No description available.

sn-Glycerol-3-phosphate acyltransferase [G3PAT, PlsB (E.coli), EC 2.3.1.15] is an enzyme involved in glycerolipid biosynthesis, catalysing the acylation of glycerol-3-phosphate (G3P) to produce lysophosphatidic acid (LPA). Chilling tolerance in plants is linked to the acyl-group composition of membranes, which is linked to acyltransferases with a higher selectivity for unsaturated acyl-substrates. Plant soluble G3PAT is located in the chloroplast and uses acyl-acyl carrier protein (acyl-ACP) as substrate. Soluble G3PAT exhibits strong substrate selectivity for acyl-ACP, the plastidial substrate in vivo, over acyl-CoA. cDNAs encoding soluble G3PATs have previously been cloned from several plant species and both oleate-selective and non-selective forms identified. The purpose of this thesis is to study the mechanism of plastidial G3PAT and attempt to identify factors important in determining substrate selectivity. An in vitro assay has been optimised to distinguish selective and non-selective enzyme forms under physiologically relevant conditions. The assay has been adapted to determine enzyme activity with a range of acyl-ACP and acyl-CoA substrates and to measure the kinetic constants Km and Vmax. Kinetic measurements have been made on a G3PAT protein from the chilling sensitive plant squash (Cucurbita moschata) and the L261F mutant protein containing a single amino acid substitution that significantly alters substrate selectivity. The mutation was found to increase selectivity by raising Km for unsaturated acyl-substrate. Mutant squash G3PAT proteins have been investigated to determine the importance of particular regions or amino acid residues. The mutations E142A, K193S, R235S and R237S resulted in enzymes that were completely inactive. The mutations H194S and L261F altered catalytic or substrate binding characteristics without enzyme inactivation. The catalytic mechanism and order of substrate binding for squash G3PAT have been determined, the reaction was found to proceed via a compulsory-ordered ternary complex with acyl-ACP binding before glycerol-3-phosphate.

Antibiotic resistance represents a real threat in the modern world. The problem of resistance is brought about by the fast evolution of bacteria, accelerated by misuse and over-prescription of antibiotics and compounded by the decline in the discovery and development of new classes of antibiotics. Consequently, new targets for antibiotics are in high demand. Flavin-dependent thymidylate synthase (FDTS), which is not present in humans and is responsible for the biosynthesis of a DNA building block in several human pathogens (e.g., M. tuberculosis, B. anthracis, H. pylori), is one such novel target. FDTS catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to produce 2'-deoxythymidine-5'-monophosphate (dTMP), with N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (CH₂H₄fol) serving as the carbon source and a nicotinamide cofactor as the electron source. No efficient inhibitors of FDTS are known, despite high-throughput screening attempts to find them. Intermediate and transition-state mimics are likely to bind the enzyme with greater affinity and hence have a better chance at inhibiting FDTS. Therefore, the understanding of the chemical mechanism of FDTS is critical to the informed design of compounds capable of disrupting its function in bacteria. We utilized various techniques, including chemical trapping of reaction intermediates, substrate isotope exchange and stopped-flow, to investigate the FDTS mechanism and determine what sets it apart from other pyrimidine methylases. We found that at least two different intermediates kinetically accumulate in the FDTS-catalyzed reaction. Both of these intermediates are trapped in acid in the form of 5-hydroxymethyl-dUMP, which has never been isolated in other uracil-methylating enzymes. Under basic conditions, however, the earlier intermediate is converted to a species with an unusual flavin-derived adduct, while the later intermediate is converted to dTMP product. Our experiments also suggest that dUMP is activated for the reaction by the reduced flavin - a substrate activation mechanism distinct from the one employed by the classical pyrimidine-methylating enzymes.

Ribonucleases play essential roles in RNA metabolism. In Escherichia coli, the extensive degradation of RNAs that are defective or no longer required by the cell is carried out by one of three processive, 3' to 5' exoribonucleases. Relatively unstructured mRNAs are typically degraded by RNase II or PNPase. In contrast, mRNAs containing extensive secondary structure, and the highly structured rRNA and tRNA molecules, are degraded by PNPase and/or RNase R. However, RNase R differs from other exoribonucleases in that it is able to degrade through these structured RNAs without the aid of a helicase activity. Consequently, its mechanism of action is of great interest. In this dissertation, using a variety of specifically designed substrates, I show that a single-stranded overhang, which must be at least 5 nucleotides in length, is required for tight binding and subsequent degradation of double-stranded RNA by RNase R. Moreover, this overhang must be 3' to the duplex indicating that an RNA substrate must thread into the enzyme with 3' to 5' polarity. Using a series of truncated RNase R proteins, I show that the cold-shock domains and the S1 domain contribute to substrate binding. The cold-shock domains appear to play a role in substrate recruitment, while the S1 domain is required to position substrates for efficient catalysis. Furthermore, the nuclease domain alone is sufficient to bind and degrade structured RNAs. This is a unique property of the nuclease domain of RNase R since this domain in RNase II, a paralogue of RNase R, stalls as it approaches a duplex. RNase R binds RNA more tightly within its nuclease domain than RNase II. Through mutagenesis studies, I identify one amino acid, R572, within the nuclease domain of RNase R that contributes to this tight binding and the ability to degrade double-stranded RNA. Furthermore, I found that degradation of structured RNA is strongly dependent on temperature. Based on these data I propose that tight binding allows RNase R to capitalize on the natural thermal breathing of an RNA duplex to degrade structured RNA.

Methionine is involved in many cellular processes, several of which produce a feedback inhibitor. 5-methylthioribose (MTR) kinase, one protein involved in the removal of this inhibitor, has a protein kinase fold with conserved kinase motifs and several unique MTR binding motifs. Site-directed mutagenesis and characterization of the Bacillus subtilis enzyme was performed to probe the role of one motif. Active site D233 mutants show an activity profile similar to other protein kinase-like enzymes, suggesting a common mechanism that does not require a catalytic acid. An ordered sequential binding mechanism, with nucleotide binding first, was seen in wild type MTR kinase. Binding studies of the mutant proteins suggest that hydrogen bonding is important for MTR binding. The structures of the mutant proteins also show more differences in MTR binding than nucleotide binding. Overall, D233 is important for increasing the nucleophilicity of MTR, and ensuring its correct position in the active site.

Methionine is involved in many cellular processes, several of which produce a feedback inhibitor. 5-methylthioribose (MTR) kinase, one protein involved in the removal of this inhibitor, has a protein kinase fold with conserved kinase motifs and several unique MTR binding motifs. Site-directed mutagenesis and characterization of the Bacillus subtilis enzyme was performed to probe the role of one motif. Active site D233 mutants show an activity profile similar to other protein kinase-like enzymes, suggesting a common mechanism that does not require a catalytic acid. An ordered sequential binding mechanism, with nucleotide binding first, was seen in wild type MTR kinase. Binding studies of the mutant proteins suggest that hydrogen bonding is important for MTR binding. The structures of the mutant proteins also show more differences in MTR binding than nucleotide binding. Overall, D233 is important for increasing the nucleophilicity of MTR, and ensuring its correct position in the active site.

No description available.

No description available.

Galactofuranose (Galf) is the five membered ring form of galactose. It is found on the cell wall and surface of many pathogens including Mycobacterium tuberculosis, Aspergillus fumigatus, Leishmania major, and Trypanosoma cruzi. Galf has been implicated in pathogenesis in these organisms; thus the biosynthetic pathway of Galf is a target for drug design. Galf is synthesized by the enzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). Solving the mechanism and structure of UGMs will aid in the development of specific inhibitors against these enzymes. Herein we present the detailed functional analysis of UGMs from A. fumigatus, T. cruzi, and L. major. The mechamism and structure these eukaryotic UGMs were examined by steady-state kinetics, rapid-reaction kinetics, trapping of reaction intermediates, fluorescence anisotropy, and X-ray crystallography. The mechanism first involves reduction of the required flavin by NADPH, followed by UDP-Galp binding and subsequent SN2 attack by the flavin on galactose displacing UDP to form a flavin N5-C1 galactose adduct. Next, the galactose ring opens forming an iminium ion allowing isomerization to occur. Lastly, the product is released and UGM is available to bind another substrate or be reoxidized by molecular oxygen. The three-dimensional structure of A. fumigatus UGM was solved using X-ray crystallography in four conformations: oxidized in complex with sulfate ions, reduced, reduced in complex with UDP, and reduced in complex with UDP-Galp, giving valuable information on the unique features of eukaryotic UGMs including features important for oligomerization and for substrate binding. The novel mechanism and structure provide valuable information for the development of specific inhibitors of eukaryotic UGMs. / Ph. D.

The relationship between protein structure, motions, and catalytic activity is an evolving perspective in enzymology. An interactive approach, where experimental and theoretical studies examine the same catalytic mechanism, is instrumental in addressing this issue. We combine various techniques, including steady state and pre-steady state kinetics, temperature dependence of kinetic isotope effects (KIEs), site-directed mutagenesis, X-ray crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations, to study the catalytic mechanisms of thymidylate synthase (TSase). Since TSase catalyzes the last step of the sole intracellular de novo synthesis of thymidylate (i.e. the DNA base T), it is a common target for antibiotic and anticancer drugs. The proposed catalytic mechanism for TSase comprises a series of bond cleavages and formations including activation of two C-H bonds: a rate-limiting C-H→C hydride transfer and a faster C-H→O proton transfer. This provides an excellent model system to examine the structural and dynamic effects of the enzyme on different C-H cleavage steps in the same catalyzed reaction. Our experiments found that the KIE on the hydride transfer is temperature independent while the KIE on the proton transfer is temperature dependent, implying the protein environment is better organized for H-tunneling in the former. Our QM/MM calculations revealed that the hydride transfer has a transition state (TS) that is invariable with temperature while the proton transfer has multiple subsets of TS structures, which corroborates with our experimental results. The calculations also suggest that collective protein motions rearrange the network of H-bonds to accompany structural changes in the ligands during and between chemical transformations. These computational results not only illustrate functionalities of specific protein residues that reconcile many previous experimental observations, but also provide guidance for future experiments to verify the proposed mechanisms. In addition, we conducted experiments to examine the importance of long-range interactions in TSase-catalyzed reaction, using both kinetic and structural analysis. Those experiments found that a remote mutation affects the hydride transfer by disrupting concerted protein motions, and Mg2+ binds to the surface of TSase and affects the hydride transfer at the interior active site. Both our experiments and computations have exposed interesting features of ecTSase that can potentially provide new targets for antibiotic drugs targeting DNA biosynthesis. The relationship between protein structure, motions, and catalytic activity learned from this project may have general implications to the question of how enzymes work.