Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nucleotide- and nucleoside-degrading enzymes are present in the surface of platelets, blood cells involved in clotting disturbances of Trypanosoma evansi-infected animals. Thus, this study was aimed at evaluating the activity of the enzymes NTPDase, 5 - nucleotidase and adenosine deaminase in platelets of rats experimentally infected by T. evansi. Animals were divided into four groups, according to the degree of parasitemia. Samples were collected at days 3 (group A: at the beginning of parasitemia), 5 (group B: high parasitemia) and 15 (group C: chronic infection). Group D (control group) was composed of non-infected animals. Blood samples with citrate as the anticoagulant were collected and used for platelet separation and enzymatic assays. NTPDase, 5 - nucleotidase and adenosine deaminase (ADA) activities were decreased (p<0.05) in platelets from rats of groups A and B, when compared to the control group. In group C, only NTPDase and 5 -nucleoside activities were decreased (p<0.001), observed by ADP and AMP hydrolysis. The correlation between platelet count and nucleotide and nucleoside hydrolysis was positive and statistically significant (p<0.05) in groups A and B. Platelet aggregation of all infected groups was decreased in comparison to the control group (p<0.05). Based upon the results, it is concluded that the alterations observed in the activity of the enzymes NTPDase, 5 -nucleotidase and adenosine deaminase in platelets of T. evansi-infected animals might be related to thrombocytopenia. / Nucleotide- and nucleoside-degrading enzymes are present in the surface of platelets, blood cells involved in clotting disturbances of Trypanosoma evansi-infected animals. Thus, this study was aimed at evaluating the activity of the enzymes NTPDase, 5 - nucleotidase and adenosine deaminase in platelets of rats experimentally infected by T. evansi. Animals were divided into four groups, according to the degree of parasitemia. Samples were collected at days 3 (group A: at the beginning of parasitemia), 5 (group B: high parasitemia) and 15 (group C: chronic infection). Group D (control group) was composed of non-infected animals. Blood samples with citrate as the anticoagulant were collected and used for platelet separation and enzymatic assays. NTPDase, 5 - nucleotidase and adenosine deaminase (ADA) activities were decreased (p<0.05) in platelets from rats of groups A and B, when compared to the control group. In group C, only NTPDase and 5 -nucleoside activities were decreased (p<0.001), observed by ADP and AMP hydrolysis. The correlation between platelet count and nucleotide and nucleoside hydrolysis was positive and statistically significant (p<0.05) in groups A and B. Platelet aggregation of all infected groups was decreased in comparison to the control group (p<0.05). Based upon the results, it is concluded that the alterations observed in the activity of the enzymes NTPDase, 5 -nucleotidase and adenosine deaminase in platelets of T. evansi-infected animals might be related to thrombocytopenia.

Adenosine deaminase has been shown to consist of three molecular weight forms, A, B, and C. In higher mammals, the A and C forms are dominant while in lower mammals, the B and C forms are dominant. In this work, the B and C forms were isolated from the frog liver and several kinetic parameters were determined.Ammonium sulfate salt fractionation, starting at 40 percent and increased by 5 percent increments to 80 percent was used to separate the two forms. The B form adenosine deaminase was predominantly found in the 50 to 60 percent precipitate fractions while the C form was predominant in precipitate fractions containing more than 60 percent ammonium sulfate. The rechromatographed B and C forms were subjected to isoelectric focusing and thin layer electrophoresis. The B form separated into three activity bands while the C form separated into two activity bands`. Michaelis constant values were determined to be 4.61 X 10-5M and 2.00 X 10-5M for the B and C forms with adenosine as a substrate, respectively. The relative substrate specificity ratio showed that the B form was very specific for adenosine.In conclusion, the B form adenosine deaminase was found to be dominant in the frog liver. The Michaelis constant, relative substrate specificity ratio, thin layer electrophoresis and isoelectric focusing distinguished between the adenosine deaminase B and C forms. The technique of ammonium sulfate fractionation gave excellent separation between the B and C forms of adenosine deaminase.Ball State UniversityMuncie, IN 47306

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. ACC deaminases break down ACC, the immediate precursor of ethylene in plants, into ammonia and α-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. It was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather that it utilizes D-cysteine as a substrate, and in fact encodes a D-cysteine desulfhydrase. Kinetic characterization of the enzyme has shown that it is similar to other previously characterized D-cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering two amino acid residues within the predicted active site changed the enzyme from D-cysteine desulfhydrase to ACC deaminase. Concomitantly, it was shown that by altering two amino acids residues at the same position within the active site of ACC deaminase from Pseudomonas putida UW4 changed this enzyme into D-cysteine desulfhydrase.

Bacteria that provide benefit to plants are considered to be plant growth-promoting bacteria (PGPB) and can facilitate plant growth by a number of different mechanisms. Plant growth-promoting bacteria that are able to utilize the plant compound 1-aminocyclopropane-1-carboxylate (ACC) as a sole source of nitrogen, as a consequence of possessing the enzyme ACC deaminase, can protect host plants from a number of environmental stresses. In addition to ACC deaminase, PGPB may utilize other mechanisms to facilitate plant growth including IAA synthesis, siderophore production, phosphate solubilization activity, ammonia production, and antibiotic production. Plant growth-promoting bacterial endophytes employ similar plant growth promotion mechanisms to those used by rhizospheric PGPB. In fact, bacterial endophytes are PGPB that go one step further and colonize the inside of the plant tissues and provide more efficient and prompted protection to their hosts compared to those that bind exclusively to the plant’s rhizosphere. Therefore, it is likely that endophytic plant growth-promoting bacteria will be superior to similar non-endophytic bacterial strains in promoting plant growth under a wide range of environmental conditions. In the work reported here, new bacterial endophytes were isolated and characterized. Among twenty-five ACC deaminase positive strains, two best strains were selected and ACC deaminase deficient mutants were constructed. The ability of two newly isolated 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant growth-promoting bacterial endophytes Pseudomonas fluorescens YsS6, Pseudomonas migulae 8R6 and their ACC deaminase deficient mutants was shown to 1) delay the senescence of mini carnation cut flowers and 2) to facilitate tomato plant growth under salinity stress. In the mini carnation flower senescence evaluation, the only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity, our results demonstrate that this enzyme is directly responsible for a significant delay in flower senescence. Despite containing ACC deaminase activity, the rhizosphere-binding PGPB Pseudomonas putida UW4 was not taken up by the cut flowers and therefore had no effect on prolonging flower shelf life. In evaluating the effect of bacterial endophytes under salt stress, tomato plants treated with either of the wild-type strains of the two selected bacterial endophytes demonstrated early flowering and fruiting and had significantly greater numbers of flowers, buds, and fruits than either the corresponding ACC deaminase mutant strain-treated plants or the control plants. Although both bacterial endophytes P. fluorescens YsS6 and P. migulae 8R6 showed significant plant growth-promotion capabilities, P. migulae 8R6 demonstrated better plant growth facilitation under salt stress than did P. fluorescens YsS6. P. migulae 8R6 treated tomato plants demonstrated the least sodium uptake, the highest chlorophyll content, and highest fresh and dry biomass. The results of the work presented here suggest that ACC deaminase containing selected bacterial endophytes could be employed as environmentally friendly adjuncts to agricultural and horticultural practice.

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. ACC deaminases break down ACC, the immediate precursor of ethylene in plants, into ammonia and α-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. It was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather that it utilizes D-cysteine as a substrate, and in fact encodes a D-cysteine desulfhydrase. Kinetic characterization of the enzyme has shown that it is similar to other previously characterized D-cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering two amino acid residues within the predicted active site changed the enzyme from D-cysteine desulfhydrase to ACC deaminase. Concomitantly, it was shown that by altering two amino acids residues at the same position within the active site of ACC deaminase from Pseudomonas putida UW4 changed this enzyme into D-cysteine desulfhydrase.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 125-139).

Bacteria that provide benefit to plants are considered to be plant growth-promoting bacteria (PGPB) and can facilitate plant growth by a number of different mechanisms. Plant growth-promoting bacteria that are able to utilize the plant compound 1-aminocyclopropane-1-carboxylate (ACC) as a sole source of nitrogen, as a consequence of possessing the enzyme ACC deaminase, can protect host plants from a number of environmental stresses. In addition to ACC deaminase, PGPB may utilize other mechanisms to facilitate plant growth including IAA synthesis, siderophore production, phosphate solubilization activity, ammonia production, and antibiotic production. Plant growth-promoting bacterial endophytes employ similar plant growth promotion mechanisms to those used by rhizospheric PGPB. In fact, bacterial endophytes are PGPB that go one step further and colonize the inside of the plant tissues and provide more efficient and prompted protection to their hosts compared to those that bind exclusively to the plant’s rhizosphere. Therefore, it is likely that endophytic plant growth-promoting bacteria will be superior to similar non-endophytic bacterial strains in promoting plant growth under a wide range of environmental conditions. In the work reported here, new bacterial endophytes were isolated and characterized. Among twenty-five ACC deaminase positive strains, two best strains were selected and ACC deaminase deficient mutants were constructed. The ability of two newly isolated 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant growth-promoting bacterial endophytes Pseudomonas fluorescens YsS6, Pseudomonas migulae 8R6 and their ACC deaminase deficient mutants was shown to 1) delay the senescence of mini carnation cut flowers and 2) to facilitate tomato plant growth under salinity stress. In the mini carnation flower senescence evaluation, the only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity, our results demonstrate that this enzyme is directly responsible for a significant delay in flower senescence. Despite containing ACC deaminase activity, the rhizosphere-binding PGPB Pseudomonas putida UW4 was not taken up by the cut flowers and therefore had no effect on prolonging flower shelf life. In evaluating the effect of bacterial endophytes under salt stress, tomato plants treated with either of the wild-type strains of the two selected bacterial endophytes demonstrated early flowering and fruiting and had significantly greater numbers of flowers, buds, and fruits than either the corresponding ACC deaminase mutant strain-treated plants or the control plants. Although both bacterial endophytes P. fluorescens YsS6 and P. migulae 8R6 showed significant plant growth-promotion capabilities, P. migulae 8R6 demonstrated better plant growth facilitation under salt stress than did P. fluorescens YsS6. P. migulae 8R6 treated tomato plants demonstrated the least sodium uptake, the highest chlorophyll content, and highest fresh and dry biomass. The results of the work presented here suggest that ACC deaminase containing selected bacterial endophytes could be employed as environmentally friendly adjuncts to agricultural and horticultural practice.

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine. Mechanistic characterization of ADE from Escherichia coli was performed along with biophysical studies. The structure of ADE was solved from A. tumefaciens. The structure, along with the biochemical and biophysical characterization, enabled the elucidation of the mechanism of the deaminase reaction of ADE. Elucidation of the origin of the oxygenation reactions within ADE led to the discovery of a promiscuous catalase reaction. The diiron ADE from all tested bacterial species exhibited this unusual reaction, along with the generation of superoxide and hydroxyl radicals, the latter being responsible for the oxygenation of the protein. The residues that were identified to be oxygenated were primarily the metal binding residues implying the origin of this reaction was the binuclear iron center. A group of bacterial enzymes that are co-localized in the same genomic operon as ADE but of unknown function were identified. The enzyme Bh0637 from Bacillus halodurans, a representative member of this group of enzymes was characterized. This enzyme was shown to preferentially catalyze the deamination of epigenetic base, N-6-methyadenine. Lastly, gram-negative bacteria have a highly conserved phn operon composed of 14 genes to break the C-P bond of inert alkylphosphonates. The genes phnGHIJKLM are absolutely critical for this activity. We discovered that methylphosphonate reacts first with MgATP to form alpha-D-ribose-1-methylphosphonate-5-triphosphate (RPnTP) and adenine by the action of PhnI, PhnG, PhnH and PhnL. PhnI by itself was shown to perform a novel nucleosidase reaction converting MgATP to ribose-5-triphosphate and adenine. The triphosphate moiety of RPnTP is then hydrolyzed to pyrophosphate and alpha-D-ribose-1-methylphosphonate-5-phosphate (PRPn) by PhnM. The carbon-phosphorus bond of PRPn is subsequently cleaved via a radical-based reaction to alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate (PRcP) and methane in the presence of S-adenosyl-L-methionine by PhnJ.

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: R.S. Gupta. Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: R.S. Gupta. Includes bibliographical references. Also available via World Wide Web.