Glucosinolate hydrolysis in plants - the role of specifier proteins
Final Report Abstract
The original objectives of the project to explore the roles of specifier proteins at an organismic level (SP1), to elucidate the biochemical mechanisms of specifier proteins based on the investigation of their structure and their interaction with myrosinase (SP2), and to investigate how the protein family has evolved (SP3) have been achieved with major focus on SP2. The most important results of the project can be summarized as follows: A. thaliana Col-0 NSPs are differentially expressed depending on development and organ. Due to NSP expression, nitriles are the major glucosinolate hydrolysis products in homogenates of seeds, seedlings and roots. NSPs are cytosolic proteins and are therefore expected to interfere with glucosinolate breakdown pathways in damaged and intact tissue. - Crystallization of TaTFP resulted in the elucidation of the first experimental structure of a plant kelch protein. This structure largely confirmed a molecular model that had been developed de novo based on predicted structural similarities with other proteins. TaTFP forms a homodimer composed of two six-blade β-propellers. The active site possesses a conserved Fe2+ binding site (EXXXDXXXH) which is essential for activity. Detection of bound iron by two independent methods identified specifier proteins of all three types as non-heme iron proteins. - Molecular modeling, semiempirical quantum-mechanical calculations and biochemical characterterization of mutant variants of TaTFP and AtNSP3 suggested mechanisms for thiocyanate, epithionitrile and simple nitrile formation by specifier proteins that are in agreement with an activity as C-S and C-S/C-C lyases, respectively. While epithionitrile and thiocyanate formation depend on Fe2+/Fe3+ as redox partner, simple nitrile formation proceeds without Fe2+ oxidation. - Phylogenetic analyses showed that NSPs are the ancestral representatives of the specifier protein family, from which ESPs evolved monophyletically. TFPs arose from ESPs, likely twice independently. Specifier proteins are subject to purifying selection which is in agreement with a selective advantage provided by the ability to form non-isothiocyanate products through specifier protein activity. - The structure of the active site has become more confined during the evolution of specifier proteins leading to more restricted aglucone conformations in ESPs and TFPs as compared to NSPs. For TaTFP, we found that confinement of the active site to enable allylthiocyanate formation is largely due to enlargement of the 3L2 loop and a conformational change of this loop that allows an unusual aglucone conformation.
Publications
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(2010) Glucosinolate breakdown in Arabidopsis - mechanism, regulation and biological significance. In: Last R et al. (eds) The Arabidopsis Book (http://www.aspb.org/publications/arabidopsis/). The American Society of Plant Biologists (14 pages)
Wittstock U, Burow M
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(2011) A thiocyanate-forming protein generates multiple products upon allylglucosinolate breakdown in Thlaspi arvense. Phytochemistry 72:1699-1709
Kuchernig J-C, Backenköhler A, Lübbecke M, Burow M, Wittstock U
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(2012) Evolution of specifier proteins in glucosinolate-containing plants. BMC Evol Biol 12:127 (14 pages)
Kuchernig J-C, Burow M, Wittstock U
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(2014) Molecular models and mutational analyses of plant specifier proteins suggest active site residues and reaction mechanism. Plant Mol Biol 84:173-188
Brandt W, Backenköhler A, Schulze E, Plock A, Herberg T, Roese E, Wittstock U
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(2015) The crystal structure of the thiocyanate-forming protein from Thlaspi arvense, a kelch protein involved in glucosinolate breakdown. Plant Mol Biol 89:67-81
Gumz F, Krausze J, Eisenschmidt D, Backenköhler A, Barleben L, Brandt W, Wittstock U
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(2016) Glucosinolate breakdown. In: Advances in Botanical Research, Vol. 80: Glucosinolates (S. Kopriva, ed.), pp. 125-169
Wittstock U, Kurzbach E, Herfurth A-M, Stauber EJ
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(2016) NSP-dependent simple nitrile formation dominates upon breakdown of major aliphatic glucosinolates in roots, seeds, and seedlings of Arabidopsis thaliana Columbia-0. Front Plant Sci 7:1821
Wittstock U, Meier K, Dörr F, Ravindran, BM
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(2018) Iron is a centrally bound cofactor of specifier proteins involved in glucosinolate breakdown. PLOS ONE 13(11): e0205755 (18 pages)
Backenköhler A, Eisenschmidt D, Schneegans N, Strieker M, Brandt W, Wittstock U
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(2019) Structural diversification during glucosinolate breakdown: mechanisms of thiocyanate, epithionitrile and simple nitrile formation. Plant J
Eisenschmidt-Bönn D, Schneegans N, Backenköhler A, Wittstock U, Brandt W
