Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF

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 a...

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Autores principales:	Christina Diederich, Mario Leypold, Martin Culka, Hansjörg Weber, Rolf Breinbauer, G. Matthias Ullmann, Wulf Blankenfeldt
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2067ab1830ad4e8891977558d2293393
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spelling	oai:doaj.org-article:2067ab1830ad4e8891977558d22933932021-12-02T12:30:17ZMechanisms and Specificity of Phenazine Biosynthesis Protein PhzF10.1038/s41598-017-06278-w2045-2322https://doaj.org/article/2067ab1830ad4e8891977558d22933932017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-06278-whttps://doaj.org/toc/2045-2322Abstract 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.Christina DiederichMario LeypoldMartin CulkaHansjörg WeberRolf BreinbauerG. Matthias UllmannWulf BlankenfeldtNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-13 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Christina Diederich Mario Leypold Martin Culka Hansjörg Weber Rolf Breinbauer G. Matthias Ullmann Wulf Blankenfeldt Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF
description	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.
format	article
author	Christina Diederich Mario Leypold Martin Culka Hansjörg Weber Rolf Breinbauer G. Matthias Ullmann Wulf Blankenfeldt
author_facet	Christina Diederich Mario Leypold Martin Culka Hansjörg Weber Rolf Breinbauer G. Matthias Ullmann Wulf Blankenfeldt
author_sort	Christina Diederich
title	Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF
title_short	Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF
title_full	Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF
title_fullStr	Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF
title_full_unstemmed	Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF
title_sort	mechanisms and specificity of phenazine biosynthesis protein phzf
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/2067ab1830ad4e8891977558d2293393
work_keys_str_mv	AT christinadiederich mechanismsandspecificityofphenazinebiosynthesisproteinphzf AT marioleypold mechanismsandspecificityofphenazinebiosynthesisproteinphzf AT martinculka mechanismsandspecificityofphenazinebiosynthesisproteinphzf AT hansjorgweber mechanismsandspecificityofphenazinebiosynthesisproteinphzf AT rolfbreinbauer mechanismsandspecificityofphenazinebiosynthesisproteinphzf AT gmatthiasullmann mechanismsandspecificityofphenazinebiosynthesisproteinphzf AT wulfblankenfeldt mechanismsandspecificityofphenazinebiosynthesisproteinphzf
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Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF

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