Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M²⁺_R), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M²⁺_A) at 2.4 Å distance. Bidentate coordination of a ketoacid substrate to M²⁺_A affords the overall lowest energy complex, which drives the transition from M²⁺_R to M²⁺_A. While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures.

Diastereomers are characterised by an intrinsic energy difference, and thermodynamics dictate their distribution within a dynamic equilibrium. The characteristic mechanistic reversibility and non-ideal stereoselectivity of catalysts therefore simultaneously promote both synthesis and epimerization of products during the formation of diastereomers. This feature can even result in the thermodynamic inversion of a chiral centre against the catalyst's stereoselectivity. Here, we provide a comprehensive experimental and theoretical study of factors that govern thermodynamic epimerization in catalysis, using enzymes as example. Our analysis highlights, that the deduction of a catalyst's stereoselectivity based on the absolute configuration of the isolated product constitutes a potential pitfall. The selective formation of either the thermodynamic-, or the kinetic product is less determined by the catalyst, but rather by the reaction conditions. Next to low temperatures, a high maximal extent of conversion was identified to promote kinetically controlled conditions. For bimolecular reactions, conversions can be conveniently modulated via the use of one substrate in excess. Quantum mechanical calculations accurately predicted the diastereomeric excess under equilibrium conditions, which opens the prospect of a rational choice between thermodynamic and kinetic reaction control at an early stage of process design. Our findings are of critical importance for multi-step syntheses of stereocomplex molecules via catalytic cascade reactions or artificial metabolic pathways, as the final stereochemistry may be determined by the absolute configuration of the product that is overall lowest in energy.