Coenzyme-binding pathway on glutamate dehydrogenase suggested from multiple-binding sites visualized by cryo-electron...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Glutamate dehydrogenase (GDH) is a crucial enzyme in cellular metabolism.
• Understanding the cofactor binding mechanism is key to enzyme function.
• GDH exists as a hexamer with distinct domains involved in catalysis and cofactor binding.

Purpose of the Study:

• To visualize the structure of hexameric GDH with nicotinamide adenine dinucleotide phosphate (NADP).
• To investigate the ligand-binding pathways of NADP to the GDH active site.
• To elucidate the role of conformational changes in NADP binding.

Main Methods:

• Cryogenic transmission electron microscopy (cryo-EM) was employed to determine the structure of the GDH-NADP complex.
• Focused classification was used to resolve conformational heterogeneity of the nucleotide-binding domain.
• Enzymatic kinetics were measured to assess the functional relevance of observed binding modes.

Main Results:

• The 2.4 Å resolution cryo-EM structure of the GDH hexamer in complex with NADP was determined.
• Five distinct NADP binding sites were identified within the active-site cleft.
• Three binding sites suggest a primary pathway for NADP entry, while two others appear less common during catalysis.

Conclusions:

• The study provides structural insights into the dynamic binding of NADP to GDH.
• Ligand-induced conformational changes facilitate NADP access to the active site.
• The findings contribute to understanding the regulation of GDH activity.