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 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 Effect of fungicides on the enzymatic activity of the antioxidant system and the chlorophyll content in lupine plants during seed dressing

2020 ◽  
pp. 3-6
Author(s):  
O. Borzykh ◽  
O. Tsurkan ◽  
L. Chervyakova ◽  
T. Panchenko
Keyword(s):  
Analytical Chemistry ◽  
Chlorophyll Content ◽  
Peroxidase Activity ◽  
Catalase Activity ◽  
Control Level ◽  
Growth Stages ◽  
Enzymatic System ◽  
Initial Growth ◽  
Seed Dressing ◽  
Catalase Peroxidase

Goal. The effect of fungicides on the dynamics of the activity of peroxidase, catalase (CAT) and chlorophyll content in lupine plants during seed dressing has been established. Methods. Laboratory and vegetation researches were conducted in the laboratory of analytical chemistry of pesticides of the Institute of Plant Protection. Yellow lupine (Lupinus luteus L.), variety Obriy has been grown. The objects of research were fungicides triticonazol (40 g/t) and its combination with prochloraz (120 g/t). Determination of the content of fungicides in plants was carried out using chromatographic methods according to officially approved methods and me­thods developed in the laboratory of analytical chemistry of pesticides. Chlorophyll content and peroxidase activity were measured by colorimetric method, catalase activity — by titrimetric method. Results. According to the research results, the varying sensitivity of the enzymatic system of antioxidant defense (catalase, peroxidase) in response to seed dressing by fungicides was recorded. It showed that on the 10th day after sowing, content of triticonazol in plants was 0.8 mg/kg, and the peroxidase activity was similar to that in untreated plants. Subsequently, against the background of a decrease in the content of the active substance, a gradual activation of the enzyme was observed. Catalase activity also gradually increased beginning from the 14th day, and on the 30th day it exceeded the corresponding control indicator by 40%. When using a combination of triticonazol with prochloraz, the disturbance in the balance of peroxidase catalase was more significant. However, by the phase of 7—8 leaves, with a minimal total content of fungicides (0.38 mg/kg), the enzyme activity approached the control level, which is associated with the restoration of plant homeostasis and the formation of its adaptive potential under stress conditions. The stimulating effect of these fungicides on chlorophyll content at the initial growth stages of lupine was established. The chlorophyll concentration in fungicides-treated plants exceeded the control indicator by 11—29%. Conclusions. The use of systemic triazole fungicides to protect seedlings, improves the photosynthetic activity of plants and at the same time acts as a stress factor that activates protecting enzymes (catalase, peroxidase), which trigger the development of protective adaptive reactions of plants.

Identification and physical characterization of a Col E1 hybrid plasmid containing a catalase gene of Escherichia coli

1983 ◽  
Vol 61 (12) ◽  
pp. 1315-1321 ◽  
Author(s):  
Peter C. Loewen ◽  
Barbara L. Triggs ◽  
Glen R. Klassen ◽  
Joel H. Weiner
Keyword(s):  
Escherichia Coli ◽  
Catalase Activity ◽  
Physical Map ◽  
Polyacrylamide Gel Electrophoresis ◽  
Hybrid Plasmid ◽  
E Coli ◽  
Catalase Gene ◽  
Restriction Sites ◽  
Catalase Peroxidase

A hybrid Escherichia coli: Col E1 plasmid, pLC36-19, containing a catalase gene has been identified in the Clarke and Carbon colony bank. Catalase activity was amplified two- to three-fold in the pLC36-19-containing strain relative to other hybrid-plasmid-containing strains and this activity could be induced three- or four-fold by hydrogen peroxide or ascorbic acid. The plasmid was transferred to a strain chromosomally deficient in catalase synthesis, resulting in a strain with high and inducible levels of catalase. The plasmid was also transferred to a minicell-producing strain and minicells harbouring the plasmid were found to synthesize a labelled protein with a molecular weight of 84 000 characteristic of catalase from E. coli. A catalase activity was also synthesized by the plasmid-containing minicells. Two catalase activities with associated peroxidase activities coded for by the plasmid were separable by polyacrylamide gel electrophoresis and migrated coincident with chromosomally encoded catalase–peroxidase activities. A third catalase activity which did not have an associated peroxidase activity was not coded for by the plasmid. A physical map of the 25.5-kilobase pair plasmid was constructed by restriction nuclease analysis and the relative positions of 38 restriction sites were defined.

scholarly journals Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

2004 ◽  
Vol 70 (3) ◽  
pp. 1570-1575 ◽  
Author(s):  
Dae Heoun Baek ◽  
Jae Jun Song ◽  
Seok-Joon Kwon ◽  
Chung Park ◽  
Chang-Min Jung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Specificity Constant ◽  
Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

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.

Functional Complementation in Escherichia coli of Different Phytoene Desaturase Genes and Analysis of Accumulated Carotenes

1991 ◽  
Vol 46 (11-12) ◽  
pp. 1045-1051 ◽  
Author(s):  
Hartmut Linden ◽  
Norihiko Misawa ◽  
Daniel Chamovitz ◽  
Iris Pecker ◽  
Joseph Hirschberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Phytoene Desaturase ◽  
Reaction Products ◽  
Double Bonds ◽  
E Coli ◽  
Synechococcus Pcc 7942 ◽  
Lycopene Cyclase ◽  
Pcc 7942 ◽  
Cis Isomer

Three different phytoene desaturase genes, from Rhodobacter capsulatus, Erwinia uredovora, and Synechococcus PCC 7942, have been functionally complemented with a gene construct from E. uredovora which encodes all enzymes responsible for formation of 15-cis phytoene in Escherichia coli. As indicated by the contrasting reaction products detected in the pigmented E. coli cells after co-transformation, a wide functional diversity of these three different types of phytoene desaturases can be concluded. The carotenes formed by the phytoene desaturase from R. capsulatus were trans-neurosporene with three additional double bonds and two cis isomers. Furthermore, small amounts of three ζ-carotene isomers (2 double bonds more than phytoene) and phytofluene (15-cis and all-trans with + 1 double bond) were detected as inter- mediates. When the subsequent genes from E. uredovora which encode for lycopene cyclase and β-carotene hydroxylase were present, neurosporene, the phytoene desaturase product of R. capsulatus, was subsequently converted to the monocyclic β-zeacarotene and its mono- hydroxylation product. The most abundant carotene resulting from phytoene desaturation by the E. uredovora enzyme was trans-lycopene together with a cis isomer. In addition, bisdehy-drolycopene was also formed. The reaction products of Synechococcus phytoene desaturase were two cis isomers of ζ-carotene and only small amounts of trans-ζ-carotene including 15-cis. The I50 values for flurtamone and diphenylamine to inhibit phytoene desaturation were determined and differential inhibition was observed for diphenylamine.

scholarly journals Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli

1989 ◽  
Vol 257 (2) ◽  
pp. 529-534 ◽  
Author(s):  
P R Alefounder ◽  
S A Baldwin ◽  
R N Perham ◽  
N J Short
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Recombinant Expression ◽  
Class Ii ◽  
Nucleotide Sequence Analysis ◽  
Chromosomal Dna ◽  
Expression Plasmid ◽  
E Coli ◽  
Fructose 1,6 Bisphosphate

Nucleotide sequence analysis of the Escherichia coli chromosomal DNA inserted in the plasmid pLC33-5 of the Clarke and Carbon library [Clarke & Carbon (1976) Cell 9, 91-99] revealed the existence of the gene, fda, encoding the Class II (metal-dependent) fructose 1,6-bisphosphate aldolase of E. coli. The primary structure of the polypeptide chain inferred from the DNA sequence of the fda gene comprises 359 amino acids, including the initiating methionine residue, from which an Mr of 39,146 could be calculated. This value is in good agreement with that of 40,000 estimated from sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified dimeric enzyme. The amino acid sequence of the Class II aldolase from E. coli showed no homology with the known amino acid sequences of Class I (imine-forming) fructose 1,6-bisphosphate aldolases from a wide variety of sources. On the other hand, there was obvious homology with the N-terminal sequence of 40 residues already established for the Class II fructose 1,6-bisphosphate aldolase of Saccharomyces cerevisiae. These Class II aldolases, one from a prokaryote and one from a eukaryote, evidently are structurally and evolutionarily related. A 1029 bp-fragment of DNA incorporating the fda gene was excised from plasmid pLC33-5 by digestion with restriction endonuclease HaeIII and subcloned into the expression plasmid pKK223-3, where the gene came under the control of the tac promoter. When grown in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside, E. coli JM101 cells transformed with this recombinant expression plasmid generated the Class II fructose 1,6-bisphosphate aldolase as approx. 70% of their soluble protein. This unusually high expression of an E. coli gene should greatly facilitate purification of the enzyme for any future structural or mechanistic studies.

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