Crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana provides insights into its product specificity

Zhang, Weiwei; Wang, Wenhe; Liu, Zihe; Xie, Ying; Wang, Hao; Mu, Yajuan; Huang, Yao; Feng, Yue

doi:10.1016/j.bbrc.2016.08.019

Cited by 12 publications

(8 citation statements)

References 24 publications

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“…In support of the proposed active site, this resulted in high quality docking arrangements with an ideal quadratic bipyramidal coordination of the iron by one oxygen of E266 and D270, a nitrogen of H274, the aglucone sulfur atom as well as two water molecules, and sufficient stabilization of the aglucone side chain [ 34 ]. Similar docking arrangements were obtained using the crystal structures of AtESP and AtNSP1 [ 35 , 36 ]. Substitution of the corresponding iron binding residues of AtESP, E260 and D264, by Gln and Asn, respectively, resulted in strongly diminished activity confirming the importance of these amino acid residues [ 33 ].…”

Section: Introductionmentioning

confidence: 64%

Iron is a centrally bound cofactor of specifier proteins involved in glucosinolate breakdown

et al. 2018

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Glucosinolates, a group of sulfur-rich thioglucosides found in plants of the order Brassicales, have attracted a lot of interest as chemical defenses of plants and health promoting substances in human diet. They are accumulated separately from their hydrolyzing enzymes, myrosinases, within the intact plant, but undergo myrosinase-catalyzed hydrolysis upon tissue disruption. This results in various biologically active products, e.g. isothiocyanates, simple nitriles, epithionitriles, and organic thiocyanates. While formation of isothiocyanates proceeds by a spontaneous rearrangement of the glucosinolate aglucone, aglucone conversion to the other products involves specifier proteins under physiological conditions. Specifier proteins appear to act with high specificity, but their exact roles and the structural bases of their specificity are presently unknown. Previous research identified the motif EXXXDXXXH as potential iron binding site required for activity, but crystal structures of recombinant specifier proteins lacked the iron cofactor. Here, we provide experimental evidence for the presence of iron (most likely Fe2+) in purified recombinant thiocyanate-forming protein from Thlaspi arvense (TaTFP) using a Ferene S-based photometric assay as well as Inductively Coupled Plasma-Mass Spectrometry. Iron binding and activity depend on E266, D270, and H274 suggesting a direct interaction of Fe2+ with these residues. Furthermore, we demonstrate presence of iron in epithiospecifier protein and nitrile-specifier protein 3 from Arabidopsis thaliana (AtESP and AtNSP3). We also present a homology model of AtNSP3. In agreement with this model, iron binding and activity of AtNSP3 depend on E386, D390, and H394. The homology model further suggests that the active site of AtNSP3 imposes fewer restrictions to the glucosinolate aglucone conformation than that of TaTFP and AtESP due to its larger size. This may explain why AtNSP3 does not support epithionitrile or thiocyanate formation, which likely requires exact positioning of the aglucone thiolate relative to the side chain.

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Section: Introductionmentioning

confidence: 64%

Iron is a centrally bound cofactor of specifier proteins involved in glucosinolate breakdown

et al. 2018

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“…In the past decades, epithionitriles and other atypical products from glucosinolates have received intense attention in the pure plant biochemistry field [11,12,13,14]. However, in food and nutritional science, epithionitriles have been neglected and here glucosinolate research mainly focussed on isothiocyanates, another important class of glucosinolate hydrolysis products that are valued especially for their potential cancer-preventing effects [15].…”

Section: Introductionmentioning

confidence: 99%

Identification Of N-Acetyl-S-(3-Cyano-2-(Methylsulfanyl)Propyl-Cysteine as a Major Human Urine Metabolite from the Epithionitrile 1-Cyano-2,3-Epithiopropane, the Main Glucosinolate Hydrolysis Product from Cabbage

et al. 2019

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Brassica vegetables such as cabbage or pak choi contain alkenyl glucosinolates which can release epithionitriles and to a lesser degree isothiocyanates upon enzymatic hydrolysis. Here, for the first time, the metabolism of an epithionitrile was investigated in humans, namely 1-cyano-2,3-epithiopropane (CETP). After consumption of Brassica oleracea var. capitata f. alba and Brassica carinata sprouts, the main urinary metabolite of CETP was identified as N-acetyl-S-(3-cyano-2-(methylsulfanyl)propyl-cysteine using an UHPLC-ESI-QToF-MS approach and synthesis of the metabolite. This urinary epithionitrile metabolite is an S-methylated mercapturic acid. No other metabolites were detected. Then, in a preliminary pilot experiment the excretion kinetics of CETP were investigated in three volunteers. After consumption of a B. carinata sprout preparation containing 50.8 µmol of CETP, urinary N-acetyl-S-(3-cyano-2-(methylsulfanyl)propyl-cysteine concentrations were the highest three hours after consumption, ranging from 23.9 to 37.2 µM, and declined thereafter. Thus, epithionitriles are bioavailable compounds that are metabolized similarly to isothiocyanates by the mercapturic acid pathway. In the future, more epithionitrile metabolites should be identified and the pharmacokinetics of these important class of dietary compounds should be assessed in more detail

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“…Brandt et al [17] reported that in the case of ESP from A. thaliana, a Glu260 -Asp264 -His274 binding triad interacts with Fe +2 while Arg94 and Arg157 are suggested to stabilize the thiohydroxamate-O-sulfonate group. Another study proposed that Gly186 and Val244 are responsible for the stabilization of aglycone in A. thaliana ESP [26]. In A. thaliana NSP (AtNSP3), iron-binding and NSP activity depend on Glu386, Asp390, and His394 [49].…”

Section: Molecular Interaction Of Nsp and Esp With Aglyconesmentioning

confidence: 99%

“…Also, broccoli ESP was cloned and expressed in Escherichia coli obtaining 43 kDa recombinant protein [6,13]. To date, only A. Thaliana ESP structure has been resolved by X-ray diffraction (PDB code 5GQ0) [26].…”

Section: Introductionmentioning

confidence: 99%

Molecular Modeling of Epithiospecifier and Nitrile-Specifier Proteins of Broccoli and Their Interaction with Aglycones

et al. 2020

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Glucosinolates are secondary plant metabolites of Brassicaceae. They exert their effect after enzymatic hydrolysis to yield aglycones, which become nitriles and epithionitriles through the action of epithiospecifier (ESP) and nitrile-specifier proteins (NSP). The mechanism of action of broccoli ESP and NSP is poorly understood mainly because ESP and NSP structures have not been completely characterized and because aglycones are unstable, thus hindering experimental measurements. The aim of this work was to investigate the interaction of broccoli ESP and NSP with the aglycones derived from broccoli glucosinolates using molecular simulations. The three-dimensional structure of broccoli ESP was built based on its amino-acid sequence, and the NSP structure was constructed based on a consensus amino-acid sequence. The models obtained using Iterative Threading ASSEmbly Refinement (I-TASSER) were refined with the OPLS-AA/L all atom force field of GROMACS 5.0.7 and were validated by Veryfy3D and ERRAT. The structures were selected based on molecular dynamics simulations. Interactions between the proteins and aglycones were simulated with Autodock Vina at different pH. It was concluded that pH determines the stability of the complexes and that the aglycone derived from glucoraphanin has the highest affinity to both ESP and NSP. This agrees with the fact that glucoraphanin is the most abundant glucosinolate in broccoli florets.

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Crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana provides insights into its product specificity

Cited by 12 publications

References 24 publications

Iron is a centrally bound cofactor of specifier proteins involved in glucosinolate breakdown

Iron is a centrally bound cofactor of specifier proteins involved in glucosinolate breakdown

Identification Of N-Acetyl-S-(3-Cyano-2-(Methylsulfanyl)Propyl-Cysteine as a Major Human Urine Metabolite from the Epithionitrile 1-Cyano-2,3-Epithiopropane, the Main Glucosinolate Hydrolysis Product from Cabbage

Molecular Modeling of Epithiospecifier and Nitrile-Specifier Proteins of Broccoli and Their Interaction with Aglycones

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