Loop dynamics and evolvability in the TIM-barrel proteins of histidine and tryptophan biosynthesis.

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Recent years have witnessed increasing interest in the role of conformational dynamics in enzyme evolution and engineering. In this context, TIM-barrel proteins are important model systems, as the TIM-barrel is ubiquitous, highly evolvable, supports a diverse range of chemistry, and the active site is typically decorated by one or more mobile loops that play an important role in catalysis and/or regulation of these enzymes. Here, the TIM-barrel proteins of histidine and tryptophan biosynthesis (HisA, TrpF and PriA) stand out, as their active sites are decorated by up to three long mobile loops, that move independently of each other, and the conformations of which are critically important for catalysis. These motions appear to be ligand-gated and occur on sufficiently slow timescales (ms or slower) to be at least partially rate-limiting. In recent work, we combined conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to investigate the conformational behavior of the catalytic loops in these enzymes, and the link between their dynamical behavior and substrate selectivity and catalysis. We have explored the role of loop dynamics in facilitating the bifunctionality of PriA towards substrates ProFAR and PRA (precursors for histidine and tryptophan biosynthesis, respectively), as well as during the real-time evolution of HisA to yield HisA-specific, TrpF-specific and bifunctional variants. Finally, the insights this provides emphasizes the potential of loop engineering as a powerful tool to manipulate the selectivity and substrate scope of the TIM-barrel scaffold, which has historically been one of the most important scaffolds for de novo enzyme design.