5IWE

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

Summary for 5IWE

• Entry DOI: 10.2210/pdb5iwe/pdb
• Descriptor: Trans-2,3-dihydro-3-hydroxyanthranilate isomerase, (5R,6R)-6-azaniumyl-5-ethoxycyclohexa-1,3-diene-1-carboxylate, DI(HYDROXYETHYL)ETHER, ... (6 entities in total)
• Functional Keywords: isomerase, complex, substrate analogue
• Biological source: Pseudomonas fluorescens
• Total number of polymer chains: 1
• Total formula weight: 32959.34

Authors

Diederich, C.,Blankenfeldt, W. (deposition date: 2016-03-22, release date: 2017-03-29, Last modification date: 2024-01-10)

Primary citation

Diederich, C.,Leypold, M.,Culka, M.,Weber, H.,Breinbauer, R.,Ullmann, G.M.,Blankenfeldt, W.
Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF.
Sci Rep, 7:6272-6272, 2017

PubMed Abstract: 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.

PubMed: 28740244
DOI: 10.1038/s41598-017-06278-w

Experimental method

X-RAY DIFFRACTION (1.71 Å)