Browsing by Subject "Biocatalysis"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Defining the catalytic and kinetic mechanism and natural function of the highly conserved acyl-amp hydrolase, HINT1(2011-12) Bardaweel, SanaaHistidine triad nucleotide binding proteins (Hints) are members of the histidine triad (HIT) protein superfamily of nucleotidyl transferases and hydrolyases. It has been recently demonstrated that Hints are efficient phosphoramidases and therefore activators of potent antiviral and anticancer pronucleotides. In spite of their high evolutionary conservation among all kingdoms of life, and the several regulatory functions in which Hints have been implicated, a clear connection between their observed function and their catalytic efficiency has not been elucidated. To gain a comprehensive understanding of the essential role of these ubiquitous enzymes, our laboratory has devoted a considerable effort toward the delineation of the principles governing Hints catalysis and cellular function. Such understanding will provide an unprecedented ability to assess the role of these highly conserved, but functionally unknown enzymes. Since Hints are found in both prokaryotes and eukaryotes, we have attempted to understand their function, mechanism, and structural determinants in prokaryotes, under the assumption that their role may be at least partially conserved among members of the tree of life. Recently, we have demonstrated by E. coli gene disruption studies that the bacterial Hint enzyme is necessary for growth under high salt conditions, and when alanine is a carbon and nitrogen source. Through a combination of phenotypic screening and complementation experiments with wild-type and ecHinT knock-out E. coli strains, we have shown that catalytically-active ecHinT is required for growth on D-alanine. In addition, using Hint-inhibitors and active-site mutants, we have demonstrated that expression of catalytically-active ecHinT is essential for the activity of the enzyme D-amino acid dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-amino acids metabolic pathway. These results are considered as the first report in literature that shows a successful connection between a discovered Hint-related phenotype and the catalytic activity of Hint. Previously, we have demonstrated that lysyl-AMP generated by LysRS is a substrate for both human and E. coli Hints. In addition, we have shown that the ability of Hint to hydrolyze lysyl-AMP depends on its enzymatic activity. Here, we demonstrate that the molecular determinants governing this regulation appear to reside in the C-terminus region of Hint. Interestingly, the ecHinT-DadA interaction appears also to be governed by both ecHinT-activity and the C-terminus loop. We have also expanded our scope to look at possible toxicity of D-alanine in E. coli strains lacking dadA or hinT. Our results demonstrate that E. coli mutants lacking dadA or hinT are highly susceptible to D-alanine toxicity and that the catalytic activity of Hint is an essential requirement to protect E. coli from the observed toxicity of D-alanine. Based on careful analysis of the combined results from the ecHinT-LysRS and ecHinT-DadA potential interactions, and comprehensive understanding of the D-alanine metabolic pathway in bacteria, we proposed a possible regulatory mechanism of Hint, LysRS and DadA on global protein translational processes to prevent D-amino acids toxicity in E. coli.Item From functional metagenomics to unique synthetic expression strategies in iron-reducing bacteria.(2012-05) Gonzalez, Tanhia DenysCellulose and hemicellulose are renewable sources of fermentable sugars. The use of fermentable sugars for the production of alternative energy sources (i.e. ethanol, butanol, etc.) is an attractive solution to alleviate the shortage and high prices of petroleum. Cellulases and hemicellulases are the two groups of glycosyl hydrolases responsible for breaking down the polysaccharide component of biomass into their respective sugar moieties. The enzymatic hydrolysis of cellulose and hemicellulose has relied on enzymes originally produced by culturable organisms. This thesis describes the use of metagenomics coupled to high-throughput screening techniques to identify glycosyl hydrolases originally encoded by uncultured organisms. The findings of this thesis include the identification and biochemical characterization of a unique endoglucanase. Besides catalyzing the hydrolysis of soluble and insoluble cellulosic substrates, this endoglucanase exhibited a domain architecture that has not been previously reported in the literature. This thesis also describes two different strategies to engineer the surface of (Fe+3)-reducing bacteria. These expression systems are a valuable tool for studying the cellular respiration of Geobacter and Shewanella. Furthermore, they have practical applications in the area of whole-cell biocatalysis in microbial fuel cells. The first strategy involved using an autodisplay system to engineer the cell envelope of Geobacter and Shewanella. The autodisplay system translocated a functional β-galactosidase enzyme to the cell envelope of G. sulfurreducens and S. oneidensis. Furthermore, this system proved to be an effective tool for catalyzing reactions in electrochemical cells using biofilms of G. sulfurreducens cells. The second strategy exploited the use of in-frame fusions with the c-type cytochrome OmcZ to translocate a recombinant protein to the outer membrane and extracellular matrix of Geobacter sulfurreducens. This is the first time that the c-type cytochrome OmcZ has been used to engineer biofilms of Geobacter sulfurreducens.Item Synthesis of carotenoid-containing polyesters and protein engineering to improve synthetic efficiency of Pseudomonas fluorescens esterase.(2009-10) Jiang, YunThe conjugated double bonds in carotenoids central chain make these compounds potential building blocks for conductive polymers. Bixin and crocetin's capability of forming carboxylester bonds at end are used to synthesis polyesters. Among 20 esterases and lipases, Candida antarctica Lipase B (CalB) is the only enzyme that catalyzes bixin esterification reaction with linear alcohols. None of these enzyme catalyzes crocetin esterification reaction. Candida antarctica Lipase B added alcohols, such as, n-propanol, polyethylene glycol 400 and 1, 10- decanediol to bixin to make bixin diesters, which worked as initiators and were added into "-caprolactone to synthesis polyesters. The corresponding polymers had the MW 10,600, 12,900 and 11,100. Esterases/lipases and some acyltransferases share a Ser-His-Asp catalytic triad and similar mechanisms. Comparison of the x-ray structures of these structurally related esterases/lipases with acyltransferases reveals a different conformation of the oxyanion loop. In esterases/lipases this loop adopts a type II ! turn conformation with C=O of the main chain facing the active site. In acyltransferases this loop adopts a type I ! turn with the N–H of the main chain facing the active site. The x-ray crystal structure of Pseudomonas fluorescens esterase containing a sulfonate transition state analog shows the C=O facing active site activate via a bridging water molecule to the attacking water molecule. While in acyltransferases an opposing interaction with the N– H may deactivate the attacking water molecule. Oxyanion turn GWLL of Pseudomonas fluorescens esterase (PFE) was engineered in order to switch to a type I # turn and favor acyltransfer reaction. Replacing GWLL with acyltransferases oxyanion turn peptides yielded several mutants with only PFE-GLRA soluble. However PFE-GLRA doesn't show activity. Saturation mutagenesis at position W28 and L29, yielded mutants L29I, L29T, L29V, L29W, with acyltransfer/hydrolysis (A/H) ratios 2.2, 2.5, 2.5, 4 fold of that of the wild type. Some of these mutants might contain type I # turns.