Switch of Coenzyme Specificity of Mouse Lung Carbonyl Reductase by Substitution of Threonine 38 with Aspartic Acid

Abstract

Mouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase (SDR) family, exhibits coenzyme specificity for NADP(H) over NAD(H). Crystal structure of the enzyme-NADPH complex shows that Thr-38 interacts with the 2'-phosphate of NADPH and occupies the position spatially similar to an Asp residue of the NAD(H)-dependent SDRs that hydrogen-bonds to the hydroxyl groups of the adenine ribose of the coenzymes. Using site-directed mutagenesis, we constructed a mutant mouse lung carbonyl reductase in which Thr-38 was replaced by Asp (T38D), and we compared kinetic properties of the mutant and wild-type enzymes in both forward and reverse reactions. The mutation resulted in increases of more than 200-fold in the Km values for NADP(H) and decreases of more than 7-fold in those for NAD(H), but few changes in the Km values for substrates or in the kcat values of the reactions. NAD(H) provided maximal protection against thermal and urea denaturation of T38D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the single mutation converted the coenzyme specificity from NADP(H) to NAD(H). Calculation of free energy changes showed that the 2'-phosphate of NADP(H) contributes to its interaction with the wild-type enzyme. Changing Thr-38 to Asp destabilized the binding energies of NADP(H) by 3.9-4.5 kcal/mol and stabilized those of NAD(H) by 1.2-1.4 kcal/mol. These results indicate a significant role of Thr-38 in NADP(H) binding for the mouse lung enzyme and provide further evidence for the key role of Asp at this position in NAD(H) specificity of the SDR family proteins.

A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases

“…-4.2 (Liang et al 2007) P. stipitis XR 1K8C (h) K270R, N272D 2.9 9.3 -1.2 (Maddock et al 2015) E. coli CaADH 1KEV (h) G198D, S199V, P201E, Y218A >1 ? -4.6 (Maurer et al 2005) B Log Relative Activity c (Medina et al 2001) A. PCC7119 FDNR 2BSA S223D 0.12 8.1x10 3 -5.4 (Nakanishi et al 1997) M. musculus CR 1CYD T38D 31 1.3x10 3 -0.51 (Paladini et al 2009 (Schepens et al 2000) S. bicolor MDH 7MDH (1CIV) G84D, S85I, R87Q, S88A 12 2.1x10 4 -0.96 (Scrutton et al 1990) E. coli GTR 1GET A179G, A183G, V197E, R198M, K199F, H200D, R204P 8.1 1.8x10 4 -1.5 (Shiraishi et al 1998) N. crassa CbR S920D, R932S 65 7.2x10 4 -2.6 (Takase et al 2014) S. sp. A1-R 3AFN H37N, G38S, R39H, K40V, A41D >1 ?…”

Crystal structure of a carbonyl reductase from Candida parapsilosis with anti‐Prelog stereospecificity

“…The fluorescence in both cases was quenched by the addition of the coenzyme. The data were analyzed by a modified model from a simple Hill equation (Sakoda and Hiromi 1976;Nakanishi et al 1997) with consideration of cooperativity. The ratio of K d values between NADH and NADPH for the S67D/H68D mutant was 0.28, indicating a stronger preference for NADH; in contrast, the ratio became 1.9 for wild type, indicating a weaker binding for NADH (Table 2).…”

Cloning and bacterial expression of monomeric short-chain dehydrogenase/reductase (carbonyl reductase) from CHO-K1 cells

“…2). CHO-CR cDNA is an ortholog of the human CBR3 [40] with high similarity in nucleotide sequence (< 86%) and also had homology to other monomeric CRs (Table 2), but showed very low similarity to the mouse tetrameric CR (, 50% in nucleotide sequence and 30% in the deduced amino-acid sequence) [20]. The deduced amino-acid sequence of CHO-CR cDNA had a typical SDR active site (S-Y-K) and NADP…”