Research Focus, David L. Jakeman, Dalhousie University

The Jakeman Research Group

Research Involving Aspects of Medicinal Chemistry, Structural Biology, Carbohydrate Enzymology and Natural Products discovery

Glycosyltransferases

Carbohydrates are transferred by glycosyltransferases onto a wide variety of biomolecules and secondary metabolites to give bioactive molecules. One class of glycosyltransferases we are investigating includes those enzymes involved in attaching carbohydrates onto macrolides, phenols and other carbohydrates to generate many of the antibiotics and anticancer agents in clinical use. We are chemically modifying enzyme substrates and bioengineering enzyme substrate pathways to prepare novel carbohydrate-containing molecules using glycosyltransferases. Protein engineering of glycosyltransferases is an important tool to provide access to novel hybrid enzymes with altered substrate specificity and chemical synthesis is of paramount importance to prepare novel sugar nucleotide diphosphate enzyme substrates. The research is funded through operating grants from the Nova Scotia Health Research Foundation and the Canadian Institutes of Health Research.

Antibiotic activities (with non-pathogenic strains) of the prepared carbohydrate-containing biomolecules are measured in-house and anticancer activities are measured in collaboration with Johnathan Blay (Pharmacology, Dalhousie University).

We have cloned, expressed and purified JadS, the putative family 1 glycosyltransferase responsible for this transformation and are currently analyzing its substrate specificity in terms of sugar nucleoside diphosphate and aglycone. We are also investigating JadS substrate specificity using blocked mutants in the dedeoxysugar biosynthetic pathway. Future studies will involve evolving jadS using directed evolution to accept a wider range of substrates and to generate different types of glycosidic linkages. Significant effort is required to synthesize the novel monosaccharides used in this study since they are derived from L-rhamnose. We have developed stereoselective coupling procedures for the formation of the b-phosphate linkage and have efficient purification procedures for these compounds using reversed-phase HPFC and ion-exchange chromatography.

Nucleotidylyltransferases

Nucleotidylyltransferases condense sugar-1-phosphates with nucleotide triphosphates releasing sugar nucleotide diphosphates. Our approaches to engineering nucleotidylyltransferases, and investigations into the mechanism of these catalysts will offer access to novel sugar nucleotide diphosphates to be used for the development of novel carbohydrate-containing molecules.

We have cloned, expressed and purified three nucleotidylyltransferases, from Streptococcus mutans (RmlA), Streptococcus pneumoniae R6 (Cps2L) and from Streptomyces venezuelae ISP5230 (JadQ). RmlA and Cps2L will be evolved to generate a novel nucleotidylyltransferase that will be screened for altered sugar specificity.

Medicinal Chemistry

The preparation of novel antibiotics is of the utmost importance for the development of drugs to combat emerging drug-resistant bacteria. We have a medicinal chemistry project funded through the Canadian Bacterial Diseases Network for the synthesis and evaluation of mechanism-based inhibitors of nucleotidylyltransferases in collaboration with Dr. Joseph Lam, CRC Tier 1 chair, University of Guelph, ON. These novel compounds mimic the transition state of the reaction catalyzed by nucleotidylyltransferases, by altering the charge state on the diphosphate. Increased negative charge on the bridging diphosphate is introduced by either inductively withdrawing substituents, or functional groups with formal negative charge.

Natural products

The Atlantic Chapter of the Canadian Breast Cancer Foundation has funded a natural products isolation project aimed at developing novel breast cancer analogues based on heat-shock derived secondary metabolites produced by Streptomyces venezualae. This project involves growing up Streptomyces venezualae ISP5230 on defined media and isolating the natural products.

The blue portion of jadomycin B is derived from L-Ile, an amino acid present in the culture media. We have improved the culture conditions such that we have been able to isolate novel jadomycins when standard, non-standard and synthetic amino acids are used in the cultures. The size of the oxazolone ring has also been changed by using b-amino acids. Electrospray ionization mass spectrometry has been particularly useful in demonstrating the existence of these compounds due to the breakdown observed in MS/MS mode, as shown below.

Glycosynthases

Glycosynthases are mutant glycosidases developed specifically for the synthesis of oligosaccharides. These mutant enzymes have significant potential to provide cheap and ready access to a wide variety of oligosaccharides. Our research aims to develop new glycosynthases with novel regio- and stereoselectivities for a wide variety of carbohydrates. The precise control over the size of biopolymers and relatively cheap starting materials ensure glycosynthases are an excellent technology for developing biocompatible materials. The research project in funded by the Mizutani Glycoscience Foundation of Japan.

The aims of our glycosynthase project are to evolve donor sugar specificity (the glycosyl fluoride). To achieve this we are developing a novel selection method (as opposed to a screening one) to evaluate libraries of mutant glycosynthases generated by error-prone PCR and DNA shuffling. Family 3 glycosidases are enzymes expressed by antibiotic producing microorganisms to activate macrolide antibiotics when excreted. We are generating glycosynthases of these enzymes and characterizing them in vitro. Subsequently libraries of mutant glycosynthases will be assessed using our selection methodology to ensure cell survival.

Structural studies of bioactive membrane-associated peptides and proteins

We have developed collaborations with Dr. Roy Duncan (Department of Microbiology), Dr. Hung-Yua Li (Department of Oral Sciences) and Dr. Sue Douglas (NRC, Halifax) to study their peptides and proteins by NMR spectroscopy.

The fusion-associated small transmembrane (FAST) proteins studied in Dr. Duncan’s laboratory are novel fusion proteins that act through an unknown fusion mechanism. Our structural studies on these proteins provides mechanistic insight into how they function.

The competence stimulating peptides studied in Dr. Li’s laboratory are responsible for quorum sensing in Streptococcus mutans, the microorganism responsible for forming a biofilm, and subsequently dental plaque on your teeth. We have determined the first structures of these peptides and provided insight into the areas of the peptide responsible for initiating quorum sensing.

Pleurocidin is a antimicrobial peptide initially discovered by Dr. Douglas and is a primary defense mechanism against bacterial infection of the winter flounder. We have determined its structure in micelles and are currently studying how it interacts with living cells using NMR spectroscopy.

Training opportunities

Organic synthesis and molecular biology are two strengths of the research group. Students will also have significant exposure to enzymology, protein chemistry and structural characterization of macromolecules by NMR spectroscopy, X-ray crystallography and mass spectrometry.

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