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Title: STRUCTURE AND FUNCTION OF THE PHENAZINE BIOSYNTHETIC PROTEIN PHZF FROM PSEUDOMONAS FLUORESCENS

Author
item BLANKENFELDT, W - MAX PLANCK INSTITUTE
item KUZIN, A - COLUMBIA UNIVERSITY
item SKARINA, T - UNIVERSITY OF TORONTO
item KORNIYENKO, Y - UNIVERSITY OF TORONTO
item TONG, L - COLUMBIA UNIVERSITY
item BAYER, P - MAX PLANCK INSTITUTE
item JANNING, P - MAX PLANCK INSTITUE
item Thomashow, Linda
item MAVRODI, D - WASHINGTON STATE UNIV

Submitted to: Proceedings of the National Academy of Sciences (PNAS)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/5/2004
Publication Date: 12/20/2004
Citation: Blankenfeldt, W., Kuzin, A.P., Skarina, T., Korniyenko, Y., Tong, L., Bayer, P., Janning, P., Thomashow, L.S., Mavrodi, D. 2004. Structure and function of the phenazine biosynthetic protein phzf from pseudomonas fluorescens. Proceedings of the National Academy of Sciences. 101:16431-16436.

Interpretive Summary: Phenazine compounds produced by certain species of bacteria have antibiotic activity against a wide range of bacterial and fungal pathogens including many that cause important root diseases of plants. The antibiotic activity of these compounds has long been known but the mechanism of synthesis is poorly understood, making it difficult to fully exploit their biological potential. This paper reports the structure of PhzF, a key enzyme in the phenazine biosynthetic pathway, and describes the probable catalytic mechanism by which the core skeleton of the phenazine molecule is synthesized. These analyses provide novel insight not only into phenazine synthesis, but also into the activity of several other genes of unknown function identified through recent genomic sequencing efforts.

Technical Abstract: Phenazines produced by Pseudomonas and Streptomyces spp. are heterocyclic nitrogen-containing metabolites with antibiotic, antitumor, and antiparasitic activity. The antibiotic properties of pyocyanin, produced by P. aeruginosa, were recognized in the 1890s, although this blue phenazine is now known to be a virulence factor in human disease. Despite their biological significance, the biosynthesis of phenazines is not fully understood. Here we present structural and functional studies of PhzF, an enzyme essential for phenazine synthesis in Pseudomonas spp. PhzF shares topology with diaminopimelate epimerase DapF but lacks the same catalytic residues. The structure of PhzF in complex with its substrate, trans-2,3-dihydro-3-hydroxyanthranilic acid, suggests that it is a novel isomerase utilizing the conserved glutamate E45 to abstract a proton from C3 of the substrate. The proton is returned to C1 of the substrate after rearrangement of the double bond system, yielding an enol that converts to the corresponding ketone. PhzF is a dimer that may be bi-functional, providing a shielded cavity for ketone dimerization via double Schiff base formation to produce the phenazine scaffold. Our proposed mechanism is supported by mass and NMR spectroscopy. The results are discussed in the context of related structures and protein sequences of unknown biochemical function.