TY - GEN

AU - Koschmieder, Julian

AU - Albert-Ludwigs-Universität Freiburg Institut für Biologie II

AU - Albert-Ludwigs-Universität Freiburg Fakultät für Biologie

AU - Albert-Ludwigs-Universität Freiburg

AU - Beyer, Peter

TI - The poly-cis pathway of carotene desaturation: enzymology, herbicide action and retrograde signaling

PB - Universität

KW - Carotinoide

KW - Phytoendesaturase

KW - Herbizid

KW - Mathematische Modellierung

KW - Enzymologie

KW - Enzymkinetik

KW - Flavoenzym

KW - (local)doctoralThesis

KW - Hochschulschrift

PY - 2017

N2 - IN COPYRIGHT http://rightsstatements.org/page/InC/1.0 rs

N2 - Abstract: Plant carotenoid biosynthesis in plastid membranes proceeds via the so-called poly-cis pathway of carotene desaturation, converting 15-cis-phytoene into all-trans-lycopene via poly-cis-configured intermediates. Its four constituent enzymes are poorly understood in terms of mechanisms and structure. In addition, the reason for the prevalence of these intricate reactions remains enigmatic considering that the bacterial carotene desaturation pathway requires only one enzyme. In this work, the crystal structure of phytoene desaturase (PDS) from O. sativa, the first desaturation pathway enzyme, in complex with its inhibitor norflurazon (NFZ) was refined and structural implications were functionally evaluated by a kinetic characterization. The kinetics of the downstream ζ-carotene desaturase (ZDS), representing a PDS homolog, were investigated for comparison and revealed pronounced similarities. The data presented support an ordered ping pong bi bi kinetic mechanism for PDS and ZDS. Desaturation of their substrates occurs via two formally identical, mechanistically independent reactions on half sides of the symmetric substrate. The intermediate is released into the membrane after the first desaturation. The enzyme-bound FADred produced by carotene desaturation is reoxidized by plastoquinone (PQ) to allow repeated rounds of catalysis. As inferred from the active site structure, data support an unprecedented “flavin only” mechanism characterized by FADox as sole catalyst of carotene desaturation. PDS and ZDS interact monotopically with membranes to access their substrates. Mathematical modelling implies substrate channeling between subunits within PDS homotetramers whereas ZDS is active as monomer. Notably, PDS does not produce ζ-carotene in the stereoconfiguration required by ZDS. In this work, a cell-free activity assay demonstrated that the membrane-integral ζ-carotene isomerase (ZISO) is a bona fide isomerase representing the link between PDS and ZDS. An additional aspect of this work relates to the mode of action of bleaching herbicides targeting PDS and containing meta-CF3-phenyl moieties, such as NFZ. It is demonstrated that they compete with PQ and NFZ, occupying the PQ binding site as suggested in crystallo. The meta-CF3 substituent provides unique non-covalent interactions with conserved residues and is crucial for inhibitory effectiveness. Mutagenesis of the conserved Arg300, forming a hydrogen bond with NFZ, allows engineering NFZ-resistant PDS but at the expense of altered enzyme kinetics and partial loss of enzymatic activity. Evidence has been presented that carotenoid cleavage dioxygenases (CCDs) might form retrograde signals from cis-configured carotene desaturation intermediates, regulating plastid development and carotenoid biosynthesis. Investigating intermediate cleavage by CCDs, Arabidopsis CCD7 was found to cleave 9-cis-ζ-carotene yielding 9-cis-ζ-apo-10’-carotenal (C27) which, in contrast to strigolactone biosynthesis, is not converted by AtCCD8. These data suggest this apocarotenoid as a candidate signal precursor requiring further modification by hitherto unidentified enzyme activities

CY - Freiburg

UR - http://slubdd.de/katalog?TN_libero_mab2

ER -