2.50
Hdl Handle:
http://hdl.handle.net/10033/621041
Title:
Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF.
Authors:
Diederich, Christina; Leypold, Mario; Culka, Martin ( 0000-0002-3944-152X ) ; Weber, Hansjörg; Breinbauer, Rolf ( 0000-0001-6009-7359 ) ; Ullmann, G Matthias ( 0000-0002-6350-798X ) ; Blankenfeldt, Wulf ( 0000-0001-9886-9668 )
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.
Affiliation:
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Citation:
Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF. 2017, 7 (1):6272 Sci Rep
Journal:
Scientific reports
Issue Date:
24-Jul-2017
URI:
http://hdl.handle.net/10033/621041
DOI:
10.1038/s41598-017-06278-w
PubMed ID:
28740244
Type:
Article
Language:
en
ISSN:
2045-2322
Appears in Collections:
Publications of the Dept. Structure and Functions of Proteins(SFPR)

Full metadata record

DC FieldValue Language
dc.contributor.authorDiederich, Christinaen
dc.contributor.authorLeypold, Marioen
dc.contributor.authorCulka, Martinen
dc.contributor.authorWeber, Hansjörgen
dc.contributor.authorBreinbauer, Rolfen
dc.contributor.authorUllmann, G Matthiasen
dc.contributor.authorBlankenfeldt, Wulfen
dc.date.accessioned2017-08-04T07:58:49Z-
dc.date.available2017-08-04T07:58:49Z-
dc.date.issued2017-07-24-
dc.identifier.citationMechanisms and Specificity of Phenazine Biosynthesis Protein PhzF. 2017, 7 (1):6272 Sci Repen
dc.identifier.issn2045-2322-
dc.identifier.pmid28740244-
dc.identifier.doi10.1038/s41598-017-06278-w-
dc.identifier.urihttp://hdl.handle.net/10033/621041-
dc.description.abstractPhenazines 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.en
dc.language.isoenen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.titleMechanisms and Specificity of Phenazine Biosynthesis Protein PhzF.en
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.en
dc.identifier.journalScientific reportsen

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