title: Structure and catalytic mechanism of monodehydroascorbate reductase, MDHAR, from Oryza sativa L. japonica.
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.
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea.
authors.forename: Ae Kyung
authors.initials: AK
authors.lastname: Park
authors.affiliation: School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu 41566, Republic of Korea.
authors.forename: Il-Sup
authors.initials: IS
authors.lastname: Kim
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea.
authors.forename: Hackwon
authors.initials: H
authors.lastname: Do
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea.
authors.forename: Byung Wook
authors.initials: BW
authors.lastname: Jeon
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea.|Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic of Korea.
authors.forename: Chang Woo
authors.initials: CW
authors.lastname: Lee
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea.
authors.forename: Soo Jung
authors.initials: SJ
authors.lastname: Roh
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea.
authors.forename: Seung Chul
authors.initials: SC
authors.lastname: Shin
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea.|Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic of Korea.
authors.forename: Hyun
authors.initials: H
authors.lastname: Park
authors.affiliation: Research Institute for Ulleung-do &Dok-do, Kyungpook National University, Daegu 41566, Republic of Korea.
authors.forename: Young-Saeng
authors.initials: YS
authors.lastname: Kim
authors.affiliation: Highland Agriculture Research Institute, National Institute of Crop Science, Rural Development Administration, Pyeongchang 25342, Republic of Korea.
authors.forename: Yul-Ho
authors.initials: YH
authors.lastname: Kim
authors.affiliation: School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu 41566, Republic of Korea.
authors.forename: Ho-Sung
authors.initials: HS
authors.lastname: Yoon
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea.|Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic of Korea.
authors.forename: Jun Hyuck
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea.|Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic of Korea.
authors.forename: Han-Woo
authors.initials: HW
authors.lastname: Kim