Protein arginine methyltransferases (PRMTs)mediate the transfer of methyl groups to arginine residues in histone and non-histone proteins. PRMT5 is an important member of PRMTs which symmetrically dimethylates arginine 8 in histone H3 (H3R8) and arginine 3 in histone H4 (H4R3). PRMT5 was reported to inhibit some tumor suppressors in leukemia and lymphoma cells and regulate p53 gene, through affecting the promoter of p53. Through methylation of H4R3, PRMT5 can recruit DNA-methyltransferase 3A (DNMT3A) which regulates gene transcription. All the above suggest that PRMT5 has an important function of suppressing cell apoptosis and is a potential anticancer target. Currently, the enzymatic activities of PRMT5 are not clearly understood. In our study, we improved the protein expression methodology and greatly enhanced the yield and quality of the recombinant PRMT5. In addition, mutagenesis and enzymatic studies implicate an interesting mechanism of PRMT5 activity regulation.

Graduation date: 1992

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.

<p><i>Lactococcus lactis,</i> prolidase (<i>Lla</i>prol) hydrolyzes Xaa-Pro dipeptides. Since Xaa-Pro is known as bitter peptides, <i>Lla</i>prol is potentially applicable to reduce bitterness of fermented foods. <i>Lla</i>prol shows allosteric behaviour and substrate inhibition, which are not reported in other prolidases. Computer models of <i>Lla</i>prol based on an X-ray structure of non-allosteric <i>Pyrococcus furiosus</i> prolidase showed that a loop structure (Loop^32-43) is located at the interface of the protomers of this homodimeric metallodipeptidase. This study investigated roles of four charged residues (Asp³⁶, His³⁸, Glu³⁹, and Arg⁴⁰) of Loop^32-43 in <i>Lla</i>prol using a combination of kinetic examinations of ten mutant enzymes and their molecular models. Deletion of the loop structure by Î36-40 mutant resulted in a loss of activity, indicating Loop^32-43 is crucial for the activity of <i>Lla</i>prol. D36S and H38S exhibited 96.2 % and 10.3 % activity of WT, whereas little activities (less than 1.0 % of WT activity) were observed for mutants E39S, D36S/E39S, R40S, R40E, R40K and H38S/R40S. These results implied that Glu³⁹ and/or Arg⁴⁰ play critical role(s) in maintaining the catalytic activity of <i>Lla</i>prol. These observations suggested that the loop structure is flexible and this attribute, relying on charge-charge interactions contributed by Arg⁴⁰, Glu³⁹ and Lys¹⁰⁸, is important in maintaining the activity of <i>Lla</i>prol. When the loop takes a conformation close to the active site (closed state), Asp³⁶ and His³⁸ at the tip of the loop can be involved in the catalytic reaction of <i>Lla</i>prol. The two active mutant prolidases (D36S and H38S) resulted in modifications of the unique characteristics; the allosteric behaviour was not observed for D36S, and H38S <i>Lla</i>prol showed no substrate inhibition. D36E/R293K, maintaining the negative charge of position 36 and positive charge of position 293, still possessed the allosteric behaviour, whereas the loss of the charges at these positions (D36S of this study and R293S of a previous study (Zhang et al., 2009 BBA-Proteins Proteom 1794, 968-975) eliminated the allosteric behaviour. These results indicated the charge-charge attraction between Asp³⁶ and Arg²⁹³ is important for the allostery of <i>Lla</i>prol. In the presence of either zinc or manganese divalent cations as the metal catalytic centre, D36S and H38S enzymes also showed different substrate preferences from WT <i>Lla</i>prol, implying the influence of Asp³⁶ and His³⁸ on the substrate binding. D36S and H38S also showed higher activities at pH 5.0 to 6.0, in which range WT <i>Lla</i>prol steeply decreased its activity, indicating Asp³⁶ and His³⁸ are involved in the active centre and influence the microenvironment of catalytic His²⁹⁶. The above observations are attributed to modifications of their local structure in the active centre since the temperature dependency and thermal denaturing temperature indicated little effects on the overall structure of the <i>Lla</i>prol mutants.</p> <p>From these results, we concluded that the unique behaviours of <i>Lla</i>prol are correlated to Loop^32-43 and Asp³⁶ and His³⁸ on it. When Loop^32-43 takes a closed conformation, Asp³⁶ interacts with Arg²⁹³ via charge-charge attraction to form an allosteric subsite. The saturation of the allosteric site with substrates further allowed the communications of His³⁸ with S₁ site residues to complete the active site. When the substrate concentration becomes higher than it is required to saturated productive S₁' site, His³⁸, Phe¹⁹⁰ and Arg²⁹³ would resemble the residue arrangement of S₁' site residues (His²⁹², Tyr³²⁹, and Arg³³⁷) and bind to the proline residue of substrates. This non-productive binding would prevent the conformational change of Loop^32-43, which further results in the substrate inhibition. For further confirmation of this mechanism, crystallographic studies will be conducted. In this thesis, we have indentified the conditions to produce crystals of <i>Lla</i>prol proteins.</p>

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 179-191).

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 82-99). Also available in electronic version. Access restricted to campus users.

Chlorate and perchlorate pollutions have become a problem in the environment in the last decades. Studies have shown that some bacteria can degrade these substances into unharmful substances such as chloride and molecular oxygen. One of these chlorate degrading bacteria is Ideonella dechloratans that uses chlorate reductase and chlorite dismutase to process chlorate. In the promoter gene sequence of chlorite dismutase there might be regulator sequences such as fumarate and nitrate reductase regulator (FNR) and aerobic respiration control protein (ArcA) that might control the transcription of this enzyme. This promoter sequence was placed in a pBBR1MCS-4-LacZ reporter vector and the possible regulatory sequences were changed through site-directed mutagenesis and tested on activity through beta-galactosidase assays. The changes in the FNR binding sequence gave beta-galactosidase activity that was close to a negative control which might give conclusions that either FNR has an important role or an important part of the promoter was hit. The changes in the ArcA regulator binding sequence did not give such big differences and no certainty can be given if this made important changes to the promoter.

Altering the substrate specificity of proteases is a powerful process with possible applications in many areas of therapeutics as well as proteomics. Although the field is still developing, several proteases have been successfully engineered to recognize novel substrates. Previously in our laboratory, eight highly active OmpT variants were engineered with novel catalytic sites. The present study examined the roles of several residues surrounding the active site of OmpT while attempting to use rational design to modulate fine specificity enough to create a novel protease that prefers phosphotyrosine containing substrates relative to sulfotyrosine or unmodified tyrosine residues. In particular, a previously engineered sulfotyrosine-specific OmpT variant (Varadarajan et al., 2008) was the starting point for rationally designing fifteen new OmpT variants in an attempt to create a highly active protease that would selectively cleave phosphotyrosine substrates. Our design approach was to mimic the most selective phosphoryl-specific enzymes and binding proteins by increasing positive charge around the active site. Sulfonyl esters have a net overall charge of -1 near neutral pH, while phosphate monoesters have a net overall charge of -2. Selected active site residues were mutated by site-directed mutagenesis to lysine, arginine, and histidine. The catalytic activities and substrate specificities of each variant were characterized. Although several variants displayed altered substrate specificity, none preferred phosphotyrosine over sulfotyrosine containing peptides. Taken together, our results have underscored the subtle nature of protease substrate specificity and how elusive it can be to engineer fine specificity. Apparently, phosphotyrosine specific variants were not possible within the context of our starting sulfotyrosine specific OmpT derivative mutated to have single amino acid changes chosen on the basis of differential charge interactions. / text

From microscopic bacteria to the giant whale, every single living organism on Earth uses the same language of life: DNA. Deoxyribonucleoside triphosphates––dNTPs (dATP, dTTP, dGTP, and dCTP)––are the building blocks of DNA and are therefore the “alphabet of life”. A balanced supply of dNTPs is essential for integral DNA transactions such as faithful genome duplication and repair. The enzyme ribonucleotide reductase (RNR) not only synthesizes all four dNTPs but also primarily maintains the crucial individual concentration of each dNTP in a cell. In this thesis we investigated what happens if the crucial balanced supply of dNTPs is disrupted, addressing whether a cell has a mechanism to detect imbalanced dNTP pools and whether all pool imbalances are equally mutagenic. To address these questions, we introduced single amino acid substitutions into loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR and obtained a collection of yeast strains with different but defined dNTP pool imbalances. These results directly confirmed that the loop 2 is the structural link between the substrate specificity and effector binding sites of RNR. We were surprised to observe that mutagenesis was enhanced even in a strain with mildly imbalanced dNTP pools, despite the availability of the two major replication error correction mechanisms: proofreading and mismatch repair. However, the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity, and the locations of the mutations in a strain with elevated dTTP and dCTP were completely different from those in a strain with elevated dATP and dGTP. We then investigated, whether dNTP pool imbalances interfere with cell cycle progression and if they are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. The S-phase checkpoint was activated by the depletion of one or more dNTPs. In contrast, when none of the dNTP pools was limiting for DNA replication, even extreme and mutagenic dNTP pool imbalances did not activate the S-phase checkpoint and did not interfere with the cell cycle progression. We also observed an interesting mutational strand bias in one of the mutant rnr1 strains suggesting that the S-phase checkpoint may selectively prevent formation of replication errors during leading strand replication. We further used these strains to study the mechanisms by which dNTP pool imbalances induce genome instability. In addition, we discovered that a high dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions, which are difficult to bypass at normal dNTP concentrations. Our results broaden the role of dNTPs beyond ‘dNTPs as the building blocks’ and suggest that dNTPs are not only the building blocks of DNA but also that their concentrations in a cell have regulatory implications for maintaining genomic integrity. This is important as all cancers arise as a result of some kind of genomic abnormality.