1998 — 2002 |
Gatti, Domenico L |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Biosynthesis of the Escherichia Coli Lipopolysaccharide
DESCRIPTION: The lipopolysaccharide (LPS), also known as endotoxin, is a unique constituent of the outer cell membrane of Gram negative bacteria and is responsible for the pathophysiological phenomena of the shock syndrome associated with Gram negative sepsis. Since LPS is essential for bacterial growth, the enzymes that catalyze its synthesis are important drug targets for antimicrobial chemotherapy. One of the principal components of LPS is 3-deoxy-D-manno-octulosonate (KD0). This 8-carbon sugar is first synthesized as a phosphorylated precursor (KDO8P) by a specific bacterial synthase. The reaction, with involves the condensation of phosphoenolpyruvate (PEP) with arabinose 5-(A5P) to yield KDO8P and Pi, is poorly understood at present. This fact has limited the suitability of KDO8P synthase as therapeutic target. The current project will employ structural analysis of KDO8P synthase from Escherichia coli to determine the details of the enzyme catalytic mechanism. The three-dimensional structure of the wild type enzyme will be solved first in the absence of bound ligands. Subsequent structure determinations will be done in the presence of the enzyme substrates (PEP+ a5P), its products (KDO8P, Pi) and analogs of these ligands, and will provide initial information on the amino acids in the active site of KDO8P synthase that are important for catalysis. The catalytic role of chemical groups of KDO8P synthase will be examined further in structures obtained at different pH values, and by employing mutant forms of the enzyme. Notably, E. coli KDO8P synthase exhibits sequence similarity to two other enzymes that recognize a pyruvyl moiety, the bifunctional 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase/chorismate mutase from Bacillus subtilis and the chorismate mutase from Staphylococcus xylosus, for which no structural information is available. Thus, studies of KDO8P synthase will provide a mechanism model for a new class of enzymes that catalyze the transfer of three- carbon units.
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1998 — 2001 |
Gatti, Domenico L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure/Function of An Ion Motive Atpase
DESCRIPTION: (Adapted from applicant's Abstract) The arsenical resistance (ars operon of the Escherichia coli plasmid R773 encodes a system for the active extrusion from cells of the toxic oxyanions arsenite (As(III)O21-) and antimonite (Sb(III)O21-) via an ATP-driven pump. The arsA and the arsB genes o the operon encode, respectively, the catalytic subunit (ATPase) and the membrane subunit of the pump. The arsC gene codes for a reductase that convert arsenate (As(V)O43) to arsenite and appears to channel it into the ArsA-ArsB pump, thus extending bacterial resistance also to the pentavalent state of arsenic. Crystals diffracting at high resolution (2.0 Angstrom) were obtained for both the catalytic subunit of the pump (ArsA) and for the reductase (ArsC) and native data sets have been collected. Structural studies are proposed to identify ArsA and ArsC binding sites for substrates and/or for allosteric effectors, and the regions of interaction with other proteins (e.g., ArsA with ArsB, ArsC and ArsA). Since expression of the arsB gene is highly toxic in E. coli, production of the protein in other hosts (Archaebacteria, yeast) will be pursued to obtain a large amount of pure protein for crystallization. Arsenical resistance is a useful model for the study of multiple drug resistance in both eukaryotic and prokaryotic cells. The ArsA-ArsB pump exhibits structural and functional similarity to the P-glycoprotein: both are efflux pumps for toxic compounds, have two nucleotide binding sites, are substrate-dependent ATPases, have 12 membrane spanning alpha-helices, and are each able to detoxify structurally distinct drugs. The latter point is illustrated for the ArsA-ArsB pump by the fact that while arsenate and arsenit are both oxyanions of arsenic, they are chemically dissimilar. The ArsA protein is also the only other ion-motive ATPase, besides the mitochondrial F1 ATPase, for which three-dimensional crystals have been obtained. Analysis of the similarities and differences between these two enzymes will further our understanding of how ions are transported across biological membranes.
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2004 — 2007 |
Gatti, Domenico L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure, Mechanism and Evolution of Kdo8p Synthase
DESCRIPTION (provided by applicant): The lipopolysaccharide (LPS) of Gram negative bacteria, also known as endotoxin, is responsible for the pathophysiological phenomena of the shock syndrome associated with Gram negative sepsis. 3-Deoxy-Dmanno-octulosonate (KDO) is an essential constituent of the LPS of all Gram negative bacteria. This eight carbon sugar is the first component of the oligosaccharide core region that links the lipid A moiety of LPS to the O-antigen. The combination of lipid A and two KDO units is the LPS component where the endotoxin activity is located. The first step in the synthesis of KDO is the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO 8-phosphate (KDO8P), the phosphorylated precursor of KDO, catalyzed by KDO8P synthase (KDO8PS). Since LPS is essential for bacterial growth, this enzyme is a potential target for new antimicrobial drugs. There are two classes of highly homologous KDO8PS's differing primarily in the requirement, or lack thereof, of a metal ion for activity. We have determined the structure of one member of the metal-free class, the Escherichia coil enzyme, and of one member of the metal-requiring class, the enzyme from the hyperthermophile Aquifex aeolicus. These studies have revealed that KDO8PS is a homotetramer with two active sites located on each face of the enzyme. There is alternation of catalysis between the two faces of the enzyme, such that when PEP and A5P bind and react in the active sites located on one face, only PEP binds at the active sites located on the other face. Despite the wealth of information derived from the initial structural and biochemical studies, the mechanism of the reaction catalyzed by KDO8PS is not clear yet. The current project will employ methods of kinetic analysis, spectroscopy, rational mutagenesis, directed evolution and X-ray diffraction to investigate (a) the function of individual residues and active site water in catalysis, and the mechanistic differences between metallo and non-metallo synthases, (b) the molecular mechanism of alternating face catalysis.
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