Diazeniumdiolate Siderophores: Biosynthesis and Reactivity

Researchers from the University of California at Santa Barbara are investigating the biosynthesis and reactivity of a newly discovered iron-binding ligand in a class of microbial natural products called siderophores. The new ligand is a diazeniumdiolate present in the amino acid graminine. The biosynthesis of this amino acid involves two new classes of metalloenzymes of potential biotechnological significance. The first biosynthetic step involves a new arginine hydroxylase enzyme, GrbE, which is part of a large family of enzymes which until recently had no known function; the second step involves a non heme iron enzyme, GrbD which oxidatively forms the nitrogen-nitrogen bond in graminine. The diazeniumdiolate group in graminine is photoreactive producing nitric oxide and oxime products, which are also of biological significance in bacterial growth, and have important pharmaceutical applications. The research team will express, characterize and investigate the reactivity of the new GrbE and GrbD metalloenzymes, and investigate the photoreactivity of gramine to determine the factors affecting this reaction. Their discoveries may reveal biotechnological routes to the production of new diazeniumdiolate compounds for potential societal benefit, as well as to uncover how the reactivity of graminine affects bacterial growth. This research provides multi-disciplinary research training to the graduate and undergraduate students across bioinorganic chemistry, bioorganic chemistry, and chemical biology. The newly discovered iron(III)-binding group bacteria use to sequester iron is a C-diazeniumdiolate group in the amino acid graminine found in microbial siderophores. Little is known about the biosynthesis of C-diazeniumdiolates in natural products, nor the reactivity of this group, which is a recognized donor of nitric oxide. The specific aims of the research are to characterize selected new graminine-containing siderophores; to investigate the biosynthesis of graminine from arginine by the heme-iron-containing enzyme, GrbE, and the non-heme-iron-containing enzyme, GrbD; and to study the photoreactivity of the C-diazeniumdiolate in graminine-containing siderophores, which the researchers have shown converts each diazeniumdiolate to a mixture of E and Z oximes with concomitant production of nitric oxide and an H-atom equivalent. The new siderophore(s) will be isolated from microbial cultures, purified and structurally characterized. The GrbE and GrbD enzymes will be heterologously expressed, purified, and characterized, and investigated in hydroxylation reactions with arginine and in the formation of graminine. The focus of photoreactions of apo and iron(III)-bound graminine siderophores is identification of the reactive nitrogen species produced. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.