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

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.

[1]  I. Tsigelny,et al.  Adding a positive charge at residue 46 of Drosophila alcohol dehydrogenase increases cofactor specificity for NADP+ , 1994, FEBS letters.

[2]  J. Hoch,et al.  Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement. , 1992, The Journal of biological chemistry.

[3]  D. Ghosh,et al.  The refined three-dimensional structure of 3α,20β-hydroxysteroid dehydrogenase and possible roles of the residues conserved in short-chain dehydrogenases , 1994 .

[4]  N S Scrutton,et al.  Creation of an NADP-dependent pyruvate dehydrogenase multienzyme complex by protein engineering. , 1993, Biochemistry.

[5]  Y. Mély,et al.  Determinants of coenzyme specificity in glyceraldehyde-3-phosphate dehydrogenase: role of the acidic residue in the fingerprint region of the nucleotide binding fold. , 1993, Biochemistry.

[6]  A. Bairoch,et al.  cis-diol dehydrogenases encoded by the TOL pWW0 plasmid xylL gene and the Acinetobacter calcoaceticus chromosomal benD gene are members of the short-chain alcohol dehydrogenase superfamily. , 1992, European journal of biochemistry.

[7]  H. Jörnvall,et al.  Pseudomonas 3β‐hydroxysteroid dehydrogenase , 1991 .

[8]  W. Pangborn,et al.  Structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase at 2.20 A resolution. , 1995, Structure.

[9]  R. Sheridan,et al.  Role of lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase. , 1990, Biochemistry.

[10]  A. Hara,et al.  Localization of pulmonary carbonyl reductase in guinea pig and mouse: enzyme histochemical and immunohistochemical studies. , 1990, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[11]  T. Nakayama,et al.  Characterization of pulmonary carbonyl reductase of mouse and guinea pig. , 1986, Biochimica et biophysica acta.

[12]  G Labesse,et al.  Structural comparisons lead to the definition of a new superfamily of NAD(P)(H)-accepting oxidoreductases: the single-domain reductases/epimerases/dehydrogenases (the 'RED' family). , 1994, The Biochemical journal.

[13]  Z. Chen,et al.  Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase. , 1991, European journal of biochemistry.

[14]  Nigel S. Scrutton,et al.  Redesign of the coenzyme specificity of a dehydrogenase by protein engineering , 1990, Nature.

[15]  M. Nakagawa,et al.  Kinetic mechanism of pulmonary carbonyl reductase. , 1988, The Biochemical journal.

[16]  M. Karplus,et al.  Crystallographic R Factor Refinement by Molecular Dynamics , 1987, Science.

[17]  M. Nakanishi,et al.  Cloning and sequence analysis of a cDNA encoding tetrameric carbonyl reductase of pig lung. , 1993, Biochemical and biophysical research communications.

[18]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[19]  M. Nakanishi,et al.  Cloning, Expression and Tissue Distribution of Mouse Tetrameric Carbonyl Reductase , 1995 .

[20]  T. Hashimoto,et al.  cDNA cloning of rat liver 2,4-dienoyl-CoA reductase. , 1990, Biochimica et biophysica acta.

[21]  T. Nonaka,et al.  Crystal structures of the binary and ternary complexes of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli. , 1996, Biochemistry.

[22]  T. Nakayama,et al.  Purification and characterization of pig lung carbonyl reductase. , 1992, Archives of biochemistry and biophysics.

[23]  T. Flynn,et al.  Studies on Human Aldose Reductase. , 1995, The Journal of Biological Chemistry.

[24]  M. Nakanishi,et al.  Involvement of two basic residues (Lys-17 and Arg-39) of mouse lung carbonyl reductase in NADP(H)-binding and fatty acid activation: site-directed mutagenesis and kinetic analyses. , 1996, Journal of biochemistry.

[25]  R. Wierenga,et al.  INTERACTION OF PYROPHOSPHATE MOIETIES WITH ALPHA-HELIXES IN DINUCLEOTIDE BINDING-PROTEINS , 1985 .

[26]  P. Terpstra,et al.  Prediction of the Occurrence of the ADP-binding βαβ-fold in Proteins, Using an Amino Acid Sequence Fingerprint , 1986 .

[27]  H. Jörnvall,et al.  Prokaryotic 20β‐hydroxysteroid dehydrogenase is an enzyme of the ‘short‐chain, non‐metalloenzyme’ alcohol dehydrogenase type , 1990, FEBS letters.

[28]  M. Nakanishi,et al.  Crystal structure of the ternary complex of mouse lung carbonyl reductase at 1.8 A resolution: the structural origin of coenzyme specificity in the short-chain dehydrogenase/reductase family. , 1997, Structure.

[29]  M. Nakanishi,et al.  Cloning, expression and tissue distribution of mouse tetrameric carbonyl reductase. Identity with an adipocyte 27-kDa protein. , 1995, European journal of biochemistry.

[30]  Yasuyuki Yamada,et al.  Two tropinone reductases with different stereospecificities are short-chain dehydrogenases evolved from a common ancestor. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[31]  T. Nakayama,et al.  Purification and characterization of a novel pyrazole-sensitive carbonyl reductase in guinea pig lung. , 1982, Archives of biochemistry and biophysics.

[32]  C. Hollenberg,et al.  Amino acid substitutions in the yeast Pichia stipitis xylitol dehydrogenase coenzyme-binding domain affect the coenzyme specificity. , 1995, European journal of biochemistry.

[33]  D. Sem,et al.  Interaction with arginine 597 of NADPH-cytochrome P-450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and NADH. , 1993, Biochemistry.

[34]  M. Nakagawa,et al.  Isolation of proteins with carbonyl reductase activity and prostaglandin-9-ketoreductase activity from chicken kidney. , 1982, Journal of biochemistry.

[35]  Short-chain dehydrogenases/reductases (SDR). , 1995 .

[36]  J. Grima-Pettenati,et al.  Site-directed mutagenesis of a serine residue in cinnamyl alcohol dehydrogenase, a plant NADPH-dependent dehydrogenase, affects the specificity for the coenzyme. , 1995, Biochemistry.

[37]  J M Whiteley,et al.  Structural and mechanistic characteristics of dihydropteridine reductase: a member of the Tyr-(Xaa)3-Lys-containing family of reductases and dehydrogenases. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[38]  R. Horton,et al.  In Vitro Recombination and Mutagenesis of DNA : SOEing Together Tailor-Made Genes. , 1993, Methods in molecular biology.

[39]  J. Samuelson,et al.  Cloning and expression of an NADP(+)-dependent alcohol dehydrogenase gene of Entamoeba histolytica. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[40]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[41]  I. Maurer-Fogy,et al.  Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli. , 1991, The Journal of biological chemistry.