Computational Design of Multisubstrate Enzyme Specificity

The engineering of multisubstrate enzyme specificity is highly desirable to foster the application of biocatalysis in industry. Here, we develop a multistate computational protein design methodology called multichemical state analysis (MCSA) that can optimize enzyme sequences on large structural ensembles for productive binding of multiple target substrates. Using MCSA, we redesigned E. coli branched-chain amino acid aminotransferase to accept both α-ketoglutarate and the non-native substrate l-histidine. Screening of a designed combinatorial library comprising 32 mutants for enhanced l-histidine transamination activity yielded four variants displaying up to ∼200-fold improvements to kcat/KM. MCSA opens the door to the design of broad-specificity biocatalysts and multisubstrate enzymes displaying tailored specificity.