Members of the tautomerase superfamily are characterized by a [beta-alpha-beta] structural fold motif as well as a catalytic N-terminal proline (Pro-1). Three members of the superfamily are involved in the degradation of the nematocide 1,3-dichloropopene; trans-3-chloroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD) and malonate semialdehyde decarboxylase (MSAD). CaaD and cis-CaaD are involved in the hydration of their respective 3-chloroacrylic acid isomers to generate malonate semialdehyde. Subsequently, MSAD is responsible for catalyzing the decarboxylation of malonate semialdehyde to generate acetaldehyde. All three of these enzymes contain an N-terminal proline (Pro-1) that functions as a general acid, in contrast to other tautomerase superfamily members, such as 4-oxalocrotonate tautomerase (4-OT) and macrophage migration inhibitory factor (MIF), where Pro-1 acts as a catalytic base. Two new members of the tautomerase superfamily have been cloned and characterized; FG41 MSAD, a homologue of MSAD from Coryneform Bacterium strain FG41, and Cg10062, a homologue of cis-CaaD from Corynebacterium glutamicum, with low-level cis-CaaD and CaaD activities. As part of an effort to delineate the mechanisms of CaaD, cis-CaaD and Cg10062, secondary activities for all three enzymes were characterized. The three enzymes function as efficient phenylpyruvate tautomerases (PPT), converting phenylenolpyruvate to phenylpyruvate. The activity also indicates that the active site of these three enzymes can ketonize enol compounds, thereby providing evidence for the presence of an enediolate intermediate. The characterization of FG41 MSAD uncovered an activity it shares with MSAD. FG41 MSAD catalyzes the hydration of 2-oxo-3-pentynoate, but at a rate that is 50-fold less efficient than that of MSAD (as assessed by kcat/Km values). Mutagenesis studies of FG41 MSAD revealed that a single mutation resulted in a 8-fold increase in the activity. The characterization of Cg10062 and attempts to enhance the low-level cis-CaaD activity demonstrated the need for a bacterial screen that could screen a library of mutants. The resulting bacterial screen could be used to screen other members of the superfamily for dehalogenase activity. An in-depth exploration of the Cg10062 and FG41 MSAD activities may lead to a better understanding of the mechanism of cis-CaaD and MSAD and further delineate the evolutionary pathway for the tautomerase superfamily. / text

published_or_final_version / abstract / Botany / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Common crystallization procedures, X-ray diffraction method and crystallographic software to determine and refine the structure of haloalkane dehalogenase enzyme were used in this thesis.

Submitted by Jordan (jordanbiblio@gmail.com) on 2017-10-04T14:12:15Z No. of bitstreams: 1 DISS_2015_Geovanny Bruno Gonçalves Dias.pdf: 1405701 bytes, checksum: 24fbe33b137779f7f8668a0dfcb69dd5 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-10-04T14:12:38Z (GMT) No. of bitstreams: 1 DISS_2015_Geovanny Bruno Gonçalves Dias.pdf: 1405701 bytes, checksum: 24fbe33b137779f7f8668a0dfcb69dd5 (MD5) / Made available in DSpace on 2017-10-04T14:12:38Z (GMT). No. of bitstreams: 1 DISS_2015_Geovanny Bruno Gonçalves Dias.pdf: 1405701 bytes, checksum: 24fbe33b137779f7f8668a0dfcb69dd5 (MD5) Previous issue date: 2015-03-03 / CAPES / CNPq / Do grupo das plantas que causa morte súbita em animais de interesse pecuário no Brasil, sete delas contém monofluoroacetato de sódio (MFA) como princípio tóxico. MFA causa bloqueio do ciclo de Krebs e consequente acúmulo de citrato nos tecidos. A movimentação dos animais intoxicados desencadeia os sinais clínicos e a morte com evolução superaguda. Uma das alternativas preconizadas para prevenção da intoxicação é a ingestão de doses não tóxicas de plantas que contém MFA. Sugere-se que a resistência ocorra devido à multiplicação de microrganismos que expressam o gene fluoroacetato dehalogenase e degradam o MFA no rúmen. Este estudo teve por objetivo verificar se E. faecalis EF09, JQ661270.1 e E. faecalis EF08, JQ661271.1, isoladas do rúmen de bovinos, degradam MFA in vivo e podem ser usadas para detoxificação de plantas que contém MFA. Para a realização deste experimento, seis ovinos com idade média de dois anos, fêmeas, entre 35-40 kg, de raça Santa Inês, foram divididos em dois grupos com três animais cada. O Grupo Inoculação recebeu durante três dias seguidos o inóculo de E. faecalis de 30 ml dividido em três doses de 10 ml. O Grupo Controle recebeu água destilada durante o mesmo período. Os animais de ambos os grupos foram mantidos em descanso por 48 horas e após esse período, o MFA foi fornecido em uma dose de 1,5 mg/Kg/PV por via oral para todos os ovinos. Os sinais clínicos, frequência cardíaca e respiratória foram obtidos em repouso a cada uma hora, e após 10 minutos de movimentação decorridas seis horas da intoxicação. Todos os animais de ambos os grupos morreram entre três e oito horas após o fornecimento de MFA. Os resultados indicam que os animais não desenvolveram resistência ao MFA após a inoculação com E. faecalis. / From the group of plants that cause sudden death in livestock in Brazil, seven of which contains sodium fluoroacetate (MFA) as toxic principle. MFA cause blockage of the Krebs cycle and subsequent accumulation of citrate in the tissues. The movement of animals that are intoxicated triggers the clinical signs and cause the death with acute evolution. One envisaged alternative to preventing poisoning is the ingestion of nontoxic doses of plants containing the MFA. It is suggested that the resistance occurs due to proliferation of microorganisms that express the MFA degrading gene fluoroacetate dehalogenase and that the MFA is degrade in the rumen. This study aimed to determine whether E. faecalis FY09, JQ661270.1 and FY08, JQ661271.1, isolated from bovine rumen, degrade MFA in vivo and can be used for detoxification of plants containing MFA. To carry out this experiment, six sheep with an average age of two years old, female, between 35-40 kg, Santa Ines, were divided into two groups of three animals each. The inoculation Group received for three consecutive days, E. faecalis - 30 ml inoculum divided into three doses of 10 ml. The control group received distilled water for the same period. The animals of both groups were kept at rest for 48 hours and after this period, the MFA is provided in a dose of 1.5 mg / kg / Live Weight orally to all sheep. Clinical signs, heart and respiratory rate were obtained at rest every hour, and after 10 minutes of elapsed drive six hours of poisoning. All animals in both groups died between three and eight hours after delivery of MFA. The results indicate that the animals have not developed resistance to MFA after inoculation with E. faecalis.

In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with ‘‘known’’ function are mis-annotated. Several global approaches are being employed to predict function, including sequence similarity searches, analysis of gene expression, protein interaction, and protein structure. Enzymes comprise a group of target proteins that require experimental characterization for accurate functional annotations. Here I applied enzyme genomics to identify new enzymes by screening individually purified proteins for enzymatic activity under relaxed reaction conditions, which allowed me to identify the subclass or sub-subclasses of enzymes to which the unknown protein belongs. Further biochemical characterization of proteins was facilitated by the application of secondary screens with natural substrates (substrate profiling). Application of general enzymatic screens and substrate profiling greatly sped up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches. As a test case, I used this approach to characterize the members of the haloacid dehalogenase (HAD)-like hydrolase superfamily, which consists mainly of uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Low sequence similarity between the members of the HAD superfamily precludes the computational prediction of their substrates and functions. Using a representative set of 80 phosphorylated substrates I characterized the phosphatase activities of 21 soluble HADs from Escherichia coli and seven soluble HADs from Saccharomyces cerevisiae. E. coli HADs show broad and overlapping substrate specificity against a wide range of phosphorylated metabolites. The yeast enzymes were more specific, and one protein also showed protein phosphatase activity. Comparison of HAD substrate profiles from two model organisms showed several “functional niches” that are occupied by HADs, which include hydrolysis of nucleotides, phosphoglycolate, phosphoserine, and pyridoxal phosphate. I proposed the cellular function for a number of HADs from both organisms based on substrate specificities. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, E. coli YniC.

In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with ‘‘known’’ function are mis-annotated. Several global approaches are being employed to predict function, including sequence similarity searches, analysis of gene expression, protein interaction, and protein structure. Enzymes comprise a group of target proteins that require experimental characterization for accurate functional annotations. Here I applied enzyme genomics to identify new enzymes by screening individually purified proteins for enzymatic activity under relaxed reaction conditions, which allowed me to identify the subclass or sub-subclasses of enzymes to which the unknown protein belongs. Further biochemical characterization of proteins was facilitated by the application of secondary screens with natural substrates (substrate profiling). Application of general enzymatic screens and substrate profiling greatly sped up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches. As a test case, I used this approach to characterize the members of the haloacid dehalogenase (HAD)-like hydrolase superfamily, which consists mainly of uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Low sequence similarity between the members of the HAD superfamily precludes the computational prediction of their substrates and functions. Using a representative set of 80 phosphorylated substrates I characterized the phosphatase activities of 21 soluble HADs from Escherichia coli and seven soluble HADs from Saccharomyces cerevisiae. E. coli HADs show broad and overlapping substrate specificity against a wide range of phosphorylated metabolites. The yeast enzymes were more specific, and one protein also showed protein phosphatase activity. Comparison of HAD substrate profiles from two model organisms showed several “functional niches” that are occupied by HADs, which include hydrolysis of nucleotides, phosphoglycolate, phosphoserine, and pyridoxal phosphate. I proposed the cellular function for a number of HADs from both organisms based on substrate specificities. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, E. coli YniC.

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-09-04T23:14:49Z No. of bitstreams: 1 EXPEDITO KENNEDY ALVES CAMBOIM - TESE PPGMV 2012..pdf: 1710872 bytes, checksum: c527311a481ea2baaf5080340bd08c0c (MD5) / Made available in DSpace on 2018-09-04T23:14:49Z (GMT). No. of bitstreams: 1 EXPEDITO KENNEDY ALVES CAMBOIM - TESE PPGMV 2012..pdf: 1710872 bytes, checksum: c527311a481ea2baaf5080340bd08c0c (MD5) Previous issue date: 2012-11-09 / O objetivo deste trabalho foi isolar e identificar bactérias capazes de degradar fluoroacetato de sódio (FS) em amostras de solo e plantas, coletadas em áreas onde são encontradas plantas que contêm fluoroacetato tais como Mascagnia rigida e Palicourea aenofusca. As amostras foram cultivadas em meio mineral acrescido de 20 mmol L−1 de fluoroacetato de sódio. Através do sequenciamento do gene 16S rRNA os sete isolados foram identificados como Paenibacillus sp. (ECPB01), Burkholderia sp. (ECPB02), Cupriavidus sp. (ECPB03), Staphylococcus sp. (ECPB04), Ancylobacter sp. (ECPB05), Ralstonia sp. (ECPB06), e Stenotrophomonas sp. (ECPB07). Todos os sete isolados degradaram o FS contido no meio, alcançando uma taxa de degradação de 20 mmol L−1 de íon flúor. Dos isolados, seis são descritos pela primeira vez como capazes de degradar FS. No futuro, alguns destes microorganismos podem ser utilizados para estabelecer no rúmen uma população bacteriana capaz de degradar o fluoroacetato de sódio e proteger ruminantes de intoxicações por este composto.

Organohalide-respiring Bacteria (OHRB) play key roles in the reductive dehalogenation of natural organohalides and anthropogenic chlorinated contaminants. Reductive dehalogenases (RDases) catalyze the cleavage of carbon-halogen bonds, enabling respiratory energy conservation and growth. Large numbers of RDase genes, a majority lacking experimental characterization of function, are found on the genomes of OHRB. In silico genomics tools were employed to identify shared sequence features among RDase genes and proteins, predict RDase functionality, and elucidate RDase evolutionary history. These analyses showed that the RDase superfamily could be divided into proteins exported to the membrane and cytoplasmic proteins, indicating that not all RDases function in respiration. Further, Hidden Markov models (HMMs) and multiple sequence alignments (MSAs) based upon biochemically characterized RDases identified previously uncharacterized members of an RDase superfamily, delineated protein domains and amino acid motifs serving to distinguish RDases from unrelated iron-sulfur proteins. Such conserved and discriminatory features among RDases may facilitate monitoring of organohalide-degrading microbial communities or improve accuracy of genome annotation. Phylogenetic analyses of RDase superfamily sequences provided evidence of convergent evolution and horizontal gene transfer (HGT) across distinct OHRB genera. Yet, the low frequency of RDase transfer outside the genus level and the absence of RDase transfer between phyla indicate that RDases evolve primarily by vertical evolution or HGT is restricted among related OHRB strains. Polyphyletic evolutionary lineages within the RDase superfamily comprise distantly-related RDases, some exhibiting activities towards the same substrates, suggesting a longstanding history of OHRB adaptation to natural organohalides. Similar functional and phylogenetic analyses provided evidence that nitrous oxide (N₂O, a potent greenhouse gas) reductase (nosZ) genes from versatile OHRB members of the Anaeromyxobacter and Desulfomonile genera comprised a nosZ sub-family evolutionarily distinct from nosZ found in non-OHRB denitrifiers. Hence, elucidation of RDase and NosZ sequence diversity may enhance the mitigation of anthropogenic organohalides and greenhouse gases (i.e., N₂O), respectively. The tetrachloroethene-respiring bacterium Geobacter lovleyi strain SZ exhibited genomic features distinguishing it from non-organohalide-respiring members of the Geobacter genus, including a conjugative pilus transfer gene cluster, a chromosomal genomic island harboring two RDase genes, and a diminished set of c-type cytochrome genes. The G. lovleyi strain SZ genome also harbored a 77 kbp plasmid carrying 15 out of the 24 genes involved in biosynthesis of corrinoid, likely related to this strains ability to degrade PCE to cis-DCE in the absence of supplied corrinoid (i.e., vitamin B₁₂). Although corrinoids are essential cofactors to RDases, the strictly organohalide-respiring Dehalococcoides mccartyi strains are corrinoid auxotrophs and depend upon uptake of extracellular corrinoids via Archaeal and Bacterial salvage pathways. A key corrinoid salvage gene in D. mccartyi, cbiZ, occurs at duplicated loci adjacent to RDase genes and appears to have been horizontally-acquired from Archaea. These comparative genome analyses highlight RDase dependencies upon corrinoids and also suggest mobile genomic elements (e.g., plasmids) are associated with organohalide respiration and corrinoid acquisition among OHRB. In summary, analyses of OHRB genomes promise to enable more complete modeling of metabolic and evolutionary processes associated with the turnover of organohalides in anoxic environments. These efforts also expand knowledge of biomarkers for monitoring OHRB activity in anoxic environments, and will improve our understanding of the fate of chlorinated contaminants.

In this study, a strategy called gene splicing by overlap extension, &ldquo / Gene SOEing&rdquo / , was used for the construction of the fusion proteins with the purpose of increasing the thermostability of mesophilic enzymes by incorporation of stability domain from a thermostable enzyme. Gene SOEing is a PCR-based approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. In fusion constructs, as the stability determinant Thermoplasma volcanium citrate synthase (CS) large domain has been used. This gene has recently been cloned in our laboratory. In two different fusions, as fusion partners, dehalogenase II (dehCII) gene of Pseudomonas sp. CBS3 and aminoglycoside-3&#039 / -phosphotransferase-II (APH(3&#039 / )-II) gene of E. coli were employed. Following the Gene SOEing, two fusion products, 1722 bp long CS Large Domain-dehCII and 1750 bp long CS Large Domain-APH(3&#039 / )-II were constructed. Also a 1586 bp long dehCII-APH(3&#039 / )-II fusion was prepared. Three fusion constructs were cloned in E. coli. Cloning was confirmed in each case, by restriction analysis of the isolated plasmids from recombinant colonies. APH(3&#039 / )-II gene associated with CS Large Domain-APH(3&#039 / )-II and dehCII-APH(3&#039 / )-II fusion constructs were successfully expressed in E. coli as revealed by enzyme assay and antibiotic agar plate assay. CS Large Domain-APH(3&#039 / )-II fusion protein retained 9.4% of the original APH(3&#039 / )-II activity after 10 minutes at 60&ordm / C. However, CS Large Domain-dehCII and dehCII-APH(3&#039 / )-II fusions did not display any dehalogenase activity.