Structural insights into the bifunctional enzyme human FAD synthase

Area of Science:

• Enzymology
• Structural Biology
• Biochemistry

Background:

• Human flavin adenine dinucleotide synthase (hFADS) is a bifunctional enzyme.
• It possesses both flavin mononucleotide adenylyltransferase and pyrophosphatase activities.
• Understanding its domain contributions is crucial for elucidating enzyme mechanisms.

Purpose of the Study:

• To determine the crystal structure of full-length human flavin adenine dinucleotide synthase 2 (hFADS2) and its C-terminal PAPS domain.
• To dissect the structural determinants of enzymatic activities within different hFADS isoforms.
• To elucidate the functional roles of individual domains in hFADS activity.

Main Methods:

• X-ray crystallography of full-length hFADS2 and its C-terminal PAPS domain.
• Structural and functional characterization of complete and truncated hFADS constructs.
• Biochemical assays to assess enzymatic activities.

Main Results:

• The crystal structure of hFADS2 in complex with flavin adenine dinucleotide (FAD) was determined.
• The C-terminal domain catalyzes FAD synthesis and binds FAD tightly.
• The N-terminal molybdopterin-binding and KH domains form the minimal active site for FAD hydrolysis.
• hFADS2 forms a stable dimer, with the KH domain facilitating the hydrolytic active site.

Conclusions:

• The C-terminal domain is responsible for FAD synthesis.
• The N-terminal domains are essential for FAD hydrolysis.
• Domain interplay within the hFADS2 dimer dictates its bifunctional catalytic activities.