Biomedical subjects

M Yohda

Publications and source records attributed to M Yohda.

At least 19 recordsLinked to original sources

PubMed1999

Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine.

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.

Amino Acid Sequence↗

PubMed1998

Characterization of homo-oligomeric complexes of alpha and beta chaperonin subunits from the acidothermophilic archaeon, Sulfolobus sp. strain 7.

The chaperonin from the acidothermophilic archaeon, Sulfolobus sp. Strain 7, is composed of two kinds of subunits designated as Scp alpha and Scp beta. In this study, we characterized the recombinant Scp alpha and Scp beta, which were separately expressed in Escherichia coli. Both of them were able to assemble to homo-oligomeric double-ring complexes, similar to subunits of group II chaperonins from Thermoplasma acidophilum and Thermococcus strain KS-1. Both complexes have no or at most trace ATPase activities. However, they could arrest spontaneous refolding of chemically denatured enzyme in the same way as the purified Sulfolobus chaperonin. We found that they dissociated in the presence of 15% ethanol to monomers, which spontaneously assembled to oligomers when concentrated in the absence of ethanol. Both the reconstituted homo-oligomers were unstable, and easily dissociated to monomers. Further structural and functional characterization is necessary to elucidate if these homo-oligomers exist and if so, their function in vivo.

3-Isopropylmalate Dehydrogenase↗

PubMed1998

Novel non-heme iron center of nitrile hydratase with a claw setting of oxygen atoms.

The iron-containing nitrile hydratase (NHase) is a photoreactive enzyme that is inactivated in the dark because of persistent association with NO and activated by photo-dissociation of NO. The crystal structure at 1.7 A resolution and mass spectrometry revealed the structure of the non-heme iron catalytic center in the nitrosylated state. Two Cys residues coordinated to the iron were post-translationally modified to Cys-sulfenic and -sulfinic acids. Together with another oxygen atom of the Ser ligand, these modifications induced a claw setting of oxygen atoms capturing an NO molecule. This unprecedented structure is likely to enable the photo-regulation of NHase and will provide an excellent model for designing photo-controllable chelate complexes and, ultimately, proteins.

Bacterial Proteins↗

PubMed1997

F0F1-ATPase genes from an archaebacterium, Methanosarcina barkeri.

It has been known that an archaebacterium Methanosarcina barkeri strain MS (DSM 800) has a V-type ATPase (Inatomi, K., et al. (1989) J. Biol. Chem. 264, 10954-10959). Here, we report cloning of a cluster of F0F1-ATPase genes from the same organism, the first ever found in archaebacteria. The cluster and encoded subunits exhibit several unusual features such that a gene for delta subunit is lacking, F0-b subunit is unusually large, and gamma subunit is split into two peptide fragments. Attempts to detect F0F1-ATPase proteins and mRNA have been unsuccessful and therefore it is not certain if this gene cluster is really expressed in the cell.

Amino Acid Sequence↗

PubMed1997

Structure of the photoreactive iron center of the nitrile hydratase from Rhodococcus sp. N-771. Evidence of a novel post-translational modification in the cysteine ligand.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the alpha subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu - Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128. The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from alphaIle107 to alphaTrp117. Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the alpha subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.

Amino Acid Sequence↗

PubMed1997

Structural and functional characterization of homo-oligomeric complexes of alpha and beta chaperonin subunits from the hyperthermophilic archaeum Thermococcus strain KS-1.

To elucidate the function of group II chaperonin, the gene for the chaperonin from the hyperthermophilic archaeum Thermococcus strain KS-1 was cloned and sequenced. Two distinct genes coding for chaperonin subunits, designated alpha and beta, were obtained, and their deduced amino acid sequences are highly homologous to those of group II chaperonins from other sources. The alpha and beta subunits were individually expressed in Escherichia coli. Both of the recombinant subunits assemble to constitute the homo-oligomeric double-ring complexes, which are prone to form large aggregates. The alpha aggregate is dissociated into the typical chaperonin ring complex by incubation in buffer containing 15% (v/v) methanol, while the beta aggregate cannot be dissociated. At high temperature, both of the recombinant complexes have weak ATPase activities. They are able to arrest refolding of a chemically denatured thermophilic enzyme in the absence of ATP, and refolding is resumed when ATP is supplemented. These results suggest that homo-oligomeric complexes of the archaeal chaperonin have activity.

3-Isopropylmalate Dehydrogenase↗

PubMed1997

K+ is an indispensable cofactor for GrpE stimulation of ATPase activity of DnaK x DnaJ complex from Thermus thermophilus.

K+ is an indispensable cofactor for ATPase activity of eukaryotic cytosolic Hsp70 chaperone systems which lack a GrpE homolog. In the case of the bacterial Hsp70 (DnaK) system, GrpE, a nucleotide exchange factor, stimulates ATPase activity but little is known about the effect of K+. Here, we have cloned a grpE gene from a thermophile, Thermus thermophilus, and purified a homodimeric GrpE protein. Using proteins of this bacterium, we found that the GrpE stimulation of ATPase activity of DnaK x DnaJ complex was absolutely dependent on the presence of K+.

Adenosine Triphosphatases↗

PubMed1997

Purification and molecular cloning of the group II chaperonin from the acidothermophilic archaeon, Sulfolobus sp. strain 7.

To elucidate the structure and functional mechanism of the group II chaperonin, molecular cloning of the gene for and purification of the group II chaperonin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 were performed. The purified Sulfolobus chaperonin exhibited weak ATPase activity and arrested the spontaneous refolding of the thermophilic lactate dehydrogenase. However, the refolding could not be resumed by addition of ATP. The chaperonin consists of two kinds of subunits, alpha and beta, the deduced amino acid sequences of which were highly homologous to those of TF56 and TF55 from Sulfolobus shibatae, respectively.

Adenosine Triphosphate↗

PubMed1997

The stabilizing residues and the functional domains in the hyperthermophilic V-ATPase of Desulfurococcus.

To clarify a universal mechanism of the intramolecular rotation of ATP-synthase, an operon encoding a stable, ancestral ATPase was cloned from a heterotrophic archaeum Desulfurococcus strain SY. The operon of about 7 kbp contained genes E, C, G, A, B and D encoding subunits with predicted molecular weights of 23,217, 41,659, 11,499, 65,476, 52,295, and 24,897, respectively. The sequence was compared with that of Na-ATPase of Enterococcus hirae, A-ATPase of Halobacterium salinarium, V-ATPase of Methanosarcina mazei, and ATP synthase of Methanococcus jannaschii, which are homologous. (1) The cause of hyperthermostability: The main exchanges in the amino acid residues of hyperthermophilic proteins included Asp --> Glu (11 residues of A subunit of E.h.) and, Ser --> Ala. (2) The domains needed for the intramolecular rotation: The domains similar to those established in F-type ATPases were also found in the V-type ATPases of species with a different energy metabolism.

Adenosine Triphosphatases↗

PubMed1997

Rapid construction of a transcription map for a cosmid contig of Arabidopsis thaliana genome using a novel cDNA selection method.

Significant progress has been made on the random sequencing of cDNAs (ESTs) and the genetic and physical mapping of the Arabidopsis thaliana genome. New techniques are now required to identify and map the expressed genes efficiently on A. thaliana chromosomes. A novel method to construct a transcription map of expressed genes or cDNAs in specific regions of the genome using DNA-latex particles has been developed. The region-specific DNA fragments prepared from six cosmid clones that constitute a contig covering the abi1 locus on chromosome 4 were covalently bound to latex particles. The DNA-latex particles were used for the selection of region-specific cDNAs. Sequence analysis of the cDNA clones revealed that ABI1, RPS2, casein kinase 1 (CK1), nucleosome assembly protein I (NAP) cDNAs and T20837 EST, which are situated within the contig near abi1 locus, were selected. These results indicate that the cDNAs in the specific region of the genome were faithfully selected with this method. Sequence analysis also indicated that 11 selected cDNAs were derived from novel genes located near the abi1 locus and that four of the selected cDNAs encode putative proteins that have sequence similarity to cationic peroxidase, phosphatidylserine decarboxylase 2 (PSD2), trans-caffeoyl CoA 3-O-methyltransferase (CCoAMT), and proteasome subunit XC3.

Arabidopsis↗

PubMed1996

Gene for aspartate racemase from the sulfur-dependent hyperthermophilic archaeum, Desulfurococcus strain SY.

Amino acid racemases are ubiquitous throughout eubacteria. However, no amino acid racemases have yet been found in eukaryotes and archaea. We cloned a gene highly homologous to that for the aspartate racemase from the sulfur-dependent hyperthermophilic archaeum, Desulfurococcus strain SY. The product of the gene showed 35.2% amino acid sequence identity with the aspartate racemase of Streptococcus thermophilus IAM10064, and was also homologous to glutamate racemases around the putative catalytic cysteine residues. The encoded protein was expressed in Escherichia coli. The recombinant protein had amino acid racemizing activity, which was highly specific for aspartate and increased with temperature from 37 degrees C to 90 degrees C. Therefore, this was identified as the first hyperthermophilic archaeal amino acid racemase. A little aspartate racemizing activity was also detected in the crude extract of Desulfurococcus strain SY. The function of this aspartate racemase might be the uptake of -aspartate formed at high temperature or the production of -aspartate as a cell component. The fact that the amino acid racemases are distributed among both eubacteria and archaea suggests that endogenous -amino acids in mammals are also synthesized by amino acid racemases.

Amino Acid Isomerases↗

PubMed1996

A novel factor required for the assembly of the DnaK and DnaJ chaperones of Thermus thermophilus.

We previously reported the isolation of T.DnaK.DnaJ chaperone complex from Thermus thermophilus. Here, we show that a novel factor is necessary for the assembly of T.DnaK and T.DnaJ into the complex. A dnaK gene cluster of T. thermophilus contained five genes, dnaK-grpE-dnaJ-orf4-clpB. Interestingly, T.DnaJ lacks the whole "cysteine-rich region" that has been postulated to be necessary to bind unfolded proteins. The orf4 gene encodes a novel 78-amino acid protein. Curiously, T.DnaK and T.DnaJ expressed in Escherichia coli did not form the complex. Careful reexamination of the T.DnaK.DnaJ complex revealed the presence of a small protein in the complex, which turned out to be a product of orf4. As expected, expression of three genes, dnaK-dnaJ-orf4, resulted in production of a T.DnaK.DnaJ complex in E. coli that was indistinguishable from the authentic complex in its ability to interact with nucleotide and denatured protein. The product of orf4 was also required for in vitro reconstitution of the complex and named T.DafA (T.DnaK.DnaJ assembly factor A). The complex comprises three copies each of T.DnaK, T.DnaJ, and T.DafA. Even though a definite homolog of T.DafA has not been found in the data base, this finding raises a possibility that interaction between DnaK and DnaJ chaperones in other organisms is also mediated by a small protein yet unnoticed.

Amino Acid Sequence↗

PubMed1996

Location of the non-heme iron center on the alpha subunit of photoreactive nitrile hydratase from Rhodococcus sp. N-771.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771, which possesses a non-heme iron center binding nitric oxide (NO), is activated by light irradiation. To localize the iron center in the protein, we quantified Fe atoms and performed FTIR measurements of the isolated alpha and beta subunits. The native NHase and the isolated alpha subunit contained about 1.0 and 0.8 mol Fe per mol protein, respectively, whereas the beta subunit contained only a trace of Fe. An NO stretching band was observed at 1852 cm-1 in the FTIR spectrum of the alpha subunit, but not in that of the beta subunit. Upon light irradiation of the alpha subunit, the affinity of the Fe atom decreased and the NO band disappeared from the FTIR spectrum. These observations indicate that the non-heme iron center, which is responsible for the photoreaction, is present in the alpha subunit.

Amino Acid Sequence↗

PubMed1996

Photoreactive nitrile hydratase: the photoreaction site is located on the alpha subunit.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 exists in active and inactive forms. The inactive NHase is immediately activated by light irradiation and changes to the active form. To characterize the photoreactive center, the inactive NHase was denatured by 6 M urea, and two kinds of subunits (alpha and beta) were separated and purified by anion-exchange chromatography. In a manner similar to the native NHase, the isolated alpha subunit showed two absorption peaks at 280 and 370 nm, which were diminished by light irradiation. However, irradiation failed to elicit the appearance of absorption peaks at around 400 nm and at 710 nm, which were characteristic of the activated enzyme. The beta subunit seemed not to possess any photoreactive chromophore because its absorption spectrum was not altered by light irradiation. Neither of the subunits showed NHase activity before and after light irradiation, but the inactive NHase was reconstituted by incubating the two subunits together in the dark at 4 degrees C for 1 h. Light irradiation of the beta subunit did not affect subsequent complex formation or NHase activity. However, the irradiated alpha subunit could not assemble with the beta subunit, and no activity was recovered. These results demonstrate that the chromophore(s) responsible for the photoactivation of NHase are entirely located on the alpha subunit, and imply that light irradiation induces conformational change of the alpha subunit.

Binding Sites↗

PubMed1995

Gene of heat shock protein of sulfur-dependent archaeal hyperthermophile Desulfurococcus.

To elucidate thermoresistance, a gene of a hyperthermophilic heat shock protein (HHSP) was isolated from the hyperthermophile Desulfurococcus strain SY which grows at 95 degrees C. The molecular weight of HHSP deduced from the open reading frame was 59,137 (545 amino acid residues). Sequence alignments of peptides reveal similarities (evolutionary distances) to the alpha (0.279) and beta (0.296) subunits of thermosome, TF55 (0.343) and human t-complex polypeptide 1. The structure of a thermophilic heat shock protein TGroEL (Tamada et al. (1991) Biochem, Biophys. Res. Commun. 179, 565) was quite different from that of HHSP. TGroEL and HSP60 have sequences identical to HHSP at its equatorial domain, while those identical to the alpha subunit of F-type ATPase are at its apical domain.

Amino Acid Sequence↗

PubMed1995

Solid-phase nested deletion: a new subcloning-less method for generating nested deletions.

We have developed a new subcloning-less method for generating nested deletions which we have termed Solid-Phase Nested Deletion. The basic procedure for this method is as follows. The target DNA fragment is cloned in the multiple cloning site of a cloning vector, pUC or its derivatives, and amplified by PCR using a set of primers, one of which is 5'-biotinylated. The amplified DNA is partially digested by a restriction enzyme with a 4-base recognition sequence. The digested DNA is ligated with a synthetic adapter DNA. Monodiverse beads coupled with streptavidin (Dynabeads M-280 streptavidin) are added to the mixture and the biotinylated DNA fragments are separated by applying magnetic field. The unidirectionally deleted DNA fragments are recovered by PCR from the magnetic beads, and size-fractionated by agarose gel electrophoresis. The DNA fragments are amplified by PCR and used for sequencing. We demonstrate the potential of this method using a 4878-bp EcoRI fragment of lambda phage DNA.

Bacteriophage lambda↗

PubMed1995

Molecular cloning, expression, and characterization of chaperonin-60 and chaperonin-10 from a thermophilic bacterium, Thermus thermophilus HB8.

The gene coding a chaperonin from a thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced. The operon structure was the same as those of other bacterial chaperonins and the deduced amino acid sequences of both subunits were highly homologous to those of other chaperonins. The cloned genes of chaperonin subunits, chaperonin-10 (T.th cpn10) and chaperonin-60 (T.th cpn60), were separately expressed in Escherichia coli cells. The expressed subunits were easily purified from other host proteins including GroE, a chaperonin of E. coli. T.th cpn60 was expressed as a tetradecameric form, like GroEL of E. coli. Since chaperonin from T. thermophilus HB8 is purified as a holochaperonin, a complex of tetradecameric T.th cpn60 and heptameric T.th cpn10, a tetradecamer of T.th cpn60 without T.th cpn10 has not been obtained before. T.th cpn60 tetradecamer tended to dissociate into monomers during storage. T.th cpn10 expressed in E. coli was purified as a stable oligomer, most likely a heptamer. The activity as holo-chaperonin was reconstituted by mixing both subunits. T.th cpn60 tetradecamer itself arrested refolding of other proteins. The monomerized T.th cpn60 was easily purified from T.th cpn60 oligomer by gel permeation chromatography. Thus-obtained T.th cpn60 monomer had an ATP-independent chaperone activity, as shown for T.th cpn60 monomer isolated from authentic holo-chaperonin.

3-Isopropylmalate Dehydrogenase↗

PubMed1994

Effects of linear polyacrylamide concentrations and applied voltages on the separation of oligonucleotides and DNA sequencing fragments by capillary electrophoresis.

Oligonucleotides and DNA sequencing fragments have been separated by capillary electrophoresis employing linear polyacrylamide (LPA) as a sieving matrix. A commercially available apparatus equipped with a laser-induced fluorescence (LIF) detection system has been utilized, but the capillary cartridge has been modified to position the capillaries without coiling. The performance of the separation, the relationship between resolution and analysis time, has been examined using poly(dT)16-500 by changing LPA concentration, capillary length, and electric field strength. It was found that, for large DNA fragments, the migration time interval between bands decreases linearly as DNA fragment size increases. This implies that there exists a maximum base number to be resolved, irrespective of the band width (we named the maximum base number Nmax). The higher value of Nmax is obtained when the applied field strength is lower, but this accompanies longer analysis time with a concomitant increase in band width. Simple experimental equations have been proposed to calculate resolution and migration times of DNA fragments separated in our system at given electrophoretic conditions. Using 9% T LPA and an electric field strength of 100 V/cm, single-base resolution of M13mp10 DNA fragments up to 520 nucleotides has been obtained.

Acrylic Resins↗