Unexpected diastereoselective chemistry on a 2D protein surface

Enantioselective enzymes feature structured catalytic sites positioned within binding pockets. In contrast, widely occurring flat protein surfaces, which have been suggested to have emerged early in ancestral protein evolution as solvent-exposed β sheets, appear unlikely to catalyze asymmetric reactions because they lack the necessary scaffold for interacting with substrates in three dimensions. At the near-flat, water-accessible surface within the α-hemolysin transmembrane pore, we now demonstrate remarkable diastereoselectivity in hemithioacetal formation between a cysteine side chain and a series of aldehyde substrates. After protein surface remodeling by mutagenesis, diastereomeric ratios of up to 95:5 (kinetic control) and 98:2 (thermodynamic control) were achieved with a range of aromatic aldehydes. Molecular dynamics simulations confirmed asymmetric interactions between adducts and nearby side chains in a two-dimensional plane. Our findings indicate that flat protein surfaces can scaffold stereoselective chemistry, thereby expanding the designable protein space for catalyst engineering and providing insight into the origin of selective enzymes. • Excellent reaction diastereoselectivities achieved on a 2D protein surface • Strong kinetic and thermodynamic diastereomeric ratios observed for >10 substrates • Molecular dynamics simulations confirm absence of binding pockets • Absence of binding pocket permits substrate and reaction promiscuity Enzymes excel at producing biologically and economically important compounds with defined stereochemistries. Conventional wisdom maintains that stereoselectivity in enzymes is conferred by densely packed binding pockets capable of interacting with substrates in three dimensions. In contrast, flat protein surfaces seem to be an improbable choice for asymmetric chemistry. Therefore, flat protein surfaces might represent a vast and largely untapped protein space for asymmetric catalysis. In this work, we demonstrate stereoselective formation of a stereogenic center on the flat, internal surface of the α-hemolysin pore. Starting from a non-selective protein surface, excellent stereoselectivity was achieved by introducing three mutations proximal to the reactive center. Molecular dynamics simulations confirmed the absence of a binding pocket on the engineered protein surface, permitting both substrate and reaction promiscuity. These findings could inform future advances in asymmetric catalyst design. The flat, internal β-barrel surface of an engineered α-hemolysin pore can support substantial stereoselectivity. This finding expands the designable chemical space of catalysts and provides insights into the origin of selective enzymes.