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.
Highlights
In several animal lungs [1,2,3,4,5], carbonyl reductase (NADPH) (CR1; EC 1.1.1.184) catalyzes the reduction of various aliphatic and aromatic carbonyl compounds and the oxidation of secondary alcohols and aliphatic aldehydes
The NAD(H)-dependent enzymes have an invariant Gly-X-Gly-X-XGly sequence and an acidic residue at the C terminus of the second  strand, whereas another consensus sequence has been proposed for the NADP(H)-dependent enzymes in which the third Gly of the NAD(H)-binding fingerprint is replaced by Ala, and a positively charged residue is usually included in the neighborhood of the C terminus of the ␣ fold [15,16,17,18,19,20]
We have recently solved the three-dimensional structure of mouse lung CR, which exhibits high coenzyme preference for NADP(H) over NAD(H), and have shown that Lys-17 and Arg39, which exist before the second Gly of the Gly-rich pattern and at the C terminus of the ␣ fold, respectively, are responsible for the coenzyme specificity [21]
Summary
Materials—Pyridine nucleotide coenzymes and pI markers were obtained from Oriental Yeast (Tokyo, Japan); restriction and DNA-modifying enzymes were from Nippon Gene (Tokyo, Japan) and Takara Shuzou (Osaka, Japan); Escherichia coli cells and plasmids were from Stratagene. The kinetic constants in the CHX oxidation with a fixed saturated concentration of substrate or coenzyme were directly determined by fit to the Michaelis-Menten equation. Thermal and Urea Stability Study—For thermal inactivation, the enzymes (0.1 mg/ml) were incubated at 34 °C in 0.1 M potassium phosphate buffer, pH 7.0, containing 0.15 M KCl and 0.1% bovine serum albumin in the presence or absence of NAD(P)(H) or substrate. For the denaturation by urea, the enzyme (30 g/ml) was incubated at 25 °C for 2 h in 0.1 M Tris-HCl buffer, pH 8.0, containing 0 –5 M urea in the presence or absence of NAD(P)ϩ or substrate. The dehydrogenase activity was expressed as a percentage of that in the absence of urea This assay is unaffected by the presence of up to 0.1 M urea. This single-subunit model with the coenzyme NADP(H) was refined through the energy minimization routine incorporated in the program X-PLOR [29]
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