Abstract
Biliverdin reductase B (BLVRB) is a redox regulator that catalyzes nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductions of multiple substrates, including flavins and biliverdin-β. BLVRB has emerging roles in redox regulation and post-translational modifications, highlighting its importance in various physiological contexts. In this study, we explore the structural and functional differences between human BLVRB and its hyrax homologue, focusing on evolutionary adaptations at the active site and allosteric regions. Using NMR spectroscopy, we compared coenzyme binding, catalytic turnover, and dynamic behavior between the two homologues. Despite lacking the arginine "clamp" present in human BLVRB, hyrax BLVRB still undergoes conformational changes in response to the oxidative state of the coenzyme. Mutations at the allosteric site (position 164) show that threonine at this position enhances coenzyme discrimination and allosteric coupling in human BLVRB, while hyrax BLVRB does not display the same allosteric effects. Relaxation experiments revealed distinct dynamic behaviors in hyrax BLVRB, with increased flexibility in its holo form due to the absence of the clamp. Our findings suggest that the evolutionary loss of the active site clamp and modifications at position 164 in hyrax BLVRB alter the enzyme's conformational dynamics and coenzyme interactions. Identified similarities and differences underscore how key regions modulate catalytic efficiency and suggest that coenzyme isomerization may represent the rate-limiting step in both homologues.