Borrowing the fold, building the site: non-catalytic protein scaffolds in the age of computational metalloenzyme design

The design of novel metalloenzymes has gained its motivation and momentum less from what enzymes already do well rather than from what they do not do at all, that is, the organic chemistry outside any biosynthetic pathways. In search for starting points to create proteins that are active in new-to-Nature transformations, computational design in particular offers powerful solutions by supplying folds made to order en route to inventing tailor-made de novo biocatalysts. Nature, however, rarely invents; it decorates. A large share of biological catalysis sits on backbones whose original role was structural or recognition-based, and in which the metal center occupies no more than a handful of side chains within an otherwise unchanged fold. In this Perspective we make the case that especially in systems addressing abiological catalysis, promising and successful approaches oftentimes adapt non-catalytic scaffolds as their foundation, taking binding modules, small disulfide-locked domains and synthetic miniproteins as hosts. We organize the discussion around the minimal functional site: the smallest ligand set that, placed on a rigid and pre-organized backbone, holds a metal in a defined geometry. Whether a given scaffold is able to offer such a site is a structural question before it is a chemical one. We examine how far current structure prediction can guide the placement of such sites, where its confidence scores mislead, and which spectroscopic and computational evidence remains indispensable. Scaffold repurposing, we conclude, offers organic synthesis a route to catalysts that enzymology alone would not deliver.