Crystal structure of NAD-dependent formate dehydrogenase

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• NAD-dependent formate dehydrogenase (FDH) from Pseudomonas sp. 101 is a key enzyme in formate metabolism.
• Understanding the structure of FDH is crucial for elucidating its catalytic mechanism and NAD binding.
• Previous studies have characterized FDH activity but lacked detailed structural information.

Purpose of the Study:

• To determine the three-dimensional structure of the ternary complex of NAD-dependent formate dehydrogenase (FDH) from Pseudomonas sp. 101.
• To identify the NAD binding site and active center residues.
• To compare the structure of FDH with other dehydrogenases and classify it within a new subfamily.

Main Methods:

• Crystallization of the enzyme-NAD-azide ternary complex.
• X-ray diffraction data collection and processing.
• Multiple isomorphous replacement phasing and electron density map calculation.
• Refinement of the atomic model using CORELS and PROLSQ packages.
• Structural comparison with other known dehydrogenase structures.

Main Results:

• The FDH ternary complex crystallized in space group P2(1)2(1)2(1).
• The refined atomic model revealed a dimeric structure with each subunit comprising coenzyme and catalytic domains.
• The active center and NAD binding site are located at the interface between the two domains.
• A unique beta sheet structure in the coenzyme binding domain and the dimeric quaternary structure distinguish FDH from other dehydrogenases, defining a new subfamily (P-oriented dimer).
• Conserved residues in the nucleotide binding region were identified and discussed.

Conclusions:

• The determined structure provides atomic-level insights into the NAD binding and catalytic mechanism of FDH.
• FDH represents a novel subfamily of NAD-dependent dehydrogenases, the P-oriented dimer, based on its distinct quaternary structure.
• The findings contribute to a broader understanding of dehydrogenase evolution and diversity.