Characterisation of recombinant epithiospecifier protein and its over-expression in Arabidopsis thaliana

Zabala, Marta de Torres; Grant, Murray; Bones, Atle M.; Bennett, Richard N.; Lim, Yin Sze; Kissen, Ralph; Rossiter, John T.

doi:10.1016/j.phytochem.2005.02.026

Cited by 57 publications

(45 citation statements)

References 18 publications

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“…The cDNA of AtESP used in this study has been described before (22). Seeds of the A. thaliana ecotypes C24 (N906), Col-0 (N1092), Cvi (N1097), Ru-0 (N1496) were obtained from the Nottingham Arabidopsis Stock Centre (Nottingham, UK).…”

Section: Methodsmentioning

confidence: 99%

“…A thiocyanate-forming protein (TFP) has recently been identified in Lepidium sativum (19). Alkenyl GSLs, a subgroup of aliphatic GSLs containing a terminal unsaturation in their side chain, can lead to the production of epithionitriles through the cooperative action of myrosinase and a protein called epithiospecifier protein (ESP (20)) in a ferrous ion-dependent way (21)(22)(23). Both TFP and ESP contain a series of Kelch repeats (19).…”

mentioning

confidence: 99%

“…Under acidic buffer conditions, a non-enzymatic production of nitriles from GSLs is observed (Ref. 29 concentrations have also been shown to favor nitrile formation over isothiocyanate formation from a number of GSLs in the presence of myrosinase and absence of ESP (21,22). Therefore, a non-enzymatic origin of this nitrile production cannot be excluded, although the presence of a nitrile-specifier protein is a tempting alternative.…”

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confidence: 99%

“…Therefore, a non-enzymatic origin of this nitrile production cannot be excluded, although the presence of a nitrile-specifier protein is a tempting alternative. Although ESP is able to generate nitriles, it has also been shown that the conversion rates of GSLs to nitriles are lower than those of GSLs to epithionitriles for ESP (21,22).…”

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Nitrile-specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana

Kissen

Bones

2009

Journal of Biological Chemistry

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127

104

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Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30 -45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinasecatalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and properties of AtNSPs and related proteins and the biological implications.Brassicaceae plants such as oilseed rape (Brassica napus), turnip (Brassica rapa), and white mustard (Sinapis alba) as well as the model plant Arabidopsis thaliana contain a group of secondary metabolites known as glucosinolates (GSLs) 2

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Nitrile-specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana

Kissen

Bones

2009

Journal of Biological Chemistry

Self Cite

127

104

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show abstract

“…In a study in Arabidiopsi tthaliana, enhanced sulforaphane nitrile production was observed in transgenic plants overexpressing ESP compared to wild type plants [189]. The ESP gene from broccoli has been cloned and the recombinant protein expressed in E. coli [103].…”

Section: Plant Breeding For Bio-fortified Cropsmentioning

confidence: 99%