scholarly journals Molecular cloning and characterization of the thiolesterase glyoxalase II from Arabidopsis thaliana

1997 ◽  
Vol 322 (2) ◽  
pp. 449-454 ◽  
Author(s):  
Marianne RIDDERSTRÖM ◽  
Bengt MANNERVIK
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Dna Sequences ◽  
Affinity Purification ◽  
Human Tissues ◽  
High Similarity ◽  
Calculated Molecular Mass ◽  
Glyoxalase Ii ◽  
Human Enzyme

cDNA encoding glyoxalase II from Arabidopsis thalianahas been cloned and sequenced. The isolated 894 bp segment included a sequence of 774 bp encoding a protein with a calculated molecular mass of 28791 Da. The amino acid sequence deduced from the A. thalianacDNA showed 54% identity with that of the human enzyme. Searches in databanks identified seven additional DNA sequences from different species with high similarity to glyoxalase II. Certain limited regions, one rich in histidine residues, shared 100% identity. A 29 kDa protein with an isoelectric point of 6.2 was obtained by heterologous expression of the A. thalianacDNA in Escherichia coli. Homogeneous enzyme was obtained by affinity purification and its catalytic parameters with thiolesters of glutathione were similar to those for human glyoxalase II. The structural and functional similarities between glyoxalase II from A. thalianaand from human tissues suggest a common evolutionary origin.

Molecular characterization of fatty acid α-hydroperoxide-forming enzyme (α-oxygenase) in rice plants

2000 ◽  
Vol 28 (6) ◽  
pp. 765-768 ◽  
Author(s):  
T. Koeduka ◽  
K. Matsui ◽  
Y. Akakabe ◽  
T. Kajiwara
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Fatty Acid ◽  
Expression Vector ◽  
Oxygen Electrode ◽  
Vector System ◽  
High Similarity ◽  
Rice Plants ◽  
Genes Encoding

Genes encoding an α-oxygenase, in Nicotiana tabacum and Arabidopsis thaliana have been recently isolated. However, the reaction mechanism of the enzyme has not so far been elucidated. In this study, a cDNA encoding the fatty acid α-oxygenase gene in rice plants was isolated. The deduced amino acid sequence showed high similarity (63.6%) to that of N. tabacum. The gene was cloned into an expression vector system, pQE-30, and expressed in Escherichia coli as a host cell. Palmitic acid as a substrate was incubated with the lysate of the cells, and the products were analysed by HPLC. A compound formed predominantly by the recombinant enzyme was shown to be n-pentadecanal. By incubating the mixture at 0 °C, 2-hydroperoxypalmitic acid was detected as a primary product and little formation of n-pentadecanal was detected. Furthermore, uptake of molecular oxygen was observed with an oxygen electrode. This indicated that the gene in rice plants encodes the α-oxygenase.

scholarly journals Characterization of the gene encoding glyoxalase II from Leishmania donovani: a potential target for anti-parasite drugs

2005 ◽  
Vol 393 (1) ◽  
pp. 227-234 ◽  
Author(s):  
Prasad K. Padmanabhan ◽  
Angana Mukherjee ◽  
Rentala Madhubala
Keyword(s):  
Substrate Specificity ◽  
Leishmania Donovani ◽  
Affinity Purification ◽  
Single Copy ◽  
Cellular Damage ◽  
Gene Encoding ◽  
Structure Based Drug Design ◽  
Calculated Molecular Mass ◽  
Glyoxalase Ii

The glyoxalase system is a ubiquitous detoxification pathway that protects against cellular damage caused by highly reactive oxoaldehydes such as methylglyoxal which is mainly formed as a by-product of glycolysis. The gene encoding GLOII (glyoxalase II) has been cloned from Leishmania donovani, a protozoan parasite that causes visceral leishmaniasis. DNA sequence analysis revealed an ORF (open reading frame) of ∼888 bp that encodes a putative 295-amino-acid protein with a calculated molecular mass of 32.5 kDa and a predicted pI of 6.0. The sequence identity between human GLOII and LdGLOII (L. donovani GLOII) is only 35%. The ORF is a single-copy gene on a 0.6-Mb chromosome. A ∼38 kDa protein was obtained by heterologous expression of LdGLOII in Escherichia coli, and homogeneous enzyme was obtained after affinity purification. Recombinant L. donovani GLOII showed a marked substrate specificity for trypanothione hemithioacetal over glutathione hemithioacetal. Antiserum against recombinant LdGLOII protein could detect a band of anticipated size ∼32 kDa in promastigote extracts. By overexpressing the GLOII gene in Leishmania donovani using Leishmania expression vector pspαhygroα, we detected elevated expression of GLOII RNA and protein. Overexpression of the GLOII gene will facilitate studies of gene function and its relevance as a chemotherapeutic target. This is the first report on the molecular characterization of glyoxalase II from Leishmania spp. The difference in the substrate specificity of the human and Leishmania donovani glyoxalase II enzyme could be exploited for structure-based drug design of selective inhibitors against the parasite.

scholarly journals Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

2005 ◽  
Vol 71 (11) ◽  
pp. 7224-7228 ◽  
Author(s):  
Tina Lütke-Eversloh ◽  
Gregory Stephanopoulos
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Molecular Level ◽  
Feedback Inhibition ◽  
Feedback Regulation ◽  
The Other ◽  
Chorismate Mutase ◽  
Amino Acid Substitutions ◽  
Prephenate Dehydrogenase

ABSTRACT In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA fbr mutants were selected by virtue of their resistance toward m-fluoro-d,l-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrAWT, but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrAfbr mutants were similar to those of the TyrAWT protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA fbr contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.

scholarly journals Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli

2008 ◽  
Vol 51 (3) ◽  
pp. 473-482 ◽  
Author(s):  
Dorismey Vieira Tokano ◽  
Marisa Emiko Kawaichi ◽  
Emerson José Venâncio ◽  
Marilda Carlos Vidotto
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Iron Uptake ◽  
Expression Vector ◽  
Iron Acquisition ◽  
Coli Strain ◽  
High Yield ◽  
High Similarity ◽  
E Coli

The aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC). The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4) and pTJ100 (AY553855.1), and one from a human invasive E. coli strain, the pColV K30. The recombinant protein IutA was over expressed in E. coli BL21(DE-3) and was solubilized with urea and purified by Ni-NTA column. This method produced a relatively high yield of r-IutA of approximately 74kDa, which was used to produce the antibody anti-IutA. This anti-IutA reacted with the protein r-IutA and native IutA of APEC 9, as demonstrated by Western blot, showing that the r-IutA conserved epitopes and its antigenicity was preserved. The anti-IutA IgY was able to inhibit the IutA biological activity, inhibiting the sensitivity to cloacin DF13 of APEC9. However, it did not inhibit the growth of APEC9 in M9 and did not protect the chickens inoculated with the APEC, suggesting that the APEC possessed another iron acquisition mechanism distinct of aerobactin.

Cloning and characterization of theddsAgene encoding decaprenyl diphosphate synthase fromRhodobacter capsulatusB10

2006 ◽  
Vol 52 (12) ◽  
pp. 1141-1147 ◽  
Author(s):  
Xinyi Liu ◽  
Haizhen Wu ◽  
Jiang Ye ◽  
Qinsheng Yuan ◽  
Huizhan Zhang
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Open Reading Frame ◽  
High Similarity ◽  
Reading Frame ◽  
Endogenous Production ◽  
Genome Library

A decaprenyl diphosphate synthase gene (ddsA, GenBank accession No. DQ191802) was cloned from Rhodobacter capsulatus B10 by constructing and screening the genome library. An open reading frame of 1002 bp was revealed from sequence analysis. The deduced polypeptide consisted of 333 amino acids residues with an molecular mass of about 37 kDa. The DdsA protein contained the conserved amino acid sequence (DDXXD) of E-type polyprenyl diphosphate synthase and showed high similarity to others. In contrast, DdsA showed only 39% identity to a solanesyl diphosphate synthase cloned from R. capsulatus SB1003. DdsA was expressed successfully in Escherichia coli. Assaying the enzyme in vivo found it made E.coli synthesize UQ-10 in addition to the endogenous production UQ-8.Key words: ubiquinone, polyprenyl diphosphate synthase, gene expression, Rhodobacter capsulatus.

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