Biochemical and Mechanistic Studies of Nitronate Monooxygenase and Roles of Histidine Residues in Select Flavoprotein Oxidases

Nitronate monooxygenase (NMO) catalyzes the flavin-dependent oxidation of propionate 3-nitronate (P3N) via the formation of an anionic flavosemiquinone. The oxidation of substrate includes the formation of a peroxy-nitro acid intermediate. P3N is activated to its radical form via a single electron transfer onto the FMN cofactor forming the anionic flavosemiquinone. Reoxidation of FMN cofactor from the anionic semiquinone has been proposed to go through two routes dependent upon which radical species oxygen reacts with first, radical P3N or the semiquinone. The recent crystallographic determination of NMO from Cyberlindnera saturnus and steady-state kinetics revealed an allosteric activation effect on the enzyme by PEG 3350 with respect to P3N. Choline oxidase (CHO) catalyzes the two-step oxidation of choline to glycine betaine via an enzyme-bound FAD cofactor. In the first redox reaction, choline is activated to it alkoxide form by means of an enzyme-derived catalytic base, H466. This histidine residue has been shown to not only act as a general base but an electrostatic catalyst stabilizing the negative charge accumulated on the reduced flavin species as shown by replacing the residue with alanine, aspartate, and glutamine using site-directed mutagenesis. CHO was also observed to catalyze excited state reactions as facilitated by H466. Evidence for the ESR comes from the observation of a pL-dependence on the fluorescence emission of CHO in H2O and D2O. Using fluorescence spectroscopy and pH effects, a hydroxy-C4a flavin intermediate was detected in the wild-type and S101A variant with and without oxygen indicating the adduct formation was with an active site hydroxide ion. The mechanism of formation has been elucidated

Establishing the Relationship Between Function and Dynamics Within the Gated Mechanism of D-arginine Dehydrogenase

Enzymes are ubiquitous in biological systems. They catalyze chemical reactions and are involved in many biochemical processes. The enzyme of interest is Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH). This flavin-dependent enzyme is composed of approximately 375 amino acid residues and has a broad substrate specificity with D-amino acids. A water recognition motif, observed in roughly 1200 non-redundant protein data bank (PDB) structures, was revealed to be embedded near the active site of PaDADH. This motif coincides with the conformational changes of the enzyme’s gated mechanism. Molecular dynamics simulations were carried out to study the gated properties and structural characteristics of PaDADH in solution. Single amino acid mutations were undertaken to further understand the dynamics of the gated mechanism of this enzyme. In addition, pKa,shift analyses were evaluated to probe for the basic catalytic amino acid residue that is suggested to trigger the catalytic mechanism of PaDADH

Computational Perspective on Intricacies of Interactions, Enzyme Dynamics and Solvent Effects in the Catalytic Action of Cyclophilin A

Cyclophilin A (CypA) is the well-studied member of a group of ubiquitous and evolutionarily conserved families of enzymes called peptidyl–prolyl isomerases (PPIases). These enzymes catalyze the cis-trans isomerization of peptidyl-prolyl bond in many proteins. The distinctive functional path triggered by each isomeric state of peptidyl-prolyl bond renders PPIase-catalyzed isomerization a molecular switching mechanism to be used on physiological demand. PPIase activity has been implicated in protein folding, signal transduction, and ion channel gating as well as pathological condition such as cancer, Alzheimer’s, and microbial infections. The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerization by CypA has been the target of intense research. Normal and accelerated molecular dynamic simulations were carried out to understand the catalytic mechanism of CypA in atomistic details. The results reaffirm transition state stabilization as the main factor in the astonishing enhancement in isomerization rate by enzyme. The ensuing intramolecular polarization, as a result of the loss of pseudo double bond character of the peptide bond at the transition state, was shown to contribute only about −1.0 kcal/mol to stabilizing the transition state. This relatively small contribution demonstrates that routinely used fixed charge classical force fields can reasonably describe these types of biological systems. The computational studies also revealed that the undemanding exchange of the free substrate between β- and α-helical regions is lost in the active site of the enzyme, where it is mainly in the β-region. The resultant relative change in conformational entropy favorably contributes to the free energy of stabilizing the transition state by CypA. The isomerization kinetics is strongly coupled to the enzyme motions while the chemical step and enzyme–substrate dynamics are in turn buckled to solvent fluctuations. The chemical step in the active site of the enzyme is therefore not separated from the fluctuations in the solvent. Of special interest is the nature of catalysis in a more realistic crowded environment, for example, the cell. Enzyme motions in such complicated medium are subjected to different viscosities and hydrodynamic properties, which could have implications for allosteric regulation and function

Investigation of Novel Functions for DNA Damage Response and Repair Proteins in Escherichia coli and Humans

Endogenous and exogenous agents that can damage DNA are a constant threat to genome stability in all living cells. In response, cells have evolved an array of mechanisms to repair DNA damage or to eliminate the cells damaged beyond repair. One of these mechanisms is nucleotide excision repair (NER) which is the major repair pathway responsible for removing a wide variety of bulky DNA lesions. Deficiency, or mutation, in one or several of the NER repair proteins is responsible for many diseases, including cancer. Prokaryotic NER involves only three proteins to recognize and incise a damaged site, while eukaryotic NER requires more than 25 proteins to efficiently recognize and incise a damaged site. XPC-RAD23B (XPC) is the damage recognition factor in eukaryotic global genome NER. The association rate of XPC to damaged DNA has been extensively studied; however, our data suggests that the dissociation of the XPC-DNA complex is the rate-limiting step in NER. The factor that verifies DNA-damage downstream of XPC is XPA. XPA also has been implicated in binding of ds-ssDNA junctions and has been found to bind at or near double-strand break sites in the premature aging syndrome Hutchinson-Gilford progeria (HGPS). This role for XPA is outside of its known function in NER and suggests that XPA may bind at collapsed replication forks in HGPS that are unprotected due to a lack of binding by replication proteins. Along with XPC and XPA, ataxia telangiectasia and Rad3-related (ATR) is activated in response to DNA damage and initiates the cell cycle checkpoint pathway to rescue cells from genomic instability. We found that ATR functions outside of its known role in the checkpoint signaling cascade. Our data demonstrate that ATR can rescue cells from apoptosis by inhibiting cytochrome c release at the mitochondria though direct interaction with the outer mitochondrial membrane and the proapoptotic protein tBid. The role of ATR in apoptosis is regulated by Pin1, which can change the structure of ATR at the backbone level. All of the results presented here suggest novel roles for DNA repair proteins in the maintenance of genome stability

Roles of auxin response factors in Arabidopsis flower development

The plant hormone auxin regulates organ initiation, growth, and development. The Auxin Response transcription Factors (ARFs) mediate transcriptional responses to auxin. Under low auxin concentration, the ARF proteins bind Aux/IAA proteins, which inhibit transcription. As auxin concentration is elevated, Aux/IAA proteins are rapidly degraded, thus allowing ARFs to activate target genes. Two closely related ARF genes, ARF6 and ARF8, regulate flower maturation by promoting stamen elongation and gynoecium development. ARF6 and ARF8 are cleavage targets of plant microRNA, miR167. Phenotypes and transcript expression patterns of miR167-insensitive mARF6 and mARF8 transgenic plants showed that miR167 patterns ARF6 and ARF8 transcript distribution in the ovule and in the anther, and this patterning activity is important for development of these two organs. Silencing ARF6 and ARF8 in the style and in the ovule funiculus by expressing a miR167 precursor gene, MIR167a, further showed that ARF6 and ARF8 promote stigmatic papillae elongation and pollen tube growth in these two different floral tissues. To further reveal functions of other ARF genes in flowers, especially during ovule formation, we repressed activity of ARF proteins by expressing a gain-of-function aux/iaa gene in the ovule outer integument and in the funiculus. We found that auxin response in the ovule is important for the asymmetric growth of the outer integument and for differentiation of the entire ovule. These results showed that auxin and the ARFmediated auxin responses regulate multiple aspects of flower development

The role of the transcription factor JAGGED in early floral organogenesis

Initiation of organ primordia from pools of undifferentiated cells requires coordinated cytoplasmic growth, oriented cell wall extension, and cell cycle progression. It is debated which of these processes are primary drivers for organ morphogenesis and directly targeted by developmental regulators. The single zinc finger transcription factor JAGGED (JAG) is a direct target of several floral organ identity genes and is expressed in early organ primordia (Dinneny et al., 2004; Ohno et al., 2004; Gomez et al., 2005; Kaufmann et al., 2009). Loss of function jag mutants have narrow floral organs with reduced distal growth. Quantitative 3D imaging has revealed that JAG is required for the transition from meristematic to organ primordium cell behaviour. The transition involves an increase in the rates of cell division and cell growth, a shift from isotropic to anisotropic growth, and modifications in cell size homeostasis in primordia (Schiessl et al., 2012). In this project, ChIP-Seq was combined with transcriptome analysis to identify global direct target genes of JAG. Consistent with the roles of JAG during organ initiation and organ growth, I found that JAG directly repressed genes involved in meristem development, such as the TALE PROTEIN BELL1 and genes involved in organ boundaries specification such as PETAL LOSS. In addition, JAG directly regulated genes involved in growth regulatory pathways, tissue polarity, cell wall modification, and cell cycle progression. For example, JAG directly repressed the cell cycle inhibitors KIP RELATED PROTEIN 2 and 4 (KRP2/4). The krp2 and krp4 mutations suppressed jag loss of function defects in organ growth and cell type patterning. In particular, loss of KRP4 rescued the defects of cell size homeostasis in the primordia of the jag loss of function mutant. In summary, this work revealed that JAG directly coordinates organ patterning with cellular processes required for tissue growth

Structural Exploration of Different Binding Pockets Suitable to Affect Protein Kinases CK2α and CK2α’ With Peptides and Small Molecules

The eukaryotic protein kinase CK2, previously known as casein kinase 2, is a ubiquitously expressed, acidophilic serine/threonine kinase belonging to a branch of the group of CMGC kinases. The enzyme features several peculiarities, one of which is its extraordinary pleiotropic character. A plethora of biological substrates have been described for CK2 to date and these are, among others, involved in cell proliferation, angiogenesis, apoptotic processes, viral infections, and DNA-damage repair. Several of these substrates play key roles in the development and progression of a diverse spectrum of diseases. The ubiquitous presence of CK2, combined with its unusual constitutive activity, presents a highly interesting pharmacological profile as a promising drug target. In particular, neoplastic diseases are significantly driven by high levels of CK2 and the importance of the search for suitable molecules to alter the enzymatic properties of CK2 is therefore evident and a subject of current research. This thesis also contributes to this field, focusing primarily on the investigation of the structural aspects of various protein-ligand interactions at different binding sites of the enzyme. An important role in these structural studies is accounted by CK2α’, a paralog of the catalytic subunit CK2α. Although the two paralogs are highly similar in many respects, CK2α’ has been neglected in CK2 research over the past decades due to its poor biochemical handleability and its insufficient crystallization properties. Therefore, in this work, for the first time, a crystallization protocol was developed that reliably yields CK2α structures with an atomic resolution of 1.0 Å and thereby outperforming all previously existing CK2α structures to date. This protocol has proven to be an extremely valuable crystallographic tool to study the precise binding mode of a wide variety of CK2 inhibitors from different substance classes, including high and low-affinity compounds. As an example, the exact binding site and binding mode of different 2 aminothiazole derivatives could be elucidated. These compounds belong to a class of CK2 inhibitors that were erroneously assumed to bind outside the cosubstrate pocket. In addition to crystallographic studies, organic syntheses were also conducted as part of the research for this thesis. This includes the synthesis of halogenated cyclic peptides which address the α/β interface area of the catalytic subunits, interfering with CK2β binding and thus with the tetrameric holoenzyme assembly. Furthermore, by conjugating with the cell-penetrating peptide sC18, it was possible to investigate the impact of some of these compounds on different cell lines. Moreover, different 4,5,6,7 tetrabromobenzimidazole derivatives were synthesized and studied. In particular, the bivalent inhibitor KN2, which simultaneously occupies the cosubstrate binding pocket as well as the recently discovered αD binding pocket, proved to be exceptionally high in affinity and outstandingly selective. The aspect of selectivity has always been a particular challenge for kinase inhibitors due to the high degree of conservation of the cosubstrate binding region among eukaryotic protein kinases. The inclusion of the αD binding pocket is currently one of the most promising approaches to overcome this challenge since the high plasticity in this region has exclusively been described for CK2. In this thesis, it was shown for the first time that this is not only true for CK2α, but rather for both paralogs. Finally, the crystallization successes with CK2α’ and an eight-month desalting procedure led to the discovery of a novel binding site, located close to the N-terminus. However, the suitability of this cryptic site for the design of future generations of CK2 inhibitors requires further studies