Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / The direct use of vegetable oils as a biofuel suffers from problems such as high viscosity, low volatility and poor cold temperature properties. 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) have lower viscosity and freezing temperature than regular vegetable oils. However, by modifying their fatty acid composition, further improvement in their fuel properties is possible. Our goal was to develop plants that synthesize seed oils with further improved fuel properties. Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes acetyl-TAGs by the acetyl-CoA dependent acylation of diacylglycerol (DAG). Knowledge of the substrate specificity of EaDAcT for its acetyl-CoA donor and DAG acceptor substrates is important to generate the required acetyl-TAG composition in seed oil. A rapid method to quantify acetyl-TAGs was developed based on electrospray ionization mass spectrometry to gain information about the substrate specificity of EaDAcT. This method is as accurate and more rapid than the traditional radiolabeled substrate based assay and additionally provides information on acetyl-TAG molecular species present. Using this assay, EaDAcT specificity for different chain length acyl-CoA and DAGs was tested. It was found that although EaDAcT can use other short chain length acyl-CoAs as acyl donors, it has high preference for acetyl-CoA. Further, EaDAcT can acetylate a variety of DAGs with short, medium and long chain length fatty acids with high preference for DAGs containing unsaturated fatty acids. To generate acetyl-TAGs with lower molecular mass, EaDAcT was transformed into transgenic Camelina sativa lines producing high amounts of medium chain fatty acids (MCFAs). EaDAcT expression was also combined with the knockdown of DGAT1 and PDAT enzymes, which compete with EaDAcT for their common DAG substrate. High acetyl-TAG yielding homozygous T3 transgenic lines were generated but the incorporation of MCFAs into acetyl-TAGs was inefficient. A small increase in the viscosity of acetyl-TAGs from these lines was observed compared to acetyl-TAGs produced in wild type Camelina plant. The combined effect of insufficient lowering of molecular mass and increased fatty acid saturation levels of acetyl-TAGs might be responsible for this increased viscosity. Overall, it was concluded that the molecular mass and the saturation levels of fatty acids of acetyl-TAGs need to be considered at the same time in future attempts to further decrease their viscosity.

The search for alternatives to oil-based monomers has sparked interest for scientists to focus on the use of renewable resources for energy production, for the synthesis of polymeric materials and in other areas. With the use of renewable resources, scientists face new challenges to first isolate interesting molecules and then to process them. Enzymes are nature’s own powerful catalysts and display a variety of activities. They regulate important functions in life. They can also be used for chemical synthesis due to their efficiency, selectivity and mild reaction conditions. The selectivity of the enzyme allows specific reactions enabling the design of building blocks for polymers. In the work presented here, a lipase (Candida antarctica lipase B (CalB)) was used to produce building blocks for polymers. An efficient route was developed to selectively process epoxy-functional fatty acids into resins with a variety of functional groups (maleimide, oxetane, thiol, methacrylate). These oligoester structures, based on epoxy fatty acids from birch bark and vegetable oils, could be selectively cured to form thermosets with tailored properties. The specificity of an esterase with acyl transfer activity from Mycobacterium smegmatis (MsAcT) was altered by rational design. The produced variants increased the substrate scope and were then used to synthesize amides in water, where the wild type showed no conversion. A synthetic procedure was developed to form mixed dicarboxylic esters by selectively reacting only one side of divinyl adipate in order to introduce additional functional groups. / <p>QC 20170823</p>

Accurate, timely replication of a DNA molecule is a pivotal moment in the life cycle of every living organism. Any temporal or spatial defect putting the fine-tuned replication machinery off balance causes the so-called replication stress. As the replication machinery consists mainly of enzymes and other proteins, it is not surprising that many of the obstacles most severely blocking the replication machinery progress are of protein origin. Therefore, specialized proteases responsible for relieving replication stress matured during evolution. However, neither the full repertoire of proteolytic enzymes and their particular substrates taking place in countering the DNA replication stress nor detailed molecular mechanisms involved remain unknown. This thesis describes how conserved putative aspartic proteases of the Ddi1-like family engage in countering DNA replication stress via a proteolysis dependent mechanism. We structurally and biophysically characterized yeast and human members of the Ddi1-like family, explored their interactions with ubiquitin and polyubiquitin chains, and identified hypersensitivity to DNA replication inhibitor hydroxyurea in a yeast strain double deleted for DDI1 gene together with a DNA dependent metalloprotease WSS1. Detailed analysis of the DDI1 role in hydroxyurea...

Intramembrane proteases from the rhomboid-like superfamily are enzymes widely distributed and conserved in all domains of life. They participate in many important processes such as membrane protein quality control or mitochondrial dynamics. Their activity is also linked with diseases like Parkinson's disease or cancer. This makes them potential therapeutic targets. In this work we tried to elucidate in more detail the mechanism of action of the main model intramembrane protease, GlpG from E. coli. We also focused on the mechanism of eukaryotic rhomboid RHBDL2, one of the four mammalian rhomboids, function of which is poorly understood. To acquire more detailed information about substrate-enzyme interaction, we synthesized a series of novel peptidyl-chloromethylketone inhibitors derived from natural rhomboid substrate TatA from P. stuartii. Crystal structure of the complex of GlpG with these inhibitors revealed four substrate binding subsites (S1 to S4) of the enzyme and explained its observed substrate specificity structurally. This study showed that substrate cleavage rate can be dramatically modified by changing the substrate sequence in positions P1 to P5. This helped us develop fluorogenic transmembrane peptide substrates for rhomboid proteases, which are usable in detergent and liposomes, and...

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first protocol tackles this problem by developing a way to obtain active untagged recombinant PRMT proteins. The second protocol describes a fast and efficient method for quantitative measurement of AdoMet-dependent methyltranseferase activity with protein substrates. In addition to being very sensitive, this method decreases the processing time for the analysis of PRMT activity to a few minutes compared to weeks by traditional methods, and generates 3000-fold less radioactive waste. We then used these methods to investigate the effect of truncating the NT of human PRMT1 variant 1 (hPRMT1-V1) on enzyme activity, protein-protein interactions, and substrate specificity. Our studies show that the NT of hPRMT1-V1 influences enzymatic activity and protein-protein interactions. In particular, methylation of a variety of protein substrates was more efficient when the first 10 amino acids of hPRMT1v1 were removed, suggesting an autoinhibitory role for this small section of the N-terminus. Likewise, as portions of the NT were removed, the altered hPRMT1v1 constructs were able to interact with more proteins. Overall, my studies suggest the the sequence and length of the NT of hPRMT1v1 is capable of enforcing specific protein interactions.

A newly discovered cysteine protease, Prp, has been shown to perform an essential, site-specific cleavage of ribosomal protein L27 in Staphylococcus aureus. In Firmicutes and related bacteria, ribosomal protein L27 is encoded with a conserved N-terminal extension that must be removed to expose residues critical for ribosome function. Uncleavable and pre-cleaved variants were unable to complement an L27 deletion in S. aureus, indicating that this N-terminal processing event is essential and likely plays an important regulatory role. The gene encoding the responsible protease (prp) has been shown to be essential, and is found in all organisms encoding the N-terminal extension of L27. Cleavage of L27 by Prp represents a new target for potential antibiotic therapy. In order to characterize this protease, Prp has been overexpressed and purified. Using an assay we have developed, based on cleavage of a fluorogenic peptide derived from the conserved L27 cleavage sequence, we have undertaken an analysis of the enzyme kinetics and substrate specificity for Prp cleavage and tested predictions made based on a structural model using active-site mutants.

The crucial part of the biosynthesis of lincosamide antibiotics lincomycin and celesticetin is the condensation of amino sugar and amino acid moieties. This reaction is catalysed by the oligomeric enzyme lincosamide synthetase (LS). One of the most important components of LS is adenylation domain recognizing and activating amino acid precursor. The substrate specificity of adenylation domain is determined by "nonribosomal code", 10 amino acids residues which side chains are in close contact with the activated substrate. The homologous adenylation domains LmbC from biosynthesis of lincomycin and CcbC from biosynthesis of celesticetin exhibit strong substrate specificity for their natural substrates (2S,4R)-4-propyl-L-proline (PPL) and L-proline, respectively. At first the effect of selected amino acid residues of LmbC nonribosomal code on the substrate specificity of the whole domain was tested. The amino acids residues, most important for preference of PPL substrate over L proline, were determined: G308, A207 and L246. Then the effect of double mutations in nonribosomal codes of both LmbC and CcbC on their substrate specificity was evaluated. The double mutants LmbC G308V + A207F and CcbC V306G + F205A were prepared and tested biochemically. The results brought new evidence of validity of homologous models...

This thesis deals with factors structuring phytoplankton flagellates. The aim of the thesis was to investigate the role of microhabitats in spatio-temporal differentiation of lentic communities of these flagellates, which has not been rigorously examined to date. The study was conducted during 2010-2013 in the shallow littoral sites of Horní rybník, Rybníčky u Podbořánek Nature Reserve. Phytoplankton flagellates showed cyclic seasonal dynamics, which was reflected by significantly different community structure and species richness during the season. In contrast, silica-scaled chrysophytes showed a non- cyclic seasonal dynamics. The impact of the season was strongly correlated with the impact of fluctuating environmental factors - pH, conductivity and temperature. Gradient of interrelated environmental factors between the northern and southern shore together with microhabitat - substrate type played the major role and accounted for 64.4 - 75.8 % variability in the data. Microhabitat type (plankton, metaphyton, epipelon) significantly affected species richness and community structure also in the course of the season. A large number of species was assessed to prefer a certain type of microhabitat. These preferences were observed at both species and generic levels, and even at the level of the main taxonomic...

English abstract Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane bound glycoprotein that belongs to the metallopeptidase M28 family. Two physiological substrates were found for GCPII. The first one, N-acetyl-aspartylglutamate (NAAG), serves as a neurotransmiter in the brain and GCPII hydrolyzes it to yield free glutamate in the synaptic cleft. Excess glutamate might be cytotoxic and eventually lead to excitoxic nerve cells death. Inhibition of NAAG hydrolyzing activity has been shown to be neuroprotective. Therefore, GCPII inhibition was suggested as a therapeutic target in treatment of neurological disorders where excess glutamate is involved. The second substrate, polyglutamyl folate, is a precursor of folic acid which is required for cell growth and development. GCPII cleaves off glutamate from dietary folates and thus facilitates their absorption in small intestine. Although GCPII biological relevance is known only in the brain and the small intestine, its role in the prostate is also important. GCPII has been described as a prostate cancer marker as it is expressed on the membrane of prostate cancer cells. Since GCPII is type II transmembrane protein, it is enzymatically active and undergoes internalization, it has been suggested as a promising tool for specific anticancer-drug...

Nitrilases are enzymes which catalyze the hydrolysis of a nitrile into the corresponding carboxylic acid and ammonia. These enzymes are potentially applicable in biocatalysis and bioremediation because of their advantages over the conventional (chemical) methods of nitrile hydrolysis (lower demand for energy, safety, simplicity, high yields, selectivity). In this work, genome mining was used to search for the sequences of hypothetical nitrilases from filamentous fungi. The amino acid sequences of previously characterized fungal nitrilases were used as the templates. Then the new synthetic genes together with other genes from our nitrilase library were expressed in E. coli and the substrate specificities of the enzymes thus produced were compared. Significant attention was focused on the relationships between the sequence of the enzyme and its substrate specificity. The arylacetonitrilases from Arthroderma benhamiae (NitAb) and Nectria haematococca (NitNh) were purified and characterized. Their substrate specificities, kinetic parameters, pH and temperature profiles and subunit and holoenzyme size were assessed. NitAb and NitNh together with other recombinant fungal nitrilases were employed in the hydrolysis of high concentrations of (R,S)-mandelonitrile in a batch or fed-batch mode. Nitrilase from...