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.