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New! OPBC-funded Preproposal Abstract

Research Interests

Structural and Mechanistic Investigations of Desaturating Enzymes

Secondary natural products benefit a host organism by providing antifeedent compounds, antimicrobial agents, phytoalexins, coloration, fragrances and hormones. The focus of the Minto research group is to elucidate bioorganic reaction mechanisms, with an emphasis on plant and fungal secondary metabolism. Nature has extended the ubiquitous desaturase enzymes, which typically dehydrogenate saturated portions of fatty acids, to produce frequently bioactive acetylenes. The remodeling of unsaturated lipids is crucial for the maintenance of many membrane functions and the resistance of plants to fungal attack and temperature stress. The central goal of our current research is to generate a detailed model of the chemical and structural factors which control desaturation in Crepis and Petroselinum plant species. We postulate that Basidomycete macrofungi employ acetylenases related to the plant microsomal desaturases to produce a complex array of polyacetylenes. Crepenynic acid is the chemical product of the C. alpina CREP-1 acetylenase, contains an alkynyl group at C-12, and is the first known committed metabolite for fatty-acid-derived acetylenic secondary natural products.

The specific objectives of our current research are:

1. to explore the yeast Pichia pastoris as an expression host for CREP-1,
2. to synthesize and test substrate analogs, competitive inhibitors and affinity labels in the biochemical characterization of CREP-1,
3. to identify unusual lipids produced by Petroselinum species,
4. to isolate acetylenase genes from Basidomycete fungi, and
5. to experimentally determine local secondary structure in D12-desaturases and acetylenases.

The goals of these projects will be attained through a combination of synthetic organic chemistry, NMR spectroscopy, molecular biology, and classical biochemistry. A heterologous yeast expression system will be established to produce quantities of CREP-1 sufficient for in vitro biochemical characterization. Substrate analogs, competitive inhibitors, and affinity labels will be synthesized and tested with microsomal preparations of CREP-1-expressing yeast. NMR spectroscopy will be used to examine the molecular interactions involved in substrate binding and catalysis by tracing the regio- and stereoselective incorporation of stable isotope-labeled metabolic precursors and inhibition by fluorinated linoleates. The microsomal enzyme CREP-1 is predicted to contain four transmembrane (TM)-spanning helices and three histidine boxes, the putative iron-binding sites conserved within the D12-desaturases. Defined substrate binding interactions may act as a "switch" controlling the regio- and chemoselectivity of the CREP-1 acetylenase/desaturase. Structural information, strategic for the alteration of membrane-bound desaturase and acetylenase regioselectivity and chemoselectivity by site-directed mutagenesis, will be obtained through collaborative experiments with Dr. Gary Lorigan. Isotope-labeled CREP-1 peptides incorporated in micelles at high magnetic fields as well as random and oriented lipid bilayers will provide structural information of the acetylenase/desaturase TM regions and residues close to the membrane surfaces as well as side-chain dynamics. Genes encoding acetylenases will be isolated from Basidomycete fungi by expression screening of cDNA libraries. Mechanistic investigations of these plant and fungal biocatalysts may make feasible the redesign of unsaturated lipid biosynthesis and the production of complex fatty-acid derived natural products in industrially important plants and microorganisms.

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