A combination of mutational and computational scanning guides the design of an artificial ligand-binding controlled lipase.

Allostery, i.e. the control of enzyme activity by a small molecule at a location distant from the enzyme's active site, represents a mechanism essential for sustaining life. The rational design of allostery is a non-trivial task but can be achieved by fusion of a sensory domain, which responds to environmental stimuli with a change in its structure. Hereby, the site of domain fusion is difficult to predict. We here explore the possibility to rationally engineer allostery into the naturally not allosterically regulated Bacillus subtilis lipase A, by fusion of the citrate-binding sensor-domain of the CitA sensory-kinase of Klebsiella pneumoniae. The site of domain fusion was rationally determined based on whole-protein site-saturation mutagenesis data, complemented by computational evolutionary-coupling analyses. Functional assays, combined with biochemical and biophysical studies suggest a mechanism for control, similar but distinct to the one of the parent CitA protein, with citrate acting as an indirect modulator of Triton-X100 inhibition of the fusion protein. Our study demonstrates that the introduction of ligand-dependent regulatory control by domain fusion is surprisingly facile, suggesting that the catalytic mechanism of some enzymes may be evolutionary optimized in a way that it can easily be perturbed by small conformational changes.