Computational design of a thermostable de novo biocatalyst for whole cell biotransformations

Abstract

Over the last decades, transformative catalytic strategies have emerged, with biocatalysis cur-rently exerting a substantial influence on the pharmaceutical and fine chemical sectors. Fast pro-gress in the design of efficient enzymatic processes, however, suffers from the lack of readily available, stable, and customizable protein scaffolds that can be adapted to different catalytic functions. Here, we detail the design and experimental characterization of computationally de-signed de novo proteins with a non-natural fold for biocatalytic applications. The initial design and several variants form a helical barrel structure comprised of six antiparallel straight helices con-nected by five loops, creating an open central channel with two accessible cavities. To demon-strate the versatility of this scaffold, we designed variants with catalytic sites positioned at differ-ent locations along the central channel. All designs show high thermal stability and excellent agreement between experimental and calculated scattering profiles from small-angle X-ray scat-tering, while a crystal structure of a surface-redesigned variant confirms the close match be-tween the designed and experimental structures. Importantly, repositioning and engineering the catalytic sites enables substantial modulation of catalytic activity, with the best variant showing an approximately 11-fold increase in catalytic efficiency compared with the original design. Finally, the designs can be used for whole-cell biotransformations and tolerate up to 20% organic sol-vent. These results establish a stable de novo protein scaffold with tunable functional sites, offer-ing a versatile platform for biocatalysis, biosensing, and biosynthetic systems.