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Structure-Function Relationships in the Oligomeric NADPH-Dependent Assimilatory Sulfite Reductase
Isabel Askenasy, Daniel T Murray, Rachel M Andrews
1Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine , University of South Florida , Tampa , Florida 33612 , United States.
Sulfur assimilation relies on NADPH-dependent assimilatory sulfite reductase (SiR). This study reveals that intrinsically disordered regions, particularly SiRHP's N-terminus, are crucial for SiR holoenzyme complex assembly and function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Sulfur assimilation is vital, with sulfite reductase (SiR) catalyzing a key six-electron reduction.
- SiR comprises two subunits: flavin-binding reductase (SiRFP) and iron-containing oxidase (SiRHP).
- While subunit structures are known, their assembly mechanism and the role of disordered regions remain unclear.
Purpose of the Study:
- To investigate the role of intrinsically disordered regions in SiR holoenzyme complex formation.
- To explore how altering amino acids in SiRFP affects its function within the complex.
- To elucidate the molecular interactions driving the assembly of this essential oxidoreductase.
Main Methods:
- Spectroscopic techniques (UV-Vis, CD) and isothermal titration calorimetry were used.
- Proteolytic sensitivity tests and electrospray ionization mass spectrometry were employed.
- Computational analysis predicted intrinsic disorder, complemented by activity assays and SiRFP variant studies.
Main Results:
- SiRHP's N-terminal region exhibits properties of intrinsic disorder and is critical for complex formation.
- Both SiRFP and SiRHP contain internal regions with characteristics of intrinsic disorder.
- Analysis of SiRFP variants provided insights into subunit interactions and holoenzyme assembly.
Conclusions:
- Intrinsically disordered regions, especially SiRHP's N-terminus, play a critical role in SiR holoenzyme assembly.
- Molecular interactions outside the globular cores of SiR subunits drive complex formation.
- This work clarifies the assembly mechanism of a key enzyme in sulfur assimilation.
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