Implication of structural constrains facilitating the functional evolution ofPseudomonas aeruginosaKPR2 into a versatile α-keto acid reductase

Protein structure and function dynamics in molecular evolution are intertwined. Theoretical concepts linking structure, function, and evolution of a protein, while often intuitive, necessitate validation through investigations in real-world systems. Our study empirically explores the implications of multiple panE2 gene copies in an organism, shedding light on the functional roles and evolutionary trajectories of Pseudomonas aeruginosa’s second copy of Ketopantoate reductase (PaKPR2) and its inactivity against the natural substrate Ketopantoate. Evolutionary changes in functional traits were examined around the active site through crystal structures and biochemical analysis. Primarily, apoKPR2 structures reveal a transformed active site cleft, forming a two-sided pocket, while substrate entry is regulated by a molecular gate. Despite cleft closure, molecular interaction properties and activity analysis of PaKPR2 suggest that it can be a versatile keto-acid reductase. However, detailed structural insights from the ligand-bound binary complex of PaKPR2-NADPH and PaKPR2-Ketoisoleucine reveal that the ligand-binding interactions at the active site are conserved and restricted to the molecules of appropriate shape and size that can be accommodated in the available space. Finally, a ternary complex structure, PaKPR2-NADP+-KIC, was solved to understand its functional evolution in terms of the residue microenvironment at the catalytic site. Collectively, the results give detailed visual experiences of different structural perspectives of the protein’s functional evolution.