No description available.

In plants, AAA-adenosine triphosphatase (ATPase) Cell Division Control Protein 48 (CDC48) uses the force generated through ATP hydrolysis to pull, extract, and unfold ubiquitylated or sumoylated proteins from the membrane, chromatin, or protein complexes. The resulting changes in protein or RNA content are an important means for plants to control protein homeostasis and thereby adapt to shifting environmental conditions. The activity and targeting of CDC48 are controlled by adaptor proteins, of which the plant ubiquitin regulatory X (UBX) domain-containing (PUX) proteins constitute the largest and most versatile family. However, few PUX proteins have been structurally or functionally characterized and how they participate in the substrate processing of CDC48A is not fully understood. Here, we first performed a comparative bioinformatic analysis, in which we found that the PUX proteins can be functionally divided into six types. We used this classification as a guide for our experimental efforts to elucidate how PUX proteins mediate client recognition and delivery for CDC48A-mediated unfolding. As a first step in this experimental analysis, we cloned and expressed a number of PUX protein constructs, we assessed their interaction features, and obtained crystals for several PUX domains. These bioinformatic and experimental results provide a basis for the in-depth structural and functional analysis of how PUX proteins control the CDC48A segregase.

No description available.

The immunologic relatedness of various cofactor-binding sites of enzymes requiring different nucleotide cofactors was examined. Chicken antibodies specific for NADPH- or CoA-binding domains were raised using an NADPH- or CoA-requiring enzyme as an immunogen. Antibodies specific for either NADPH- or CoA-binding domains were isolated by immunoaffinity chromatography of the respective antisera using unrelated NADPH- or CoA-requiring enzymes as affinity ligands. The reactivities of the NADPH- and CoA-binding-site-specific antibodies with a variety of enzymes that required different cofactors was shown on Western blots of SDS-PAGE of the enzymes. Variable cross-reactivities were observed among all nucleotide-cofactor requiring enzymes with each specific cofactor-domain-antibody population. Numerous proteins not physiologically associated with nucleotide cofactors, including acyl carrier protein, were completely unreactive. Proteins that bound phosphoryl compounds either as substrates or cofactors showed varying degrees of reactivity with each population of specific antibodies. These included aldolase, ribulose-1,5-bisphosphate carboxylase/oxygenase, ribonuclease A, carbonic anhydrase and triosephosphate isomerase. The immunologic cross-reactivity suggested that these proteins share a common structural feature, probably a primary structure epitope, since the proteins had been subjected to denaturing polyacrylamide gel electrophoresis. A candidate for this common structural feature is a glycine-rich sequence comprising a phosphate binding loop.

The iron-molybdenum cofactor (FeMo-cofactor) of nitrogenase is the subject of one the most intensive biochemical/genetic detective cases of modern science. At the active site of nitrogenase, the FeMo-cofactor not only represents the heart of biological nitrogen fixation, but its synthesis also serves as a model for complex metallocluster biosynthesis. Research in the Dean Lab is focused on furthering the understanding of Fe-S cluster biosynthesis in the nitrogenase enzyme system. Throughout the years, scientists from a broad range of disciplines have focused their intellectual might on deciphering not only the chemistry of the FeMo-cofactor, but also the biosynthesis of this unique metallocluster. Recent advances in the study of FeMo-cofactor biosynthesis have produced considerable insight regarding the complex series of biological reactions necessary for the synthesis of this metallocluster. The work contained within this dissertation represents my efforts to further the understanding of FeMo-cofactor biosynthesis. The concept of a molecular scaffold in FeMo-cofactor biosynthesis is generally accepted in the field of nitrogenase. Previous work has implicated the products of nifE and nifN as providing the assembly site for FeMo-cofactor synthesis. Researchers were able to purify this molecular scaffold, commonly referred to as the NifEN complex, however, detailed characterization was precluded by the inability to obtain sufficient quantities of NifEN. In an effort to fully characterize the NifEN complex, we initiated a gene fusion approach for the high level production NifEN. In addition to gene fusion, a poly-histidine tag was incorporated into NifEN, allowing purification through the application of immobilized metal-affinity chromatography (IMAC). NifEN obtained in this way was characterized using a variety of biophysical techniques and found to contain two [4Fe-4S] clusters in each NifEN tetramer. These clusters were also shown to be completely ligated by cysteine residues. With the information obtained from this study, it is concluded that the [4Fe-4S] clusters of the NifEN complex are likely to play either a structural or a redox role rather than being transferred and becoming incorporated into the FeMo-cofactor. In addition to the biophysical characterization of the NifEN complex, a separate study was started to characterize the apo-MoFe protein. In this study we used IMAC to purify a poly-histidine-tagged apo-MoFe protein produced by a nifB-deletion mutant of A. vinelandii. Using the poly-histidine fusion approach, apo-MoFe protein was obtained in sufficient quantities for detailed catalytic, kinetic and spectroscopic analyses. This multidisciplinary approach confirmed that apo-MoFe protein contained intact P clusters and P cluster environments, as well as the ability to interact with the Fe protein. It was also shown for the first time that this tetrameric form of purified apo-MoFe protein could be activated by the addition of preformed FeMo-cofactor. The NifEN complex was further characterized to investigate the presence of bound FeMo-cofactor intermediates. NifEN purified by IMAC is produced in the absence of the nitrogenase structural genes (nifHDK). In this genetic background, it is believed that the FeMo-cofactor biosynthetic machinery will become obstructed with unprocessed FeMo-cofactor intermediates, such as the Fe-S precursors of FeMo-cofactor, NifB-cofactor. Previous work indicated that NifEN can exist in either a charged or discharged form, based on the presence or absence of the FeMo-cofactor precursor, NifB-cofactor. EPR and VTMCD spectroscopies showed the presence of a new paramagnetic signal associated with NifEN that is believed to be in the charged or precursor bound state. This represents the first spectroscopic evidence for a precursor to the FeMo-cofactor. Furthermore, an interaction of NifEN and NifX was examined by size exclusion chromatography. From this study, NifX exhibited the capacity to bind a chromophore, presumably an FeMo-cofactor precursor, from the NifEN complex. NifX was also capable of binding to isolated FeMo-cofactor and the FeMo-cofactor precursor, NifB-cofactor. Finally, preliminary investigations involving interaction between the Fe protein and NifEN were initiated. Recent findings indicate that NifEN and the Fe protein have the capacity to interact specifically with one another. The interaction of NifEN and Fe protein appears to be dependent on the association of FeMo-cofactor precursor with NifEN. The NifEN complex also has the capacity to accept electrons from the Fe protein in a MgATP dependent manner. The ability of NifEN to accept electrons from the Fe protein may be involved in the role of Fe protein in FeMo-cofactor biosynthesis. / Ph. D.

Cysteine dioxygenase (CDO) is a non-heme iron enzyme that can be found in mammalian tissue. It is mainly localized in the liver but is also present in the brain, kidney, and adipose tissue. CDO converts cysteine to cysteine sulfinic acid, which is the first step in cysteine metabolism in the human body. CDO contains a novel cofactor located near the metal binding site that is present in another enzyme, galactose oxidase, where it is essential for redox function. This suggests that the linkage may play an important role in CDO as well. The cofactor consists of Y157 and C93. Mutation of the C93S causes a drop in activity to 57.1% and a mutation of the Y157F causes a drop to 8.1%. The metal center was studied using XAS revealing that the addition of cysteamine, an activator of CDO, changes the conformation of the binding site significantly. CDO differs from the rest of the cupin super family in that it does not contain a 2-his-1-carboxylate binding motif but rather the carboxylate is replaced with another histidine. A mutation of one of the binding residues, H140D, caused the enzyme to be non-active. Also the mechanism of the CDO was studied by conducting activity assays with various inhibitors and activators that yielded contradicting results with previously published work.

Nitrogenase catalyzes the biological reduction of N2 to ammonia (nitrogen fixation). The metalloclusters associated with the nitrogenase components include the [4Fe-4S] cluster of the Fe protein, and the P-cluster [8Fe7S] and FeMo-cofactor [7Fe-9S-Mo-X-homocitrate], both contained within the MoFe protein. These metal-complexes play a vital role in enzyme activity during electron transport and substrate reduction. It is known that the FeMo-cofactor provides the site of substrate reduction, but the exact site of substrate binding remains a topic of intense debate. Some models for the substrate binding location favor the molybdenum atom, while other models favor one or more iron atoms within FeMo-cofactor. We have shown that the a-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site: a-70 approaches one 4Fe-4S face of the FeMo-cofactor. Substitutions at this position alter enzyme specificity for reduction of alternative alkyne substrates. These altered MoFe proteins and alternative alkyne substrates, such as propargyl alcohol, were used to trap an intermediate during substrate reduction. Further studies involving the effect of pH on substrate reduction of these altered MoFe proteins pinpointed the location of the bound substrate-derived intermediate on the FeMo-cofactor to a specific Fe atom, designated Fe6. In addition to understanding how substrates are bound and reduced at the active site, understanding how these clusters are biologically assembled is a second point of interest. Inactivation of NifU or NifS has been shown to affect the activity of both nitrogenase components. NifS is a cysteine desulfurase that provides the sulfur for cluster formation and NifU serves as a molecular scaffold during [Fe-S] cluster assembly. Genetic and biochemical experiments involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific [Fe-S] cluster formation. Furthermore, the NifU and NifS protein appear to have specialized functions in the maturation of metalloclusters of nitrogenase and cannot functionally replace the isc [Fe-S] cluster system used for the maturation of other [Fe-S] proteins. These results indicate that, in certain cases, [Fe-S] cluster biosynthetic machineries have evolved to perform only specialized functions. / Ph. D.

<p>The molybdenum cofactor (Moco) is an essential enzyme cofactor found in all kingdoms of life. Moco plays central roles in many vital biological processes, and must be biosynthesized de novo. During its biosynthesis, the characteristic pyranopterin ring of Moco is constructed by a complex rearrangement of guanosine 5'-­triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC. However, the mechanisms and the functions of the two enzymes are under significant debate. To elucidate their physiological roles, I took a multidisciplinary approach to functionally characterize MoaA and MoaC in vivo and in vitro. In this dissertation, I report the first isolation and characterization of the physiological MoaC substrate, 3',8-­ cyclo-­7,8-­dihydro-­guanosine 5'-triphosphate (3',8-cH2GTP). I also report the first X-­ray crystal structures of MoaC in complex with this highly air sensitive substrate, and its product cPMP. These studies, combined with in vitro experiments using substrate analogs, catalytically impaired mutants, and synthetic peptides, have enabled me to delineate the functions of the Moco biosynthetic enzymes, MoaA and MoaC, and proposed mechanistic models for their roles in the formation of cPMP.</p> / Dissertation

Development of enzymatic labelling methods has been driven by the importance of studying molecular structures and interactions to comprehend cellular processes. Methyltransferases (MTases), which regulate genetic expression by transferring a methyl group from the cofactor S-adenosyl-L-methionine (SAM) to DNA, histones and various proteins, have been shown to accept SAM analogues with an alternative alkyl group on the sulfonium centre. These alkyl groups can be transferred to the substrate, and with a further reaction can be selectively functionalized. Thus, MTases together with SAM analogues have emerged as novel labelling tools. The project aims to use MTases to obtain an orthogonal system that can selectively use a SAM cofactor analogue to transfer functional chains to proteins with a specific motif. To achieve selectivity of the system, the SAM analogue cofactor was modified on the ribose ring; to obtain a new transferase activity of the system, the transferable methyl on the sulfonium centre was changed to a different substituent. SAM analogues were produced enzymatically with hMAT2A by using 3'-deoxy-ATP and methionine or ethionine. Mutants of SET8 and novel substrates were designed to have modifications at residues in the active site, within the vicinity of the ribose ring of SAM, and were assessed for selective activity with the new analogue cofactor. The results showed that the new cofactor 3'-deoxy-S-adenosyl-L-methionine (3'dSAM) was efficient in the mono-methylation of the substrate peptide RFRKVL, and that the mutant SET8 C270V exhibited over 13 fold MTase activity in presence of 3'dSAM and the RFRKVL substrate, in comparison with the activity with the WT sequence RHRKVL and the SAM cofactor. In addition, glutathione S-transferase (GST) was used as a model protein to express the motif RFRKVL, to transform it into a potential substrate for SET8. Assessment of the MTase activity of SET8, 3'dSAM and the novel GST substrate indicated mono-methylation of the substrate. Moreover, the motif showed no interference with GST native activity. Based on the observations, a new enzymatic system shows higher selectivity with a new analogue cofactor over SAM to effectively methylate proteins expressing the consensus RFRKVL. Work on substrates, enzymes and cofactors should continue to obtain a functional-chain transferase activity of the enzymatic system.

Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS. Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS.