title: Catalytic properties and crystal structure of thermostable NAD(P)H-dependent carbonyl reductase from the hyperthermophilic archaeon Aeropyrum pernix K1.
abstract: A gene encoding NAD(P)H-dependent carbonyl reductase (CR) from the hyperthermophilic archaeon Aeropyrum pernix K1 was overexpressed in Escherichia coli. Its product was effectively purified and characterized. The expressed enzyme was the most thermostable CR found to date; the activity remained at approximately 75% of its activity after incubation for 10min up to 90°C. In addition, A. pernix CR exhibited high stability at a wider range of pH values and longer periods of storage compared with CRs previously identified from other sources. A. pernix CR catalyzed the reduction of various carbonyl compounds including ethyl 4-chloro-3-oxobutanoate and 9,10-phenanthrenequinone, similar to the CR from thyroidectomized (Tx) chicken fatty liver. However, A. pernix CR exhibited significantly higher Km values against several substrates than Tx chicken fatty liver CR. The three-dimensional structure of A. pernix CR was determined using the molecular replacement method at a resolution of 2.09Å, in the presence of NADPH. The overall fold of A. pernix CR showed moderate similarity to that of Tx chicken fatty liver CR; however, A. pernix CR had no active-site lid unlike Tx chicken fatty liver CR. Consequently, the active-site cavity in the A. pernix CR was much more solvent-accessible than that in Tx chicken fatty liver CR. This structural feature may be responsible for the enzyme's lower affinity for several substrates and NADPH. The factors contributing to the much higher thermostability of A. pernix CR were analyzed by comparing its structure with that of Tx chicken fatty liver CR. This comparison showed that extensive formation of the intrasubunit ion pair networks, and the presence of the strong intersubunit interaction, is likely responsible for A. pernix CR thermostability. Site-directed mutagenesis showed that Glu99 plays a major role in the intersubunit interaction. This is the first report regarding the characteristics and three-dimensional structure of hyperthermophilic archaeal CR.
authors.affiliation: Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto 869-1404, Japan.
authors.forename: Yudai
authors.initials: Y
authors.lastname: Fukuda
authors.affiliation: Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Sakuraba
authors.affiliation: Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto 869-1404, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Araki
authors.affiliation: Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, 5-16-1, Ohmiya, Asahi-ku, Osaka 535-8585, Japan.
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
authors.affiliation: Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto 869-1404, Japan. Electronic address: kyoneda@agri.u-tokai.ac.jp.
authors.forename: Kazunari
authors.initials: K
authors.lastname: Yoneda
mesh_terms: D041522:Aeropyrum / Q000201:enzymology* / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002645:Chickens; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D019766:Genes, Archaeal; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D019843:Archaeal Proteins; D030161:Avian Proteins; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases
keywords: (S)-CHBE; 9,10-PQ; 9,10-phenanthrenequinone; Aeropyrum pernix K1; Archaea; COBE; CR; Enzyme stability; Hyperthermophile; NAD(P)H-dependent carbonyl reductase; SDR; carbonyl reductase; ethyl (S)-4-chloro-3-hydroxybutanoate; ethyl 4-chloro-3-oxobutanoate; short-chain dehydrogenase/reductase