Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF

Christina Diederich, Mario Leypold, Martin Culka, Hansjörg Weber, Rolf Breinbauer, G. Matthias Ullmann, Wulf Blankenfeldt

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Article number6272
JournalScientific reports
Volume7
Issue number1
DOIs
Publication statusPublished - 1 Dec 2017

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Molecular mechanics
Quantum theory
Biosynthesis
Hydrogen
Protons
Water
Phenazines
Enzyme kinetics
Stereochemistry
Proteins
Substrates
Computational methods
Isotopes
Catalysis
Glutamic Acid
Chemical activation
Derivatives
Molecules
Acids
anthranilic acid

ASJC Scopus subject areas

  • General

Fields of Expertise

  • Human- & Biotechnology

Cite this

Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF. / Diederich, Christina; Leypold, Mario; Culka, Martin; Weber, Hansjörg; Breinbauer, Rolf; Ullmann, G. Matthias; Blankenfeldt, Wulf.

In: Scientific reports, Vol. 7, No. 1, 6272, 01.12.2017.

Research output: Contribution to journalArticleResearchpeer-review

Diederich, Christina ; Leypold, Mario ; Culka, Martin ; Weber, Hansjörg ; Breinbauer, Rolf ; Ullmann, G. Matthias ; Blankenfeldt, Wulf. / Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF. In: Scientific reports. 2017 ; Vol. 7, No. 1.
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