Structural insights into the evolution of ABH-fold luciferases

Abstract

The alpha/beta-hydrolase (ABH) superfamily is a widespread and functionally versatile protein fold recognized for its ability to adapt to diverse molecular functions across all three domains of life. One such spectacular example of evolutionary adaptation at the ABH-fold represents an acquisition of oxygenolytic luciferase reaction that occurred within the hydrolytic haloalkane dehalogenase family. The molecular details of this evolution remain puzzling. In this work, we determine crystal structures and explore dynamical behavior of a bifunctional ancestral ABH-fold enzyme, highlighting molecular features associated with the transition from hydrolytic to oxygenolytic catalysis at this fold. Structures showed a canonical {beta}-sandwich shielded with a helical cap domain. The catalytic pocket is voluminous enough to accommodate a bulky substrate. Molecular docking showed productive binding of the coelenterazine substrate, as well as the product coelenteramide. Molecular dynamics simulations demonstrated that the coelenterazine entry does not present a major energetic barrier and identified a preferred binding orientation important for oxygenolytic catalysis. Comparisons between ancestral and extant enzymes highlighted specific amino acids and sequence motifs characteristic for oxygenolytic luciferases. Collectively, our results provide an expanded view of the evolutionary transition in which ABH-fold enzymes, originally using water to cleave chemical bonds, adapted to utilize dioxygen for bioluminescence.