The engineering of multi-substrate enzyme specificity is highly desirable to foster the application of biocatalysis in industry. Here, we develop a multistate computational protein design methodology called multi-chemical 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 multi-substrate enzymes displaying tailored specificity.
ID: DddCjJY34
Submitter: Antony St-Jacques
Submission Date: May 7, 2020, 7:42 a.m.
Version: 1
Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
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Percent Identity | Matching Chains | Protein | Accession | Entry Name |
---|---|---|---|---|
300.0 | A,B,C | Branched-chain-amino-acid aminotransferase | P0AB82 | ILVE_ECO57 |
300.0 | A,B,C | Branched-chain-amino-acid aminotransferase | P0AB81 | ILVE_ECOL6 |
300.0 | A,B,C | Branched-chain-amino-acid aminotransferase | P0AB80 | ILVE_ECOLI |
294.29999999999995 | A,B,C | Branched-chain-amino-acid aminotransferase | P0A1A6 | ILVE_SALTI |
294.29999999999995 | A,B,C | Branched-chain-amino-acid aminotransferase | P0A1A5 | ILVE_SALTY |