It is proposed to investigate the biosynthesis of two cyclopentanoid natural products. The first is the nucleoside antibiotic aristeromycin, a cyclopentyl analog of adenosine produced by Streptomyces citricolor and accompanied by the closely related antitumor and antiviral agent neplanocin A. Previous investigations of aristeromycin biosynthesis revealed that the adenine ring is biosynthesized along familiar lines, while the cyclopentane ring of the antibiotic is generated by C-C bond formation between C-2 and C-6 of glucose. The mechanism of cyclopentane ring formation was probed using specifically tritiated forms of glucose. Isotope dilution experiments provided evidence for the presence of the cyclopentyl analogs of ribose-5-phosphate and 5-phosphoribosyl-1-amine in extracts of S. citricolor. Future studies will investigate the formation of the cyclopentyl analogs of ribose-5-phosphate, PRPP, and 5- phosphoribosyl-1-amine in cell-free extracts of S. citricolor. The formation of the cyclopentyl analog of AMP (aristeromycin-5'- monophosphate) in cell-free extracts will also be investigated, since previous studies indicate that aristeromycin can be biosynthesized via the purine salvage pathway. Investigations will also be carried out with purified forms of three enzymes involved in the biosynthesis of adenosine in bacteria. The goal of these studies will be to determine whether these enzymes can utilize the cyclopentyl analogs of their normal substrates. The results should provide insight into the nature of some of the enzymes involved in aristeromycin biosynthesis and may allow the enzymatic synthesis of one or more of the intermediates in the aristeromycin pathway. If the purine biosynthetic enzymes will utilize the cyclopentyl analogs, then these enzymes could also be used in coupled assays for the formation of cyclopentyl compounds in S. citricolor extracts. The second cyclopentanoid to be investigated is the phytotoxin coronatine, which is produced by Pseudomonas syringae. Previous investigations have revealed that the hydrindane portion of the molecule, coronafacic acid, is a novel polykeytide derived from five acetate units and one pyruvate unit. The cyclopropyl amino acid moiety of the toxin, coronamic acid, has been found to be biosynthesized from L-alloisoleucine by an unusual process that appears to be related to the oxidative cyclizations associated with the biosynthesis of the beta- lactam antibiotics. Future studies of coronatine biosynthesis will include an attempt to clarify the mode of assembly of the hydrindane ring and will examine the mechanism of the biosynthesis of coronamic acid in further detail. The investigations should be greatly facilitated by a collaboration with Dr. Carol Bender of Oklahoma State University, who is cloning the genes for the coronatine biosynthetic pathway. Finally, the investigations of coronamic acid biosynthesis will be extended to an investigation of the mechanism of biosynthesis of 3- ethylidene-L-azetidine-2-carboxylic acid (polyoximic acid) from L- isoleucine. Polyoximic acid is a component of the polyoxins, a group of antifungal antibiotics produced by S. cacaoi var. asoensis.
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