5iwe

E45Q mutant of phenazine biosynthesis protein PhzF in complex with (5R,6R)-6-azaniumyl-5-ethoxycyclohexa-1,3-diene-1-carboxylate

spinqualitylabelsall models 5iwe, resolution 1.71Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5iwe is a 1 chain structure with sequence from Pseudomonas fluorescens. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.71Å Ligands: EDO, GOL, PEG, W81 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function PHZF_PSEFL Isomerase that catalyzes the condensation of two molecules of trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) into the phenazine ring system. The final product is not yet known. Publication Abstract from PubMed Phenazines are bacterial virulence and survival factors with important roles in infectious disease. PhzF catalyzes a key reaction in their biosynthesis by isomerizing (2 S,3 S)-2,3-dihydro-3-hydroxy anthranilate (DHHA) in two steps, a [1,5]-hydrogen shift followed by tautomerization to an aminoketone. While the [1,5]-hydrogen shift requires the conserved glutamate E45, suggesting acid/base catalysis, it also shows hallmarks of a sigmatropic rearrangement, namely the suprafacial migration of a non-acidic proton. To discriminate these mechanistic alternatives, we employed enzyme kinetic measurements and computational methods. Quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the activation barrier of a proton shuttle mechanism involving E45 is significantly lower than that of a sigmatropic [1,5]-hydrogen shift. QM/MM also predicted a large kinetic isotope effect, which was indeed observed with deuterated substrate. For the tautomerization, QM/MM calculations suggested involvement of E45 and an active site water molecule, explaining the observed stereochemistry. Because these findings imply that PhzF can act only on a limited substrate spectrum, we also investigated the turnover of DHHA derivatives, of which only O-methyl and O-ethyl DHHA were converted. Together, these data reveal how PhzF orchestrates a water-free with a water-dependent step. Its unique mechanism, specificity and essential role in phenazine biosynthesis may offer opportunities for inhibitor development. Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF.,Diederich C, Leypold M, Culka M, Weber H, Breinbauer R, Ullmann GM, Blankenfeldt W Sci Rep. 2017 Jul 24;7(1):6272. doi: 10.1038/s41598-017-06278-w. PMID:28740244 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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