# Directed evolution of a TNA polymerase identifies independent paths to fidelity and catalysis

## TL;DR

Scientists evolved a polymerase to synthesize TNA and found that accuracy and efficiency in enzyme function can develop independently.

## Contribution

The study reveals that fidelity and catalysis in a TNA polymerase evolved through distinct structural and biochemical pathways.

## Key findings

- Fidelity and catalytic activity in the polymerase evolved independently through different structural adaptations.
- Crystal structures of enzyme intermediates show distinct discrimination events for ground-state and transition-state substrates.
- The findings provide a model for understanding how new enzyme functions emerge in synthetic biology.

## Abstract

Directed evolution facilitates functional adaptations through stepwise changes in sequence that alter protein structure. While most campaigns yield solutions that maintain the framework of a rigid protein architecture, a few have produced enzymes with more notable structural differences. One example is a polymerase that was evolved to synthesize threose nucleic acid (TNA) with near-natural activity. Understanding how this enzyme arose provides a model for studying pathways that guide enzymes toward more productive regions of the fitness landscape. Here, we trace the evolutionary trajectory of an unnatural polymerase by solving crystal structures of key intermediates along the pathway and evaluating their biochemical activity. Contrary to the view that fidelity is a product of increased catalytic efficiency, we find that accuracy and catalysis are decoupled activities guided by separate ground-state and transition-state discrimination events. Together, these results offer a glimpse into the forces responsible for shaping the emergence of new enzyme functions.

Engineering polymerases to synthesize alternative genetic polymers remains a challenging problem in synthetic biology. The current study offers insights into the structural and biochemical changes responsible for improving the fidelity and catalytic activity of a laboratory evolved TNA polymerase.