Structure of monkey dimeric dihydrodiol dehydrogenase in complex with isoascorbic acid.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Mammalian dimeric dihydrodiol dehydrogenase (DD) is functionally identical to NADP+-dependent D-xylose dehydrogenase.
• DD shares structural similarities with prokaryotic glucose-fructose oxidoreductase (GFO) and 1,5-anhydro-D-fructose reductase (AFR), but exhibits distinct coenzyme-binding and catalytic residues.
• Dimeric DD demonstrates a high affinity for NADP(H) and possesses broad specificity for various substrates, including sugars and hydrophobic xenobiotics.

Purpose of the Study:

• To elucidate the three-dimensional structure of monkey dimeric DD complexed with an inhibitor.
• To propose a coenzyme binding mode and understand the roles of active site residues using molecular modeling and site-directed mutagenesis.
• To identify key residues responsible for high coenzyme affinity and broad substrate specificity.

Main Methods:

• X-ray crystallography was used to determine the structure of monkey dimeric DD with isoascorbic acid at 2.59 angstroms resolution.
• Molecular modeling of coenzyme binding was performed.
• Site-directed mutagenesis was employed to investigate the function of specific amino acid residues.

Main Results:

• The crystal structure revealed the complex of monkey dimeric DD with isoascorbic acid.
• Key residues (Arg37, Arg41, His76, His79) in the coenzyme-binding domain were identified, with Arg37 and Arg41 crucial for high NADP(H) affinity.
• Mutations in residues Trp125, Phe154, Trp254, and Phe279 affected substrate specificity and inhibitor potency, while Lys97 mutation impacted catalytic efficiency.

Conclusions:

• Specific arginine and histidine residues are critical for the high affinity of dimeric DD for NADP(H).
• Residues Trp125, Phe154, Trp254, and Phe279 contribute to the enzyme's broad substrate specificity and inhibitor binding.
• Lys97 plays a significant role in the catalytic mechanism of dimeric DD.