scholarly journals Enzymic and molecular characterization of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC 7942: resistance of the enzyme to hydrogen peroxide

1996 ◽  
Vol 316 (2) ◽  
pp. 685-690 ◽  
Author(s):  
Masahiro TAMOI ◽  
Takahiro ISHIKAWA ◽  
Toru TAKEDA ◽  
Shigeru SHIGEOKA
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Higher Plants ◽  
Sequence Similarity ◽  
Native Enzyme ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
Synechococcus Pcc 7942 ◽  
Pcc 7942

NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been purified to electrophoretic homogeneity from Synechococcus PCC 7942 cells. The native enzyme had a molecular mass of 160 kDa and consisted of four subunits with a molecular mass of 41 kDa. The activity was 6-fold higher with NADPH than with NADH; the apparent Km values for NADPH and NADH were 62±4.5 and 420±10.5 μM respectively. The gene encoding NADP-dependent GAPDH was cloned from the chromosomal DNA of Synechococcus 7942. A 1140 bp open reading frame, encoding an enzyme of 380 amino acid residues (approx. molecular mass of 41.3 kDa) was observed. The deduced amino acid sequence of the gene had a greater sequence similarity to the NADP-dependent and chloroplastic form than to the NAD-dependent and cytosolic form. The Synechococcus 7942 enzyme lacked one of the cysteines involved in the light-dependent regulation of the chloroplast enzymes of higher plants. The recombinant enzyme expressed in Escherichia coli as well as the native enzyme purified from Synechococcus 7942 cells were resistant to 1 mM H2O2.

scholarly journals The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli

1996 ◽  
Vol 316 (1) ◽  
pp. 251-257 ◽  
Author(s):  
Michinori MUTSUDA ◽  
Takahiro ISHIKAWA ◽  
Toru TAKEDA ◽  
Shigeru SHIGEOKA
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Molecular Mass ◽  
Peroxidase Activity ◽  
Catalase Activity ◽  
Native Enzyme ◽  
Nucleotide Sequence Analysis ◽  
Synechococcus Pcc 7942 ◽  
Catalase Peroxidase ◽  
Pcc 7942

Synechococcus PCC 7942, a cyanobacterium, possesses catalase–peroxidase as the sole hydrogen peroxide-scavenging system. The enzyme has been purified to electrophoretic homogenenity from the cells. The native enzyme had a molecular mass of 150 kDa and was composed of two identical subunits of molecular mass 79 kDa. The apparent Km value of the catalase activity for H2O2 was 4.2±0.27 mM and the kcat value was 2.6×104 s-1. The enzyme contained high catalase activity and an appreciable peroxidase activity with o-dianisidine and pyrogallol. The catalase activity was not inhibited by 3-amino-1,2,4-triazole but by KCN and NaN3 (apparent Ki values 19.3±0.84 and 20.2±0.95 μM respectively). The enzyme showed an absorption spectrum of typical protohaem and contained one protohaem molecule per dimer. The gene encoding catalase–peroxidase was cloned from the chromosomal DNA of Synechococcus PCC 7942. A 2160 bp open reading frame (ORF), coding a catalase–peroxidase of 720 amino acid residues (approx. 79.9 kDa), was observed. The deduced amino acid sequence coincided with that of the N-terminus of the purified enzyme and showed a remarkable similarity to those of a family of catalase–peroxidases of prokaryotic cells. Escherichia coli BL21(DE3)plysS, harbouring a recombinant plasmid containing the catalase–peroxidase gene, produced a large amount of proteins that co-migrated on SDS/PAGE with the native enzyme. The recombinant enzyme showed the same ratio of catalase activity to peroxidase activity with o-dianisidine and the same Km for H2O2 as the native enzyme.

scholarly journals The First Archaeal ATP-Dependent Glucokinase, from the Hyperthermophilic Crenarchaeon Aeropyrum pernix, Represents a Monomeric, Extremely Thermophilic ROK Glucokinase with Broad Hexose Specificity

2002 ◽  
Vol 184 (21) ◽  
pp. 5955-5965 ◽  
Author(s):  
Thomas Hansen ◽  
Bianca Reichstein ◽  
Roland Schmid ◽  
Peter Schönheit
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Divalent Cations ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Enzyme ◽  
Start Codon ◽  
Aeropyrum Pernix

ABSTRACT An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa. The apparent Km values for ATP and glucose (at 90°C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent V max was about 35 U/mg. The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (k cat/Km ), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg2+, which was most effective, could partially be replaced with Co2+, Mn2+, and Ni2+. The enzyme had a temperature optimum of at least 100°C and showed significant thermostability up to 100°C. The coding function of open reading frame (ORF) APE2091 (Y. Kawarabayasi, Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, M. Takahashi, M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui, Y. Nagai, K. Nishijima, H. Nakazawa, M. Takamiya, S. Masuda, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, and H. Kikuchi, DNA Res. 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A. pernix, was proved by functional expression in Escherichia coli. The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A. pernix. N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091. The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors. This is the first report of the characterization of an ATP-dependent glucokinase from the domain of Archaea, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses.

scholarly journals Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae

1996 ◽  
Vol 317 (1) ◽  
pp. 285-290 ◽  
Author(s):  
Kenneth A. CORNELL ◽  
R. W. WINTER ◽  
Paula A. TOWER ◽  
Michael K. RISCOE
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Sequence Similarity ◽  
Affinity Purification ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
Sds Page ◽  
High Degree ◽  
Putative Translation Product ◽  
Substrate Kinetics

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiellapneumoniae. Chromatography using a novel 5´-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46–50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 μM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 μM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.

scholarly journals Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

2003 ◽  
Vol 69 (7) ◽  
pp. 3791-3797 ◽  
Author(s):  
Nobuyuki Horinouchi ◽  
Jun Ogawa ◽  
Takafumi Sakai ◽  
Takako Kawano ◽  
Seiichiro Matsumoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Cell Extract ◽  
Predicted Amino Acid Sequence ◽  
Enzymatic Reactions ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

scholarly journals Characterization and Molecular Cloning of a Novel Enzyme, Inorganic Polyphosphate/ATP-Glucomannokinase, of Arthrobacter sp. Strain KM

2003 ◽  
Vol 69 (7) ◽  
pp. 3849-3857 ◽  
Author(s):  
Takako Mukai ◽  
Shigeyuki Kawai ◽  
Hirokazu Matsukawa ◽  
Yuhsi Matuo ◽  
Kousaku Murata
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Cell Extract ◽  
Amino Acid Sequences ◽  
Nad Kinase ◽  
Inorganic Polyphosphate ◽  
Homologous Proteins ◽  
Gene Encoding ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT A bacterium exhibiting activities of several inorganic polyphosphate [poly(P)]- and ATP-dependent kinases, including glucokinase, NAD kinase, mannokinase, and fructokinase, was isolated, determined to belong to the genus Arthrobacter, and designated Arthrobacter sp. strain KM. Among the kinases, a novel enzyme responsible for the poly(P)- and ATP-dependent mannokinase activities was purified 2,200-fold to homogeneity from a cell extract of the bacterium. The purified enzyme was a monomer with a molecular mass of 30 kDa. This enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing poly(P) as well as ATP, and was designated poly(P)/ATP-glucomannokinase. The K m values of the enzyme for glucose, mannose, ATP, and hexametaphosphate were determined to be 0.50, 15, 0.20, and 0.02 mM, respectively. The catalytic sites for poly(P)-dependent phosphorylation and ATP-dependent phosphorylation of the enzyme were found to be shared, and the poly(P)-utilizing mechanism of the enzyme was shown to be nonprocessive. The gene encoding the poly(P)/ATP-glucomannokinase was cloned from Arthrobacter sp. strain KM, and its nucleotide sequence was determined. This gene contained an open reading frame consisting of 804 bp coding for a putative polypeptide with a calculated molecular mass of 29,480 Da. The deduced amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of the poly(P)/ATP-glucokinase of Mycobacterium tuberculosis H37Rv (level of homology, 45%), ATP-dependent glucokinases of Corynebacterium glutamicum (45%), Renibacterium salmoninarum (45%), and Bacillus subtilis (35%), and proteins of bacteria belonging to the order Actinomyces whose functions are not known. Alignment of these homologous proteins revealed seven conserved regions. The mannose and poly(P) binding sites of poly(P)/ATP-glucomannokinase are discussed.

scholarly journals A Major Secreted Elastase Is Essential for Pathogenicity of Aeromonas hydrophila

2000 ◽  
Vol 68 (6) ◽  
pp. 3233-3241 ◽  
Author(s):  
Alberto Cascón ◽  
Javier Yugueros ◽  
Alejandro Temprano ◽  
María Sánchez ◽  
Carmen Hernanz ◽  
...  
Keyword(s):  
Amino Acid ◽  
Aeromonas Hydrophila ◽  
Sequence Similarity ◽  
Opportunistic Pathogen ◽  
Aeromonas Salmonicida ◽  
Nucleotide Sequence Analysis ◽  
Gene Encoding ◽  
High Sequence Similarity ◽  
Reading Frame ◽  
Amino Acid Signal Peptide

ABSTRACT Aeromonas hydrophila is an opportunistic pathogen and the leading cause of fatal hemorrhagic septicemia in rainbow trout. A gene encoding an elastolytic activity, ahyB, was cloned from Aeromonas hydrophila AG2 into pUC18 and expressed inEscherichia coli and in the nonproteolytic speciesAeromonas salmonicida subsp. masoucida. Nucleotide sequence analysis of the ahyB gene revealed an open reading frame of 1,764 nucleotides with coding capacity for a 588-amino-acid protein with a molecular weight of 62,728. The first 13 N-terminal amino acids of the purified protease completely match those deduced from DNA sequence starting at AAG (Lys-184). This finding indicated that AhyB is synthesized as a preproprotein with a 19-amino-acid signal peptide, a 164-amino-acid N-terminal propeptide, and a 405-amino-acid intermediate which is further processed into a mature protease and a C-terminal propeptide. The protease hydrolyzed casein and elastin and showed a high sequence similarity to other metalloproteases, especially with the mature form of thePseudomonas aeruginosa elastase (52% identity),Helicobacter pylori zinc metalloprotease (61% identity), or proteases from several species of Vibrio (52 to 53% identity). The gene ahyB was insertionally inactivated, and the construct was used to create an isogenic ahyB mutant ofA. hydrophila. These first reports of a defined mutation in an extracellular protease of A. hydrophila demonstrate an important role in pathogenesis.

scholarly journals Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi

1993 ◽  
Vol 292 (3) ◽  
pp. 901-906 ◽  
Author(s):  
M Montemartini ◽  
J A Santomé ◽  
J J Cazzulo ◽  
C Nowicki
Keyword(s):  
Amino Acid ◽  
Trypanosoma Cruzi ◽  
Molecular Mass ◽  
Human Liver ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Tyrosine Aminotransferase ◽  
Apparent Molecular Mass ◽  
Native Enzyme

Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.

scholarly journals Acid-Inducible Transcription of the Operon Encoding the Citrate Lyase Complex of Lactococcus lactis Biovar diacetylactis CRL264

2004 ◽  
Vol 186 (17) ◽  
pp. 5649-5660 ◽  
Author(s):  
Mauricio G. Martín ◽  
Pablo D. Sender ◽  
Salvador Peirú ◽  
Diego de Mendoza ◽  
Christian Magni
Keyword(s):  
Lactococcus Lactis ◽  
Acid Stress ◽  
Nucleotide Sequence Analysis ◽  
Transcriptional Level ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
Polycistronic Mrna ◽  
Citrate Transporter ◽  
Citrate Lyase

ABSTRACT Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex. In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region. Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization. The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb. This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG). We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs. Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI. This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase. It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions.

scholarly journals Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

2003 ◽  
Vol 50 (1) ◽  
pp. 269-278
Author(s):  
Amr M Shabaan ◽  
Magdy M Mohamed ◽  
Mohga S Abdallah ◽  
Hayat M Ibrahim ◽  
Amr M Karim
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Vaccine Development ◽  
Complete Sequence ◽  
Cdna Clones ◽  
Western Blots ◽  
Calculated Molecular Mass ◽  
Reading Frame

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes

1994 ◽  
Vol 14 (2) ◽  
pp. 1137-1146
Author(s):  
J H Lammers ◽  
H H Offenberg ◽  
M van Aalderen ◽  
A C Vink ◽  
A J Dietrich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Similarity ◽  
Sodium Dodecyl ◽  
Coiled Coil ◽  
Amino Acid Identity ◽  
Male Rat ◽  
Amino Acid Sequence Similarity ◽  
Gene Encoding ◽  
Synaptonemal Complexes ◽  
Complex Protein

The lateral elements of synaptonemal complexes (SCs) of the rat contain major components with relative electrophoretic mobilities (M(r)S) of 30,000 and 33,000. After one-dimensional separation of SC proteins on polyacrylamide-sodium dodecyl sulfate gels, these components show up as two broad bands. These bands contain closely related proteins, as judged from their peptide maps and immunological reactivity. Using affinity-purified polyclonal anti-30,000- and anti-33,000-M(r) component antibodies, we isolated a cDNA encoding at least one of the 30,000- or 33,000-M(r) SC components. The protein predicted from the nucleotide sequence of the cDNA, called SCP3 (for synaptonemal complex protein 3), has a molecular mass of 29.7 kDa and a pI value of 9.4. It has a potential nucleotide binding site and contains stretches that are predicted to be capable of forming coiled-coil structures. In the male rat, the gene encoding SCP3 is transcribed exclusively in the testis. SCP3 has significant amino acid similarity to the pM1 protein, which is one of the predicted products of an X-linked lymphocyte-regulated gene family of the mouse: there are 63% amino acid sequence similarity and 35% amino acid identity between the SCP3 and pM1 proteins. However, SCP3 differs from pM1 in several respects, and whether the proteins fulfill related functions is still an open question.

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