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Heterologous expression of cDNAs encoding monodehydroascorbate reductases from the moss, Physcomitrella patens and characterization of the expressed enzymes

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Abstract

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) catalyses the reduction of the monodehydroascorbate (MDHA) radical to ascorbate, using NADH or NADPH as an electron donor, and is believed to be involved in maintaining the reactive oxygen scavenging capability of plant cells. This key enzyme in the ascorbate-glutathione cycle has been studied here in the moss Physcomitrella patens, which is tolerant to a range of abiotic stresses and is increasingly used as a model plant. In the present study, three cDNAs encoding different MDHAR isoforms of 47 kDa were identified in P. patens, and found to exhibit enzymic characteristics similar to MDHARs in vascular plants despite low-sequence identity and a distant evolutionary relationship between the species. The three cDNAs for the P. patens MDHAR enzymes were expressed in Escherichia coli and the active enzymes were purified and characterized. Each recombinant protein displayed an absorbance spectrum typical of flavoenzymes and contained a single non-covalently bound FAD coenzyme molecule. The K m and k cat values for the heterologously expressed PpMDHAR enzymes ranged from 8 to 18 μM and 120–130 s−1, respectively, using NADH as the electron donor. The K m values were at least an order of magnitude higher for NADPH. The K m values for the MDHA radical were ∼0.5–1.0 μM for each of the purified enzymes, and further kinetic analyses indicated that PpMDHARs follow a ‘ping–pong’ kinetic mechanism. In contrast to previously published data, site-directed mutagenesis indicated that the conserved cysteine residue is not directly involved in the reduction of MDHA.

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Abbreviations

APX:

Ascorbate peroxidase

β-CMB:

Beta-chloromercuribenzoate

DHAR:

Dehydroascorbate reductase

GR:

Glutathione reductase

MDHA:

Monodehydroascorbate

MDHAR:

MDHA reductase

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Acknowledgments

This work was funded by grants from the Australian Research Council, the Grains Research and Development Corporation, and the State government of South Australia. Christina Lunde was funded by the Danish Agricultural and Veterinary Research Council and the Villum Kann Rasmussen Foundation. We also thank Naser Farrokhi, Mark Tester and Maria Hrmova for assistance with various aspects of the work.

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Author notes

  1. Christina Lunde

    Present address: Department of Plant Biology, Plant Biochemistry Laboratory, The Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, Frederiksberg C, 1871, Copenhagen, Denmark

Authors and Affiliations

  1. Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA, 5064, Australia

    Damian P. Drew, Jelle Lahnstein & Geoffrey B. Fincher

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  1. Damian P. Drew
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  2. Christina Lunde
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  4. Geoffrey B. Fincher
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Correspondence to Geoffrey B. Fincher.

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Drew, D.P., Lunde, C., Lahnstein, J. et al. Heterologous expression of cDNAs encoding monodehydroascorbate reductases from the moss, Physcomitrella patens and characterization of the expressed enzymes. Planta 225, 945–954 (2007). https://doi.org/10.1007/s00425-006-0394-x

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  • DOI: https://doi.org/10.1007/s00425-006-0394-x

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