Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites.

Phosphotriesterase (PTE) isolated from the soil bacterium Flavobacterium sp. is a member of the amidohydrolase superfamily. PTE catalyzes the hydrolysis of a broad spectrum of organophosphate triesters including the insecticide paraoxon, and the chemical warfare agents; GF, sarin, and soman. In addition, PTE has been shown to catalytically hydrolyze the lethal nerve agent, VX. However, the rate of VX hydrolysis is significantly slower. PTE was subjected to directed evolution studies to identify variants with enhanced activity towards VX hydrolysis. First generation libraries targeted amino acid residues in the substrate binding site. The H254A mutation displayed a 4-fold enhancement in kcat and a 2-fold enhancement in kcat/Km over wild type PTE. The double mutant H254Q/H257F was isolated from the second generation libraries and displayed a 10-fold enhancement in kcat and a 3-fold enhancement in kcat/Km. In addition, H254Q/H257F displayed a 9-fold enhancement in kcat/Km for the hydrolysis of the VX analog, demeton-S. An in vivo selection approach utilizing organophosphate triesters as the sole phosphorus source is discussed. The selection is based on co-expressing PTE with the phosphodiesterase (GpdQ) from E. aerogenes. Substrate specificity of GpdQ was investigated using a small library of structurally diverse organophosphate diesters and phosphonate monoesters. Results obtained from the in vivo growth assays showed that GpdQ enabled E. coli to utilize various organophosphate diesters and phosphonate monoesters as the sole phosphorus source. Cells co-expressing PTE and GpdQ were tested for their ability to utilize two different organophosphate triesters as the sole phosphorus source. The results from this experiment indicate that the growth rate is limited by the phosphotriesterase activity. Protein translocation to the periplasm was proven advantageous for in vivo selection since it overcomes the limitation of intercellular delivery of the substrate of interest. Translocation of PTE to the periplasmic space of E. coli was examined. Two signal peptides were tested; the native leader peptide from Flavobacterium sp. and the signal sequence of alkaline phosphatase. The results obtained from cellular fractionation indicated that neither signal peptides were able to translocate PTE to the periplasm and that the protein remained in the cytoplasm.

Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites.

A major hurdle facing in vitro protein characterization is obtaining soluble protein from targets that tend to aggregate and form insoluble inclusion bodies. Soluble protein is essential for any biophysical data collection and new methods are needed to approach this significant problem. Directed evolution can be used to discover mutations which lead to improved solubility using an appropriate screening method. Green fluorescent protein (GFP) has been shown to be an effective solubility reporter which can be used to screen for soluble protein variants. We have chosen three diverse enzymes as targets for improving protein solubility using this technique: arachidonate 5-lipoxygenase—an enzyme which converts fatty acids into leukotrienes, PhnG—an enzyme belonging to the bacterial carbon-phosphorus lyase pathway, and RebG—a glycosyltransferase. Error-prone PCR and DNA shuffling were used to generate libraries of mutants which were subsequently cloned into a GFP-fusion screening vector. From the evolution of 5LO and RebG, much was learned about the optimization of the protocols involved in this methodology, including valuable information about how to avoid common “false-positive” results in which fluorescent colonies arise while screening but do not represent an improvement of the target. Evolution of these two targets did not result in an improvement of solubility, however truncation strategies may still prove to be effective, and more work needs to be done in this area. Evolution of PhnG successfully produced one variant, named clone B6, which showed both an improvement in expression and folding over wild type PhnG. It was also discovered that GFPuv can act as an effective solubility enhancing fusion tag for PhnG. Prior to the current studies PhnG had not been effectively expressed and purified in E. coli , however purification and refolding of resolubilized inclusion bodies of the clone B6 PhnG-GFP fusion construct was shown to yield enough soluble protein for future crystallographic studies. / Thesis (Master, Chemistry) -- Queen's University, 2009-10-09 12:26:03.353

Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Acyl Coenzyme A: Isopenicillin N Acyl Transferase (AT) is a key enzyme in the biosynthesis of β-lactam antibiotics in penicillin producing organisms such as P. chrysogenum and A. nidulans. Its natural activity is to exchange the side chain of the low activity antibiotic IPN [18] for the phenylacetyl side chain resulting in the more active antibiotic Penicillin G [5]. The biosynthesis of β-lactams has been exploited towards producing these compounds for therapeutic use. However, increasing bacterial resistance means new analogues in this compound class are constantly sought.As well as improving current production methods of β-lactam antibiotics, AT's broad substrate specificity means it could potentially play a role in the development and production of alternative β-lactam antibiotics that are able to overcome resistance.This thesis describes the identification of an AT mutant with improved acylation activity (AAT activity) via screening of an AT library using a previously developed screening method. Approaches towards the development of a method for the identification of AT mutants with improved hydrolysis activity were also explored. The main problem to overcome in developing such a screen is the inhibitory effect of 6-APA [1], the product of hydrolysis, on AT's IAT activity. The first approach investigated the potential of increasing the sensitivity of an assay by targeting AT to the periplasm. A second approach using β-lactamases to hydrolyse 6-APA [1] thus freeing up the active site of AT was also investigated.

Gene therapy holds the unprecedented potential to treat disease by manipulating the underlying genetic blueprints of phenotypic behavior. Targeting of gene delivery is essential to achieve specificity for the intended tissue, which is especially critical in cancer gene therapy to avoid destruction of healthy tissue. Adeno-associated virus (AAV) is considered the safest viral vector and, compared to non-viral vectors, offers several advantages: higher efficiency, genetic modification, combinatorial panning, and high monodispersity. Classic viral targeting has focused on engineering ligand-receptor interactions, but many cell surface targets do not support post-binding transduction events. Furthermore, many potential target tissues – such as triple negative breast cancer – may not display a single, unique identifying surface receptor, so new methods of targeting are needed. Alternatively, many pathological states, including most cancers, exhibit upregulation of proteolytic enzymes in the extracellular milieu. The present work describes the development of an AAV platform that has been engineered to activate in response to disease-related proteases. The specificity and sensitivity of these protease-activatable viruses (PAVs) can be tuned to meet the demands of various clinical scenarios, giving the platform some therapeutic versatility. This work represents the first demonstration of a protease-controlled, non-enveloped virus for genetic therapy. These results extend the therapeutic value of AAV, the safest gene vector currently being explored in 73 clinical trials worldwide.

Thesis advisor: Abhishek Chatterjee / Thesis advisor: Eranthie Weerapana / In nature, just twenty canonical amino acids are responsible for the creation of nearly all proteins. Genetic code expansion (GCE), or the incorporation of noncanonical amino acids (ncAAs) into living cells, is a powerful tool that expands the studies we are capable of performing using proteins. This technology relies on engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that are orthogonal to the host cells’ endogenous aaRS/tRNA pairs, and one of the main limitations of GCE arises from the inefficiency of these suppressor tRNAs when expressed in a foreign host cell. To address this limitation, we have previously reported a strategy for the virus-assisted directed evolution of tRNAs (VADER) which is uniquely capable of addressing the specific needs of tRNA evolution. In order to advance the capabilities of VADER, we made a number of modifications to the VADER selection scheme. First, we designed and executed a modified VADER selection that enabled the evolution of a new class of tRNAs, and with this VADER selection, we were able to generate a first-generation E. coli tyrosyl tRNA (tRNATyr) variant that was three times as active as its wild-type equivalent. Next, we introduced a number of refinements to the VADER strategy to generate VADER 2.0, an improved workflow capable of screening larger libraries and libraries encoding more active variants. Using VADER 2.0, we created second-generation tRNAPyl and tRNATyr mutants that achieved incorporation efficiencies that were greater than five-fold higher than their wild-type equivalents across a wide variety of substrates, enabling exciting GCE experiments that would not be possible otherwise. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

No description available.

The thermophilic enzyme, Thermus aquaticus (Taq) DNA polymerase, is an essential tool in molecular biology because of its ability to synthesis DNA in vitro and its inherent thermal stability. Taq DNA polymerase is widely used in the polymerase chain reaction (PCR), an essential technique in a broad range of different fields from academic research to clinical diagnostics. The use of PCR-based tests in diagnostic testing is ever increasing; however, many of the samples being tested contain substances that inhibit PCR and prevent target amplification. Many attempts have been made to engineer polymerases not only to increase resistance to overcome the problem of inhibition, but also to enhance other characteristics such as fidelity, processivity and thermostability. Heparin, found in blood samples, and phytate, found in faecal samples, are two examples from a number of known PCR inhibitors. The mode of action of most PCR inhibitors is not well understood, but inhibition is thought to occur by enzyme binding or through the chelation of Mg2+ ions essential for PCR. In this project, a system of directed evolution by compartmentalised self-replication (CSR) was established and successfully employed to screen a mutant library for Taq DNA polymerase variants with enhanced resistance to the inhibitors heparin and phytate. CSR is a recently-established high-throughput method for the creation of novel polymerases, based on a feedback loop whereby polymerase variants replicate their own encoding gene. A mutant library of 106 variants was produced by random mutagenesis error-prone PCR, in which only the polymerase domain of Taq was mutagenised. Firstly, the CSR system was established and tested by performing a screen in the presence of heparin to select for heparin-resistant variants. Characterisation of selected variants revealed that a single round of CSR had produced a Taq variant (P550S, T588S) with a 4-fold increase in heparin resistance. The IC50 was increased from 0.012U/ml heparin to 0.050U/ml heparin. The study with heparin was followed by a phytate screen, in which two rounds of CSR were performed with an initial round of error-prone PCR followed by re-diversification (recombination) of the mutant library using the staggered extension process (StEP). The two rounds of CSR yielded a Taq variant with a 2-fold increase in phytate-resistance compared to the wild-type, with IC50 increased from 360μM phytate to 700μM phytate. The best phytate mutant (P685S, M761V, A814T) was further characterised and it was found that the catalytic activity, thermostability and fidelity of the mutant were comparable to the wildtype enzyme. The position of resistance-conferring mutations of the novel Taq variants evolved in this study provided some evidence for the inhibitors’ predicted modes of action in the case 2 of both phytate and heparin. As phytate’s mode of action is poorly understood, further investigations were performed to elucidate its role in PCR inhibition. A thorough investigation into the importance of relative phytate and Mg2+ levels on PCR was conducted and revealed for the first time convincing evidence that the primary mode of phytatemediated PCR inhibition is by chelation. Further work led to the successful crystallisation of Taq in the presence of phytate, although subsequent X-ray diffraction data to 2.5Å did not reveal phytate bound within the enzyme structure. Site-directed mutagenesis studies were used to probe cross-over between heparin and phytate-conferring mutations. Thus, in addition to providing valuable information for novel Taq variants with a potential application in fecal-based PCR diagnostic tests, this project has begun to provide insight into the fundamental aspects of the mode of action of phytate as a polymerase and PCR inhibitor.