Progress 10/01/06 to 09/30/10
Outputs
OUTPUTS: We have made substantial progress this year on the structural studies of enzymes involved in the biosynthesis of D-forosamine. We have recently solved the structure of the dehydrogenase that reduces the keto moiety at C-3 to a hydroxyl group. We have also crystallized the 2,3 dehydratase that catalyzes the first step in the pathway. Work continues on these enzymes. PARTICIPANTS: Rachel Kubiak, graduate student - has learned about all aspects of structural analysis. Nate Bruender, graduate student - has learned about all aspects of structural analysis. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our investigations have and will continue to reveal unprecedented chemistries and provide important and fundamental contributions to mechanistic enzymology.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The biochemical importance of carbohydrates cannot be overstated for they are essential elements in nearly every physiological process and represent the most abundant biomolecules in living systems. Apart from their role in providing metabolic energy, carbohydrates are involved in a wide range of biological processes including the immune response, cell-cell interactions, fertilization, cell adhesion, and drug efficacy, among others. The focus of our research is on the unusual di- and trideoxyhexoses, which are found attached to important antibiotics such erythromycin A, antitumor agents such adriamycin, or insecticides such as spinosad. The laboratory is also interested in the unusual sugars found in the O-antigens of Gram-negative bacteria. O-antigens differ among bacteria with respect to sugar content and linkages, are highly immunogenic, and serve as important virulence factors. Specifically, our work is aimed at understanding the structure and function of the enzymes involved in the biosynthesis of forosamine. General techniques utilized in the laboratory include x-ray crystallography, site-directed mutagenesis, enzymatic synthesis of appropriate nucleotide-linked sugar ligands, and kinetic analyses. Our investigations have revealed unprecedented chemistries and have provided important and fundamental contributions to mechanistic enzymology. This year, we have made considerable progress in purifying the proteins involved in the biosynthesis of forosamine. X-ray crystallographic analyses are underway. PARTICIPANTS: Rachel Kubiak - research associate TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This research focuses on enzymes involved in the biosynthesis of unusual 2,3(4),6-trideoxyhexoses. In the long term, by understanding the structures and functions of these enzymes, it will eventually be possible to design modified proteins with altered substrate specificities thereby yielding new "designer" nucleotide-linked sugars not normally encountered in Nature. These may have important ramifications for the development of new therapeutics.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Thus far we have cloned, over-expressed, and partially purified all of the enzymes involved in forosamine biosynthesis. To ensure adequate soluble protein, some of the genes have been cloned from Streptomyces ambofaciens, some from Streptomyces neyagawaensis, and some from Saccharopolyspora spinosa. Crystallization trials for some of these proteins have been initiated, and small crystals of SpnS have been obtained. In addition, a preliminary structure of SpnR has been determined. SpnR belongs to the aspartate aminotransferase family. Enzymes in this group are typically homodimers with two key features: a PLP cofactor attached to a lysine residue via a Schiff base and an aspartate that promotes protonation of the PLP ring nitrogen to enhance its electron sink properties. These are Lys 215 and Asp 186 in SpnR. Electron density corresponding to the PLP moiety in SpnR demonstrates that the internal aldimine has been trapped in the active site cleft. The overall architecture of the subunit is dominated by a seven-stranded mixed beta-sheet and several rather long alpha-helices. In addition, there are two other layers of beta-sheet, both of which are anti-parallel. With respect to the active site, the pyridoxal ring is anchored to the protein by hydrogen bonds with Asp 186, Tyr 356, and a water molecule, whereas the phosphoryl group lies within hydrogen bonding distance to the side chains of Thr 90, Ser 210, and Asp 212. PARTICIPANTS: James B. Thoden and Paul D. Cook TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This research focuses on enzymes involved in the biosynthesis of unusual 2,3(4),6-trideoxyhexoses. In the long term, by understanding the structures and functions of these enzymes, it will eventually be possible to design modified proteins with altered substrate specificities thereby yielding new "designer" nucleotide-linked sugars not normally encountered in Nature. These may have important ramifications for the development of new therapeutics.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Thus far we have cloned, over-expressed, and partially purified all of the enzymes involved in forosamine biosynthesis Crystallization trials for some of these proteins have been initiated, and small crystals of SpnS have been recently obtained. In addition, a preliminary structure of SpnR has been determined.
PARTICIPANTS: James B. Thoden, senior scientist Paul D. Cook, graduate student
TARGET AUDIENCES: The goal of this work is to provide a structural foundation for the development of new antibiotics. It is simply a matter of time before new bacterial species arise that are resistant to all known antimicrobial reagents. The judicious use of antibiotics will certainly prolong the clinical lifetime of a drug, but in the end the development of new and novel compounds is critically important. The information gleaned from these studies will yield detailed three-dimensional descriptions of protein:ligand interactions in enzymes involved in deoxysugar biosynthesis. Such information is paramount for designing new drugs with differing pharmacological properties.
PROJECT MODIFICATIONS: no major changes
Impacts
The structure of SpnR has been solved. The SpnR dimer has an extensive subunit:subunit interface and the active sites are widely separated. The overall architecture of the individual subunit is dominated by a seven-stranded mixed beta-sheet and several rather long alpah-helices. In addition, there are two other layers of beta-sheet, both of which are anti-parallel. The pyridoxal ring of the cofactor is anchored to the protein by hydrogen bonds with Asp 186, Tyr 356, and a water molecule, whereas the phosphoryl group lies within hydrogen bonding distance to the side chains of Thr 90, Ser 210, and Asp 212. There is an additional water molecule located within hydrogen bonding distance of a phosphoryl oxygen.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
There are five enzymes required for the formation of forosamine. Our goal is to solve the three-dimensionals of each of them. We have recently solved the structure of one of these, namely SpnR. A paper describing this work is in progress.
Impacts
We will better understand structural/functional issues in forosamine biosynthesis.
Publications
- No publications reported this period
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