Structure-Based Design of Versatile Biosensors for Small Molecules Based on the PAS Domain of a Thermophilic Histidine Kinase

The development of biosensors for in vitro quantification
of small molecules such as metabolites or man-made chemicals is still
a major challenge. Here we show that engineered variants of the sensory
PAS domain of the histidine kinase CitA of the thermophilic bacterium Geobacillus thermoleovorans represent promising alternatives
to established biorecognition elements. By combining binding site
grafting and rational design we constructed protein variants binding l-malate, ethylmalonate, or the aromatic compound phthalate
instead of the native ligand citrate. Due to more favorable entropy
contributions, the wild-type protein and its engineered variants exhibited
increased (nano- to micromolar) affinities and improved enantioselectivity
compared to CitA homologues of mesophilic organisms. Ligand binding
was directly converted into an optical signal that was preserved after
immobilization of the protein. A fluorescently labeled variant was
used to quantify ethylmalonate, an urinary biomarker for ethylmalonic
encephalopathy, in synthetic urine, thereby demonstrating the applicability
of the sensor in complex samples.