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.