Plant-type phytoene desaturase: Functional evaluation of structural implications.

Phytoene desaturase (PDS) is an essential plant carotenoid biosynthetic enzyme and a prominent target of certain inhibitors, such as norflurazon, acting as bleaching herbicides. PDS catalyzes the introduction of two double bonds into 15-cis-phytoene, yielding 9,15,9'-tri-cis-ζ-carotene via the...

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Main Authors: Julian Koschmieder, Mirjam Fehling-Kaschek, Patrick Schaub, Sandro Ghisla, Anton Brausemann, Jens Timmer, Peter Beyer
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2017-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC5703498?pdf=render
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spelling doaj-fab7c6b6c59e40b29afa1ab954c711d62020-11-25T01:41:52ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-011211e018762810.1371/journal.pone.0187628Plant-type phytoene desaturase: Functional evaluation of structural implications.Julian KoschmiederMirjam Fehling-KaschekPatrick SchaubSandro GhislaAnton BrausemannJens TimmerPeter BeyerPhytoene desaturase (PDS) is an essential plant carotenoid biosynthetic enzyme and a prominent target of certain inhibitors, such as norflurazon, acting as bleaching herbicides. PDS catalyzes the introduction of two double bonds into 15-cis-phytoene, yielding 9,15,9'-tri-cis-ζ-carotene via the intermediate 9,15-di-cis-phytofluene. We present the necessary data to scrutinize functional implications inferred from the recently resolved crystal structure of Oryza sativa PDS in a complex with norflurazon. Using dynamic mathematical modeling of reaction time courses, we support the relevance of homotetrameric assembly of the enzyme observed in crystallo by providing evidence for substrate channeling of the intermediate phytofluene between individual subunits at membrane surfaces. Kinetic investigations are compatible with an ordered ping-pong bi-bi kinetic mechanism in which the carotene and the quinone electron acceptor successively occupy the same catalytic site. The mutagenesis of a conserved arginine that forms a hydrogen bond with norflurazon, the latter competing with plastoquinone, corroborates the possibility of engineering herbicide resistance, however, at the expense of diminished catalytic activity. This mutagenesis also supports a "flavin only" mechanism of carotene desaturation not requiring charged residues in the active site. Evidence for the role of the central 15-cis double bond of phytoene in determining regio-specificity of carotene desaturation is presented.http://europepmc.org/articles/PMC5703498?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Julian Koschmieder
Mirjam Fehling-Kaschek
Patrick Schaub
Sandro Ghisla
Anton Brausemann
Jens Timmer
Peter Beyer
spellingShingle Julian Koschmieder
Mirjam Fehling-Kaschek
Patrick Schaub
Sandro Ghisla
Anton Brausemann
Jens Timmer
Peter Beyer
Plant-type phytoene desaturase: Functional evaluation of structural implications.
PLoS ONE
author_facet Julian Koschmieder
Mirjam Fehling-Kaschek
Patrick Schaub
Sandro Ghisla
Anton Brausemann
Jens Timmer
Peter Beyer
author_sort Julian Koschmieder
title Plant-type phytoene desaturase: Functional evaluation of structural implications.
title_short Plant-type phytoene desaturase: Functional evaluation of structural implications.
title_full Plant-type phytoene desaturase: Functional evaluation of structural implications.
title_fullStr Plant-type phytoene desaturase: Functional evaluation of structural implications.
title_full_unstemmed Plant-type phytoene desaturase: Functional evaluation of structural implications.
title_sort plant-type phytoene desaturase: functional evaluation of structural implications.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2017-01-01
description Phytoene desaturase (PDS) is an essential plant carotenoid biosynthetic enzyme and a prominent target of certain inhibitors, such as norflurazon, acting as bleaching herbicides. PDS catalyzes the introduction of two double bonds into 15-cis-phytoene, yielding 9,15,9'-tri-cis-ζ-carotene via the intermediate 9,15-di-cis-phytofluene. We present the necessary data to scrutinize functional implications inferred from the recently resolved crystal structure of Oryza sativa PDS in a complex with norflurazon. Using dynamic mathematical modeling of reaction time courses, we support the relevance of homotetrameric assembly of the enzyme observed in crystallo by providing evidence for substrate channeling of the intermediate phytofluene between individual subunits at membrane surfaces. Kinetic investigations are compatible with an ordered ping-pong bi-bi kinetic mechanism in which the carotene and the quinone electron acceptor successively occupy the same catalytic site. The mutagenesis of a conserved arginine that forms a hydrogen bond with norflurazon, the latter competing with plastoquinone, corroborates the possibility of engineering herbicide resistance, however, at the expense of diminished catalytic activity. This mutagenesis also supports a "flavin only" mechanism of carotene desaturation not requiring charged residues in the active site. Evidence for the role of the central 15-cis double bond of phytoene in determining regio-specificity of carotene desaturation is presented.
url http://europepmc.org/articles/PMC5703498?pdf=render
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