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 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.

scholarly journals Characterization of a Leishmania donovani gene encoding a protein that closely resembles a type IB topoisomerase

1999 ◽  
Vol 27 (13) ◽  
pp. 2745-2752 ◽  
Author(s):  
S. Broccoli ◽  
J.-F. Marquis ◽  
B. Papadopoulou ◽  
M. Olivier ◽  
M. Drolet
Keyword(s):  
Leishmania Donovani ◽  
Gene Encoding

scholarly journals Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P.

1991 ◽  
Vol 11 (2) ◽  
pp. 721-730 ◽  
Author(s):  
J Y Lee ◽  
C E Rohlman ◽  
L A Molony ◽  
D R Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Rnase P ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Gene Encoding ◽  
Processing Steps ◽  
Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

scholarly journals Molecular cloning and characterization of the structural gene for the major iron-regulated protein expressed by Neisseria gonorrhoeae.

1990 ◽  
Vol 171 (5) ◽  
pp. 1535-1546 ◽  
Author(s):  
S A Berish ◽  
T A Mietzner ◽  
L W Mayer ◽  
C A Genco ◽  
B P Holloway ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Neisseria Gonorrhoeae ◽  
Biochemical Characterization ◽  
Iron Acquisition ◽  
Regulatory Region ◽  
Single Copy ◽  
Oligonucleotide Probes ◽  
Gene Encoding

This report describes the cloning and sequencing of the major iron-regulated protein (termed Fbp) of Neisseria gonorrhoeae strain F62. Attempts to identify recombinants expressing the Fbp using specific antibody proved unsuccessful. Therefore, an alternative cloning strategy using oligonucleotide probes derived from NH2-terminal and tryptic fragments of this protein was used to identify short fragments of the gene. Using this methodology, the gene encoding the precursor of Fbp was cloned on three separate overlapping fragments and sequenced, and the amino acid sequence was deduced. These data were unambiguously confirmed by the known NH2-terminal amino acid sequence and were supported by the sequences from tryptic fragments that lie outside of this region. Using oligonucleotide probes, we were unable to obtain clones encoding the potential regulatory region of this protein. Therefore, the technique of inverse polymerase chain reaction was used to amplify a fragment containing an additional 200 bp. This fragment was cloned and sequenced and found to contain a consensus ribosome binding site and potential -10 and -35 sequences. Hybridization analysis of genomic DNA from gonococcal strain F62 indicated that only a single copy of the Fbp gene exists per genome. These results complement the biochemical characterization of the Fbp expressed by gonococci and further suggest that it has a role in iron-acquisition.

Molecular characterization of a gene encoding N-myristoyl transferase (NMT) from Triticum aestivum (bread wheat)

Genome ◽  
2004 ◽  
Vol 47 (6) ◽  
pp. 1036-1042 ◽  
Author(s):  
Tim Dumonceaux ◽  
Raju V.S Rajala ◽  
Rajendra Sharma ◽  
Gopalan Selvaraj ◽  
Raju Datla
Keyword(s):  
Triticum Aestivum ◽  
Protein Modification ◽  
Wheat Plant ◽  
Single Copy ◽  
Gene Encoding ◽  
Growth And Survival ◽  
Reading Frame ◽  
Eukaryotic Proteins ◽  
Taxonomic Groups

Myristoyl-CoA:protein N-myristoyl transferase (NMT; EC 2.3.1.97) acylates the Gly residue abutting the N-terminal Met with a myristic acid following the removal of the Met residue in certain eukaryotic proteins, and in some cases myristoylation is essential to cell growth and survival. We report the cloning of a full-length cDNA encoding NMT from Triticum aestivum (TaNMT). The cDNA included a predicted open reading frame of 1317 nucleotides, which encoded a predicted protein of 438 amino acids containing all of the residues that are important for NMT activity. The TaNMT amino acid and nucleotide sequences were compared with NMTs from 14 other species encompassing a wide array of taxonomic groups. Among the experimentally validated NMTs, TaNMT was most similar to that of Arabidopsis thaliana. Southern blot analysis of wheat genomic DNA showed that TaNMT is encoded by a single copy gene, with one copy per haploid genome. We expressed TaNMT in Escherichia coli cells and determined that the recombinant protein possessed NMT activity, catalyzing the N-myristoylation of peptides from known or putatively myristoylated proteins from plants and animals without a strong preference for the plant peptides. TaNMT is the second experimentally validated plant NMT sequence and the first from a monocotyledonous species.Key words: N-myristoyl transferase, myristoylation, protein modification, wheat, plant development.

Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P

1991 ◽  
Vol 11 (2) ◽  
pp. 721-730 ◽  
Author(s):  
J Y Lee ◽  
C E Rohlman ◽  
L A Molony ◽  
D R Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Rnase P ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Gene Encoding ◽  
Processing Steps ◽  
Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

scholarly journals Cloning and characterization of Arabidopsis thaliana AtNAP57--a homologue of yeast pseudouridine synthase Cbf5p.

2001 ◽  
Vol 48 (3) ◽  
pp. 699-709 ◽  
Author(s):  
J Maceluch ◽  
M Kmieciak ◽  
Z Szweykowska-Kulińska ◽  
A Jarmołowski
Keyword(s):  
Arabidopsis Thaliana ◽  
Bone Marrow Failure ◽  
Skin Pigmentation ◽  
Single Copy ◽  
Dyskeratosis Congenita ◽  
Gene Encoding ◽  
Bone Marrow Failure Syndrome ◽  
Calculated Molecular Mass ◽  
Pseudouridine Synthases ◽  
Characteristic Features

Rat Nap57 and its yeast homologue Cbf5p are pseudouridine synthases involved in rRNA biogenesis, localized in the nucleolus. These proteins, together with H/ACA class of snoRNAs compose snoRNP particles, in which snoRNA guides the synthase to direct site-specific pseudouridylation of rRNA. In this paper we present an Arabidopsis thaliana protein that is highly homologous to Cbf5p (72% identity and 85% homology) and NAP57 (67% identity and 81% homology). Moreover, the plant protein has conserved structural motifs that are characteristic features of pseudouridine synthases of the TruB class. We have named the cloned and characterized protein AtNAP57 (Arabidopsis thaliana homologue of NAP57). AtNAP57 is a 565 amino-acid protein and its calculated molecular mass is 63 kDa. The protein is encoded by a single copy gene located on chromosome 3 of the A. thaliana genome. Interestingly, the AtNAP57 gene does not contain any introns. Mutations in the human DKC1 gene encoding dyskerin (human homologue of yeast Cbf5p and rat NAP57) cause dyskeratosis congenita a rare inherited bone marrow failure syndrome characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia.

scholarly journals Identification and Characterization of the Trichoderma harzianum Gene Encoding α-1,3-Glucanase Involved in Streptococcal Mutan Degradation

2011 ◽  
Vol 60 (4) ◽  
pp. 293-301 ◽  
Author(s):  
ADRIAN WIATER ◽  
MONIKA JANCZAREK ◽  
MAŁGORZATA PLESZCZYŃSKA ◽  
JANUSZ SZCZODRAK
Keyword(s):  
Amino Acid ◽  
Trichoderma Harzianum ◽  
Transcriptional Control ◽  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gene Encoding ◽  
T7 Promoter ◽  
Calculated Molecular Mass ◽  
E Coli

alpha-1,3-Glucanases (mutanases) are currently of great interest due to their potential use in the field of dental care. These enzymes have been reported in several bacteria, yeasts and fungi, but up to now, characterization of this family of proteins has been relatively poor. In this study, we identify and characterize a mutanase gene from Trichoderma harzianum CCM F-340. Sequence analysis, on the nucleotide and amino acid levels reveals that this alpha-1,3-glucanase is highly homologous to alpha-1,3-glucanases from T harzianum isolate CBS 243.71 and T asperellum CECT 20539. T. harzianum CCM F-340 mutanase is a 634-aa residue protein with a calculated molecular mass of 67.65 kDa, composed of two distinct, highly conserved domains (a long N-terminal catalytic domain and a short C-terminal polysaccharide-binding domain) separated by a less conserved Pro-Ser-Thr-rich linker region. The mutanase gene was expressed in an E. coli BL21 (DE3) host, under the transcriptional control of T7 promoter. The purified enzyme migrated as a band of about 68 kDa after SDS-polyacrylamide gel electrophoresis, which coincided with the predicted size based on the amino acid sequence. Our data indicate that this e

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