Computational Design of Multisubstrate Enzyme Specificity

Abstract

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.

Submission Details

ID: DddCjJY34

Submitter: Antony St-Jacques

Submission Date: May 7, 2020, 7:42 a.m.

Version: 1

Publication Details
Antony D. St-Jacques, Marie-Ève C. Eyahpaise, Roberto A. Chica,ACS Catalysis (2019) Computational Design of Multisubstrate Enzyme Specificity
Additional Information

Study Summary

Number of data points 65
Proteins Branched-chain-amino-acid aminotransferase
Unique complexes 4
Assays/Quantities/Protocols Experimental Assay: kcat α-Ketoglutarate ; Experimental Assay: Km α-Ketoglutarate ; Experimental Assay: kcat/Km α-Ketoglutarate ; Experimental Assay: Ki α-Ketoglutarate ; Experimental Assay: Km L-Histidine ; Experimental Assay: kcat L-Histidine ; Experimental Assay: kcat/Km L-Histidine ; Experimental Assay: Km L-Leucine ; Experimental Assay: kcat L-Leucine ; Experimental Assay: kcat/Km L-Leucine ; Experimental Assay: Km L-Threonine ; Experimental Assay: kcat L-Threonine ; Experimental Assay: kcat/Km L-Threonine
Libraries Apparent Kinetic Parameters of E. coli BCAT and Its Mutants for Transamination of Various Amino-Acid Donors with α-Ketoglutarate as the Acceptor

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1A3G 1998-01-21T00:00:00+0000 2.5 BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE FROM ESCHERICHIA COLI
1I1M 2001-02-02T00:00:00+0000 2.4 CRYSTAL STRUCTURE OF ESCHERICHIA COLI BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE.
1I1K 2001-02-02T00:00:00+0000 2.1 CRYSTAL STRUCTURE OF ESCHELICHIA COLI BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE.
1I1L 2001-02-02T00:00:00+0000 2.4 CRYSTAL STRUCTURE OF ESCHELICHIA COLI BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE.
1IYE 2002-08-07T00:00:00+0000 1.82 CRYSTAL STRUCTURE OF ESCHELICHIA COLI BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE
1IYD 2002-08-07T00:00:00+0000 2.15 CRYSTAL STRUCTURE OF ESCHELICHIA COLI BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
294.29999999999995 A,B,C Branched-chain-amino-acid aminotransferase P0A1A5 ILVE_SALTY
294.29999999999995 A,B,C Branched-chain-amino-acid aminotransferase P0A1A6 ILVE_SALTI
300.0 A,B,C Branched-chain-amino-acid aminotransferase P0AB80 ILVE_ECOLI
300.0 A,B,C Branched-chain-amino-acid aminotransferase P0AB81 ILVE_ECOL6
300.0 A,B,C Branched-chain-amino-acid aminotransferase P0AB82 ILVE_ECO57