Dr Caitlin Hatton

Talk: Engineering catalysis through dimer interface mutations

Precise, tunable control over enzyme activity is a central goal in synthetic and engineering biology. While active-site mutations are commonly used to regulate or disrupt catalysis, predicting the effects of non-active-site mutations remains challenging. Such allosteric mutations, however, offer additional opportunities for controlling enzyme activity. Fluoroacetate dehalogenase (FAcD) is a homodimeric enzyme that exhibits half-of-the-sites reactivity, in which catalysis in one active site modulates the other, indicating strong functional coupling between protomers.

Previous work has implicated the dimer interface in allosteric communication. Here we show that single, conservative mutations at the dimer interface are sufficient to dramatically alter enzyme activity without disrupting overall structure or oligomeric state. These substitutions produce opposing functional outcomes, with one enhancing catalytic turnover and the other halting catalysis. High-resolution crystal structures (0.85 Å) enable visualisation of hydrogen atoms, hydrogen-bonding networks that drive catalysis, and low-population conformational states.

Combined hydrogen bond and rigidity analyses show that these mutations rewire the hydrogen-bond networks, providing a mechanistic explanation for the observed change in activity. Our work highlights how minimal, rational perturbations outside the active site can be leveraged to design enzymes with tailored functional properties.