The crystal structure of the aldose reductase.NADPH binary complex

Area of Science:

• Biochemistry
• Structural Biology
• Enymology

Background:

• Aldose reductase (AR) is an NADPH-dependent oxidoreductase implicated in diabetic complications.
• Understanding AR structure and mechanism is crucial for developing effective inhibitors.
• Previous research has focused on AR inhibitors for treating diabetes mellitus complications.

Purpose of the Study:

• To elucidate the 2.75 Å crystal structure of a recombinant human aldose reductase (Cys-298 to Ser mutant) in complex with NADPH.
• To investigate the unusual kinetic properties and inhibitor sensitivity of this specific AR mutant.
• To gain insights into the coenzyme binding mechanism and conformational dynamics of aldose reductase.

Main Methods:

• X-ray crystallography at 2.75 Å resolution.
• Expression and purification of recombinant human aldose reductase (Cys-298 to Ser mutant).
• Co-crystallization with NADPH and structural analysis.

Main Results:

• The crystal structure reveals a β/α-barrel fold with the coenzyme-binding domain at the carboxyl-terminal end.
• NADPH binding induces a significant conformational change, including reorientation of loop 7, locking the coenzyme.
• The mutant enzyme exhibits high Km/high Vmax kinetics and reduced sensitivity to certain inhibitors.
• NADPH binding mode is unusual, resembling FAD-dependent oxidoreductases more than NAD(P)-dependent ones.

Conclusions:

• The determined structure provides a detailed view of NADPH binding in aldose reductase.
• The unusual binding and conformational changes offer mechanistic insights relevant to enzyme function.
• Findings may inform the design of novel aldose reductase inhibitors for diabetic complications.