|Li, Qi Ang -|
|Mavrodi, Dmitri -|
|Roessle, Manfred -|
|Blankenfeldt, Wulf -|
Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 2, 2011
Publication Date: March 29, 2011
Citation: Li, Q., Mavrodi, D.V., Thomashow, L.S., Roessle, M., Blankenfeldt, W. 2011. Ligand binding induces an ammonia channel in 2-amino-2-desoxyisochorismate (ADIC) synthase PhzE. Journal of Biological Chemistry. 286(20):18213-18221. Interpretive Summary: Phenazine compounds are natural antibiotics produced in the environment by certain bacterial species that can protect crop plants against fungal root pathogens. Despite their antibiotic activity and their role in the natural protection of plants, it still is unclear how phenazines are synthesized. This study focused on PhzE, the first enzyme in the biosynthesis pathway of phenazines. The reaction carried out by the enzyme utilizes two simple cellular metabolites, chorismic acid and glutamine, to synthesize 2-amino-2-desoxyisochorismate (ADIC). PhzE is similar to another enzyme, anthranilate synthase, which is involved in the synthesis of the amino acid tryptophan, but PhzE has several differences that were revealed by crystallizing the PhzE protein. The active form of PhzE consists of two intertwined molecules that perform a novel reaction involving the transfer of an NH3 from glutamine to chorismic acid. Large structural rearrangements accompany the binding of chorismic acid. It was shown that Zn2+, Mn2+ and Ni2+ reduce the activity of PhzE. The results of this study provide new information about the enzymes that synthesize phenazines, which are an important component in the ability of some bacteria to protect plants from soilborne pathogens.
Technical Abstract: PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. At variance with the related anthranilate synthase, the monomer of PhzE consists of a single chain that contains both a chorismate-converting domain of the menaquinone, siderophore, tryptophan biosynthesis (MST) type and a type 1 glutamine amidotransferase domain (GATase1) connected by a 45-residue linker. We present here the crystal structure of PhzE from Burkholderia lata 383 in a ligand-free open and ligand-bound closed conformation at 2.9 and 2.1 Å resolution, respectively. PhzE arranges in an intertwined dimer such that GATase1 of one chain provides NH3 to the MST domain of the other monomer. This quaternary structure was also confirmed by small angle x-ray scattering. The dimer possesses relatively weak interactions along the dyad axis but makes more intimate contacts between the GATase1 and MST domains of the two opposite chains, which leads to the formation of an ammonia transport channel with approx. 25 Å length within the MST/GATase1 functional unit of the closed form. Large structural rearrangements accompany the binding of chorismic acid, which was found converted to benzoate and pyruvic acid in the closed MST active center. Unlike anthranilate synthase, no allosteric inhibition was observed in PhzE. This can be attributed to a tryptophan residue of the protein blocking the potential regulatory site. Additional electron density in the GATase1active center was identified as zinc and it could be demonstrated that Zn2+, Mn2+ and Ni2+ indeed reduce the activity of PhzE.