Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Biliverdin reductase B (BLVRB) family members are crucial flavin reductases involved in cellular redox balance.
• Recent research indicates BLVRB's role in dictating cellular fate, yet its active site evolution remains poorly understood.
• The BLVRB family presents unique challenges due to an evolving active site and unclear structural-functional relationships.

Purpose of the Study:

• To elucidate the structural and functional basis of the evolutionarily changing BLVRB active site.
• To investigate the mechanism of coenzyme binding and release in BLVRB isoforms.
• To understand the relationship between active site variations, coenzyme release, and enzyme turnover.

Main Methods:

• Utilized a multi-faceted approach combining X-ray crystallography, Nuclear Magnetic Resonance (NMR), and enzyme kinetics.
• Studied three BLVRB isoforms (human, lemur, hyrax) and multiple human BLVRB mutants.
• Measured coenzyme release rates, substrate binding affinity, and hydride transfer kinetics.

Main Results:

• Identified a novel evolutionary mechanism involving arginine side chains forming coenzyme 'clamps' at positions 14 and 78, slowing coenzyme release.
• Observed that weaker substrate binding further impedes coenzyme release, leading to slower turnover numbers.
• Discovered an inverse relationship between coenzyme release rate and substrate turnover across different BLVRB active sites, independent of hydride transfer rate.

Conclusions:

• The evolutionarily dynamic BLVRB active site modulates coenzyme release.
• Coenzyme release is intrinsically coupled to substrate turnover in the BLVRB family.
• Understanding these mechanisms provides insight into enzyme evolution and function.