Abstract
Ascorbic acid (AsA) maintains redox homeostasis by scavenging reactive oxygen species from prokaryotes to eukaryotes, especially plants. The enzyme monodehydroascorbate reductase (MDHAR) regenerates AsA by catalysing the reduction of monodehydroascorbate, using NADH or NADPH as an electron donor. The detailed recycling mechanism of MDHAR remains unclear due to lack of structural information. Here, we present the crystal structures of MDHAR in the presence of cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), and complexed with AsA as well as its analogue, isoascorbic acid (ISD). The overall structure of MDHAR is similar to other iron-sulphur protein reductases, except for a unique long loop of 63–80 residues, which seems to be essential in forming the active site pocket. From the structural analysis and structure-guided point mutations, we found that the Arg320 residue plays a major substrate binding role, and the Tyr349 residue mediates electron transfer from NAD(P)H to bound substrate via FAD. The enzymatic activity of MDHAR favours NADH as an electron donor over NADPH. Our results show, for the first time, structural insights into this preference. The MDHAR-ISD complex structure revealed an alternative binding conformation of ISD, compared with the MDHAR-AsA complex. This implies a broad substrate (antioxidant) specificity and resulting greater protective ability of MDHAR.
Highlights
Ascorbic acid (AsA) maintains redox homeostasis by scavenging reactive oxygen species from prokaryotes to eukaryotes, especially plants
Overexpression of dehydroascorbate reductase (DHAR) protected the plants against drought and ozone stress, whereas high expression of monodehydroascorbate reductase (MDHAR) increased their tolerance to osmotic stress[7,8]
In order to elucidate the electron transfer mechanism employed by OsMDHAR, we determined the crystal structure of OsMDHAR and complexes of the enzyme with NAD and NADP
Summary
We observed significant conformational changes only in the residues around FAD and NAD binding sites, on comparing the overall structure of OsMDHAR-NAD complex to the NAD-free form. Our speculation was confirmed from the structure of the OsMDHAR-NAD complex, where we observed hydrogen bonding between Glu[196] and the O2B molecule in the adenosine ring of NAD (Fig. 4c). Both apo and NADH complex structure, Tyr[349] formed hydrogen bonds with two water molecules, whose position was corresponded to the bound substrate ligand in the AsA complex structure (Fig. S4b). The yeast mutant cells expressing OsMDHAR were more tolerant to oxidative stress, compared to the control cells without OsMDHAR These results suggest that both antioxidants, AsA and ISD, are able to substitute D-erythroascorbic acid, and their reduced forms are regenerated by MDHAR activity in yeast cells. Given the structural and functional analysis, we conclude that the binding site of OsMDHAR may be able to accommodate variable substrate compositions including MDHA
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