Heterologous expression of cDNAs encoding monodehydroascorbate reductases from the moss, Physcomitrella patens and characterization of the expressed enzymes.
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 Km and kcat values for the heterologously expressed PpMDHAR enzymes ranged from 8 to 18 microM and 120-130 s(-1), respectively, using NADH as the electron donor. The Km values were at least an order of magnitude higher for NADPH. The Km values for the MDHA radical were approximately 0.5-1.0 microM 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.
Drew
Damian P
DP
Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia.
Lunde
Christina
C
Lahnstein
Jelle
J
Fincher
Geoffrey B
GB
D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000096:biosynthesis*; D019068:Bryopsida / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003545:Cysteine / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014169:Transformation, Bacterial
D018076:DNA, Complementary; D017382:Reactive Oxygen Species; D005182:Flavin-Adenine Dinucleotide; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH); D003545:Cysteine; D001205:Ascorbic Acid