Thioredoxin reductase from barley: Structure, recognition of thioredoxin, protein engineering and catalytic mechanism

Kristine Groth Kirkensgaard

    Research output: Book/ReportPh.D. thesisResearch

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    In this thesis the crystal structure of one of the two isoforms of NADPH‐dependent thioredoxin reductase (HvNTR2) from barley was determined. The overall structure of HvNTR2 resembled other low molecular weight (LMW) NTRs. However, the relative orientation of the NADPH and FAD domains of HvNTR2, is different from other LMW NTRs in the flavin oxidising (FO) and flavin reducing (FR) conformations. The difference can be described by a 38.2% closure, a 1.0 Å translation and a 24.7° rotational twist when compared to the only other plant NTR structure of AtNTR‐B from Arabidopsis thaliana (which is in the FO conformation). It was thus suggested that the HvNTR2 structure represents an intermediate between the FO and the FR conformation indicating that the conformational change does not only involve domain rotation, but also bending of one domain relative to the other. Potentially there is room for binding HvTrxh2 to the FAD domain of HvNTR2 in the FO conformation, after which a conformational change of HvNTR2 could bring the active site cysteines of the NADPH domain in proximity to the active site of HvTrxh2. A reaction mechanism was proposed, which included binding of thioredoxin (Trx) in the FO conformation of HvNTR2. A complex was produced of HvNTR2 bound covalently to one of the two isoforms of Trx from barley seeds, HvTrxh2. The complex is assumed to represent a reaction intermediate with NTR locked in the FR conformation through an intermolecular disulfide bond between active site cysteines of the two proteins. Attempts to crystallise the HvNTR2:HvTrxh2 complex were unfruitful, possibly due to sample heterogeneity as indicated by molecular weight and isoelectric point determinations. Since no crystal structure of the HvNTR2:HvTrxh2 complex was obtained, a homology model based on the crystal structures of HvNTR2 and HvTrxh2 was built, using the structure of Escherichia coli NTR covalently bound to Trx (EcNTR:EcTrx) as a template (Lennon et al., 2000). The model provides new insight into how eukaryotic LMW NTRs in general bind Trx and suggests major differences in the NTR:Trx binding interface of HvNTR2:HvTrxh2 and EcNTR:EcTrx. Notably a large loop in HvNTR2 with the sequence EGWMANDIAAGG in the FAD domain (thus termed the FAD‐loop), was predicted to have tight interactions with HvTrxh2 through hydrophobic contacts. Sequence alignments suggest that this loop is present in LMW NTRs from other eukaryotes but appears to be absent in most prokaryotes. The crystal structure of HvTrxh2 covalently bound to the barley α‐amylase/subtilisin inhibitor (BASI, Maeda et al., 2006a), allowed comparison of the molecular features involved in the interactions between HvTrxh2 and its electron donor (NTR) and a protein disulfide substrate, respectively. This comparison suggested some overlapping interactions as amino acid residues from HvTrxh2 involved in the binding of BASI are also interacting with the FAD and NADPH domains of HvNTR2. The model of HvNTR2:HvTrxh2 served as a guideline for a comprehensive mutational study of some of the residues and loops, which were indicated to be important for the binding between the two proteins. Enzyme kinetics analyses of these mutants suggest that the FAD‐loop is critical for binding of HvTrxh2. Especially, Trp42HvNTR2 and Met43HvNTR2 appears to be important for the binding of HvTrxh2. Met43HvNTR2 is the only HvNTR2 residue from the FAD‐loop, which is not conserved in the HvNTR1 isoform, where it is replaced by a leucine. Surprisingly the FAD‐loop appears to be less critical for binding of several Trx isoforms from A. thaliana. The residue Arg73EcTrx is essential for the binding of EcTrx to the NADPH domain of EcNTR2, and was predicted to be involved in the conformational change from FO to FR (Negri et al., 2009). However, a mutant of the corresponding residue Glu86 in HvTrxh2 showed retained activity with HvNTR2, indicating that this residue plays different roles in Trx from barley and E. coli Finally, the expression levels of HvNTR1, HvNTR2, HvTrxh1, HvTrxh2 and α‐amylase during imbibition was examined in different tissues of barley seeds by Q‐PCR and micro‐ array data analysis. The effects of the plant hormone gibberellic acid, dormancy/after‐ ripening, light/darkness on these expression levels were examined as well as the role of the GA‐receptor GID1. The expression of α‐amylase in aleurone layers was significantly up‐ regulated by GA, and the response was dependent on GSE1. HvNTR1 seemed to be down‐ regulated by GA independently on the dose and GSE1. HvNTR2 seemed to be upregulated by GA leading to expression levels of HvNTR2 that were 10―40 times higher than of HvNTR1. Both HvTrxh1 and HvTrxh2 were expressed in higher amounts than the NTRs and seemed unaffected by GA. Micro‐array analysis was performed on available data from root and coleorhiza. Coleorhiza is a sheath‐like structure that acts as a protective covering enclosing the plumule (growing point of embryo) and radicle (from which the root is developed). These were either dormant or after‐ripened. It was observed that after‐ripening leads to increased expression levels of both HvNTR1 (in coleorhiza) and HvNTR2 (in both roots and coleorhiza). HvNTR2 and HvTrxh1 were the most abundant isoforms in root and coleorhizae after imbibition and both showed increased expression levels in after‐ripened seeds compared with dormant. For whole embryo the levels of Trxs was higher than of NTRs. While the expression of Trxs seemed unaffected by both after‐ripening and light/darkness, the average levels of the NTRs increased in the after‐ripened samples. Light had no effect on the expression levels. All together the results presented in this thesis provide valuable new insights into the structure and function of the NTR/Trx system.
    Original languageEnglish
    Place of PublicationKgs. Lyngby, Denmark
    PublisherTechnical University of Denmark
    Publication statusPublished - Nov 2011


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