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Natural Product Bio-synthesis


Polyketide Cross-Bridging and Natural Product Biosynthesis

The Diels-Alder reaction is an efficient cyclization reaction between a conjugated diene and a dienophile to form a substituted cyclohexene ring with high regio- and stereoselectivity, and has been employed extensively in organic synthesis. This [4+2]-cycloaddition reaction has also been proposed to be a key transformation in the biosynthesis of many natural products. Several enzymes whose functions may be considered as “Diels-Alderases”, such as lovastatin nonaketide synthase, solanapyrone synthase, and macrophomate synthase, have been identified and studied. However, doubts about whether their mechanisms are true Diels-Alder type reaction remain. In an attempt to find and present more evidence for the existence of “Diels-Alderases” in Nature, we are currently investigating the biosynthesis of two bacterial metabolites, spinosyn A (1) and kijanimicin (2), in which the cross-bridging reaction in each case may be a Diels-Alder reaction.


Diels-Alder reaction mechanism involves concerted cycloaddition of a diene and dienophile via a single transition state to yield a cyclohexene.

Spinosyns are macrolide-type natural products isolated from Saccaropolyspora spinosa. The basic structural components of spinosyns consist of a tetracyclic macrolactone and two deoxy sugars, D-forosamine and methylated L-rhamnose. A mixture of spinosyn A and D, bearing a trade name Tracer, a commercial product of Dow AgroSciences, is used as an insect control agent. Spinosyns have been shown to be benign to non-target organisms. Our work studying the biosynthesis of spinosyn A has led to a complete description of its biosynthetic pathway and the unusual [4+2]-cycloaddition that can proceed both uncatalyzed and catalyzed by the enzyme SpnF. While this reaction is reminiscent of a Diels-Alder reaction to produce the cyclohexene ring of spinosyn A, our mechanistic studies including measurement of kinetic isotope effects combined with prior computational results by other groups instead suggest a stepwise reaction with an atypical and transient intermediate.


Spinosyn A biosynthesis begins with polyketide biosynthesis followed by reactions catalyzed by SpnJ, SpnM and SpnF. Mechanistic studies imply an intermediate in the SpnF catalyzed reaction. The SpnF product is then glycosylated with D-forosamine and methylated L-rhamnose to yield spinosyn A.

Kijanimicin is a spirotetronate class polyketide antibiotic isolated from Actinomadura kijaniata. It is effective against some gram-positives bacteria, anaerobes, and malaria. It also possesses antitumor activity. Kijanimicin is composed of a pentacyclic core containing an unusual spirotetronic acid moiety, which is decorated by five deoxysugars, including four digitoxose and the unusual nitro sugar D-kijanose. Analysis of the possible biosynthetic pathway for kijanimicin indicates the potential involvement of two Diels-Alder cyclization reactions in the construction of the pentacyclic aglycone.


Kijanimicin

Using a combination of chemical, biochemical, spectroscopic, and genetic methods, we have cloned and sequenced the kijanimicin biosynthetic gene cluster and are now working on biochemical characterization of enzymes involved in polyketide cross-bridging reactions. In particular, we would like to gain evidence for enzyme-catalyzed Diels-Alder reactions. More generally, we are interested in the biosynthesis of bioactive compounds possessing novel structural features for which the biosynthetic pathways and mechanisms of enzymatic transformations are not well understood. A multi-faceted approach is employed to study these problems.