificity of aspartate aminotransferase was successfully modified by directed molecular evolution using a combination of DNA shuffling and selection in an auxotrophic Escherichia coli strain. After five rounds of selection, one of the evolved mutants showed a 10(5)-fold increase in the catalytic efficiency (kcat/Km) for beta-branched amino and 2-oxo acids and a 30-fold decrease in that for the native substrates compared with the wild-type enzyme. The mutant had 13 amino acid substitutions, 6 of which contributed 80-90% to the total effect. Five of these six substitutions were conserved among the five mutants that showed the highest activity for beta-branched substrates. Interestingly, only one of the six functionally important residues is located within a distance of direct interaction with the substrate, supporting the idea that rational design of the substrate specificity of an enzyme is very difficult. The present results show that directed molecular evolution is a powerful technique for enzyme redesign if an adequate selection system is applied.

Amino Acid Sequence↗
PubMed1998

Analysis of the pH- and ligand-induced spectral transitions of tryptophanase: activation of the coenzyme at the early steps of the catalytic cycle.

Tryptophanase has an absorption maximum at 338 nm at high pH and 422 nm at low pH. The 422-nm absorption species has been considered to be the catalytically competent ketoenamine form of the Schiff base of pyridoxal 5'-phosphate with a lysine residue. The 338-nm absorption band showed an intense fluorescence band at 390 nm and not around 500 nm, indicating that the 338-nm absorption species is the substituted aldamine rather than an enolimine form of the Schiff base which has been suggested previously. To explore the mechanism of the enzyme that can exert its catalytic ability at high pH where most of its coenzyme exists as the catalytically incompetent aldamine structure, the reaction of tryptophanase with 3-indolepropionate, a substrate analogue that stops the reaction at the step of the Michaelis complex, was studied at various pH values and analogue concentrations. Kinetic analysis was done based on a scheme involving eight forms of the enzyme, i.e., the liganded and unliganded forms of the ketoenamine, the substituted aldamine structures, and their protonated and deprotonated forms. Kinetic parameters were obtained for each interconversion step. The results showed that the binding of 3-indolepropionate to tryptophanase shifts the equilibrium from the substituted aldamine to the ketoenamine structure over the entire pH region studied. This implies that in the reaction of tryptophanase with tryptophan at high pH, where the enzyme shows maximum activity, the binding of the substrate to the enzyme converts the inactive aldamine form of the coenzyme to the active ketoenamine form. Mechanisms for the activation process, in which a nucleophile is expelled from the aldamine either by steric hindrance of the nucleophile with the ligand or by the negative charge of the ligand alpha-carboxylate group that stabilizes the aldimine structure, were discussed.

Catalysis↗
PubMed1998

Aromatic L-amino acid decarboxylase: conformational change in the flexible region around Arg334 is required during the transaldimination process.

Aromatic L-amino acid decarboxylase (AADC) catalytic mechanism has been proposed to proceed through two consecutive intermediates (i.e., Michaelis complex and the external aldimine). Limited proteolysis of AADC that preferentially digested at the C-terminal side of Arg334 was slightly retarded in the presence of dihydroxyphenyl acetate that formed a stable Michaelis complex. On the contrary, AADC was scarcely digested in the presence of L-dopa methyl ester that formed a stable external aldimine. Similar protection by the substrate analogs was observed in the chemical modification experiment. From these results, we concluded that the region around Arg334 must be exposed and flexible in the unliganded state, and forming the Michaelis complex generated a subtle conformational change, then underwent marked conformational change during the subsequent transaldimination process prerequisite to forming the external aldimine. For further analyses, we constructed a mutant gene encoding in tandem the two peptides of AADC cleaved at the Asn327-Met328 bond inside the putative flexible region. The gene product, fragmentary AADC, was still active with L-dopa as substrate, but its k(cat) value was decreased 57-fold, and the Km value was increased 9-fold compared with those of the wild-type AADC. The absorption spectra of the fragmentary AADC in the presence of L-dopa methyl ester showed shift in the equilibrium of the transaldimination from the external aldimine to the Michaelis complex. Tryptic digestion of the fragmentary AADC removed seven amino acid residues, Met328-Arg334, and resulted in complete inactivation. Susceptibility of the fragmentary enzyme to trypsin was not changed by L-dopa methyl ester revealing the loss of appropriate conformational change in the flexible region induced by substrate binding. From these results we propose that the conformational change in the flexible region is required during the transaldimination process.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1997

Transient-state kinetics of the reaction of aspartate aminotransferase with aspartate at low pH reveals dual routes in the enzyme-substrate association process.

In aspartate aminotransferase, the coenzyme pyridoxal 5'-phosphate forms a Schiff base with the epsilon-amino group of Lys258. The pH dependency of the steady-state kinetics of the overall reaction had indirectly suggested that the Schiff-base-unprotonated form of the enzyme (EL) is the active species that binds the monoanionic form of aspartate (SH+), the predominant species of the substrate in solution. In order to obtain direct information on the association process, we carried out transient-phase kinetics of the first half-reaction of the enzyme with aspartate at various pH. The disappearance of EL (lambdamax = 358 nm) was fast and independent of pH, but the disappearance of ELH+ (Schiff-base-protonated form, lambdamax = 430 nm) was slow and dependent on pH. At pH values below 6.8 and low concentrations of aspartate, the results could be interpreted to indicate that EL reacts rapidly with SH+ to form the pyridoxamine 5'-phosphate form of the enzyme (EM), and the reaction of ELH+ proceeds via the route ELH+ right arrow over left arrow EL right arrow over left arrow EM, where the first step was found to be rate limiting from the pH jump/drop study of the enzyme. At higher pH values, the rate of disappearance of ELH+ became larger than expected from the above scheme. This deviation became apparent with increasing pH, and could be excellently explained if we consider that it is due to the reaction of ELH+ with the dianionic form of aspartate (S). Thus, the formation of the Michaelis complex of aspartate aminotransferase and aspartate can proceed via two routes; route A is the association of EL with SH+ to form EL.SH+, which converts intramolecularly to ELH+.S, and route B is the association of ELH+ with S to form ELH+.S directly. ELH+.S is the prerequisite structure for further processing of the substrate by the enzyme. The reactions of EM and oxo acids yielded almost exclusively EL and SH+, and therefore route B does not seem to play an essential role in the overall reactions of the enzyme. Route B, however, may be important in the reaction mechanisms of other pyridoxal 5'-phosphate enzymes which have only the ELH+ form.

Aspartate Aminotransferases↗
PubMed1997

OCAM: A new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomeronasal axons.

Zone-to-zone projection of olfactory and vomeronasal sensory axons underlies the topographic and functional mapping of chemoreceptor expression zones of the sensory epithelia onto zonally arranged glomeruli in the main and accessory olfactory bulbs. Here we identified OCAM (R4B12 antigen), an axonal surface glycoprotein expressed by subsets of both olfactory and vomeronasal axons in a zone-specific manner. OCAM is a novel homophilic adhesion molecule belonging to the immunoglobulin superfamily with striking structural homology to neural cell adhesion molecule. In both the main and accessory olfactory systems, OCAM mRNA is expressed by sensory neurons in restricted chemoreceptor expression zones, and OCAM protein-expressing axons project to the glomeruli in the corresponding zones of the main and accessory bulbs. OCAM protein is expressed on subsets of growing sensory axons in explant cultures even in the absence of the target bulb. These results demonstrate a precisely coordinated zonal expression of chemoreceptors and OCAM and suggest that OCAM may play important roles in selective fasciculation and zone-to-zone projection of the primary olfactory axons.

Amino Acid Sequence↗
PubMed1997

Use of 1H-15N heteronuclear multiple-quantum coherence NMR spectroscopy to study the active site of aspartate aminotransferase.

Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, was uniformly and selectively enriched with 15N and was studied by heteronuclear multiple-quantum coherence NMR spectroscopy in H2O. Good resolution was obtained for the downfield region (above 9.5 ppm chemical shift in the 1H dimension) for NH protons in the amide, indole, imidazole, and guanidinium group regions and several resonances were tentatively assigned. Two downfield resonances, at 12.6 and 11.36 ppm, appear to belong to oxygen- or sulfur-bound protons. The most downfield amide resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 A away from the negatively charged carboxyl group of aspartate 199. Large downfield shifts (up to 1.15 ppm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-phosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-phosphate (PMP) form of the enzyme. We discuss these striking differences in the light of the available crystallographic data. Active sites of proteins, as well as specific inhibitory molecules, often contain negatively charged groups. These may be able to form hydrogen-bonds to NH groups and to shift the NH resonances downfield into a less crowded and therefore more readily observable region for many large proteins. Our approach, which makes use of both HMQC spectroscopy and NOE observations, should be widely applicable.

Aspartate Aminotransferases↗
PubMed1997

cDNA cloning and chromosomal localization of the human telencephalin and its distinctive interaction with lymphocyte function-associated antigen-1.

We have isolated cDNA encoding human telencephalin (TLN), a brain segment-specific neuronal adhesion molecule. Human TLN comprises an NH2-terminal signal peptide, an extracellular region with nine Ig-like domains, a single transmembrane region, and a COOH-terminal cytoplasmic tail. The NH2-terminal five Ig-like domains of TLN were closely related to those of intercellular adhesion molecules (ICAMs)-1 and -3. The TLN gene was mapped to the human chromosome 19p13.2, where the ICAM-1, -3, and -4 (LW) genes are located. Furthermore, we observed lymphocyte function-associated antigen-1 (LFA-1)-mediated adhesion of HL-60 cells on recombinant TLN protein, as well as on ICAM-1. However, the interaction of TLN with LFA-1 on HL-60 cells was divalent cation-independent and phorbol 12-myristate 13-acetate stimulation-independent. We conclude that TLN is a unique neuronal member of ICAM subgroup of the Ig superfamily and propose a novel type of interaction between the Ig superfamily molecule and integrin, which does not require the activation of integrin. TLN on the surface of telencephalic neurons may be a target molecule in the brain for LFA-1-expressing microglia and leukocytes in physiological or pathological conditions.

Amino Acid Sequence↗
PubMed1997

Identification of autophosphorylation sites in c-Yes purified from rat liver plasma membranes.

c-Yes was purified 322-fold from a rat liver plasma membrane fraction to a single 60-kDa band on SDS-PAGE. The purified protein contained essentially no phosphotyrosine residues and was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues with a concomitant increase in the protein-tyrosine kinase activity. The autophosphorylated c-Yes was extensively digested by trypsin and the resultant two major phosphopeptides, peptides I and II, were purified by HPLC on a reversed-phase C-18 column. The amino acid sequence of peptide I was determined to be LIEDNEYTAR, which is identical with the sequence from Leu-418 through Arg-427 of mouse c-Yes, indicating that one of the autophosphorylation sites corresponds to Tyr-424 of the mouse c-Yes. After partial determination of the N-terminal sequence of 10 amino acid residues of peptide II, the 230 bp sequence of rat cDNA that encodes the N-terminal 76 amino acid residues of c-Yes covering peptide II, was determined. From the predicted amino acid sequence, the sequence of peptide II was assumed to be from Tyr-16 through Lys-46, YTPENPTEPVNTSAGHYGVEHATAATTSSTK. The purified c-Yes phosphorylated the tyrosine residue of synthetic peptides covering Tyr-32 and its surrounding sequence but did not phosphorylate peptides covering Tyr-16 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.

Amino Acid Sequence↗
PubMed1997

Paracoccus denitrificans aromatic amino acid aminotransferase: a model enzyme for the study of dual substrate recognition mechanism.

The gene for aromatic amino acid aminotransferase (ArAT) from Paracoccus denitrificans was cloned, sequenced, and overexpressed in Escherichia coli cells. The sequence differed from that reported previously [Takagi, T., Taniguchi, T., Yamamoto, Y., and Shibatani, T. (1991) Biotechnol. Appl. Biochem. 13, 112-119]. The enzyme (pdArAT) was purified to homogeneity, and characterized. It was similar to aspartate aminotransferase (AspAT) and ArAT of E. coli (ecArAT) in many respects, including gross protein structure and spectroscopic properties. pdArAT showed activities toward both dicarboxylic and aromatic substrates, and analysis of the binding of substrate analogs and quasisubstrates to the enzyme showed that both dicarboxylic and aromatic substrates take a similar orientation in the active site of pdArAT; these properties are essentially identical with those of ecArAT. As in the case of ecArAT, neutral amino acids with larger side chains are better substrates for pdArAT, suggesting that hydrophobic interaction between the substrate and the enzyme is important for the recognition of substrates with neutral side chains. pdArAT catalyzed transamination of phenylalanine and tyrosine far more efficiently (10(2)-fold in terms of kcat/Km) than those of straight-chain aliphatic amino acids with similar side-chain surface area, whereas ecArAT did not show significant preference for aromatic amino acids over aliphatic amino acids. This shows that the substrate-side-chain-binding pocket of pdArAT, as compared with the pocket of ecArAT, is well suited in shape for interaction with the phenyl and hydroxyphenyl rings of substrates. Thus, pdArAT is an ideal enzyme among ArATs for the study of the high-specificity recognition of two different kinds of substrates, the one having a carboxylic side chain and the other having an aromatic side chain.

Amino Acid Sequence↗
PubMed1997

An anomalous side reaction of the Lys303 mutant aromatic L-amino acid decarboxylase unravels the role of the residue in catalysis.

Aromatic L-amino acid decarboxylase (AADC) in which the pyridoxal 5'-phosphate (PLP)-binding residue Lys303 was replaced by an alanine residue is virtually inactive as a catalyst. On reaction of the normal substrate L-dopa with this mutant AADC, the absorption at around 330 nm gradually increased with concomitant decrease of the absorption of the free PLP molecule at 390 nm. Analysis of the 330-nm absorbing species on HPLC and spectrophotometry showed that it is a 1:1 adduct of PLP and dopamine, probably the Pictet-Spengler type adduct formed from the PLP-dopamine Schiff base. The product dopamine was not found outside of the enzyme, showing that the PLP-dopamine Schiff base undergoes adduct formation rather than yields product dopamine. The Pictet-Spengler adduct of PLP with L-dopa was not detected, whereas the corresponding adduct was formed when the carboxylate group of L-dopa was esterified with a methyl group to block the decarboxylation reaction. This suggests that the PLP-L-dopa Schiff base may undergo the Pictet-Spengler reaction, but its rate is much smaller than that of decarboxylation. These results indicate that Lys303 is not essential for the decarboxylation step, but has an important role in the product release, and possibly the transaldimination process.

Animals↗