Casadevall_2023_bioRxiv__

Reference

Title : Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase - Casadevall_2023_bioRxiv__
Author(s) : Casadevall G , Pierce C , Guan B , Iglesias-Fernandez J , Lim HY , Greenberg LR , Walsh ME , Shi K , Gordon W , Aihara H , Evans RL , Kazlauskas R , Osuna S
Ref : Biorxiv , : , 2023
Abstract :

Hydroxynitrile lyase from rubber tree ( Hb HNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of Hb HNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. Hb HNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both Hb HNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

PubMedSearch : Casadevall_2023_bioRxiv__
PubMedID: 37662272
Gene_locus related to this paper: hevbr-hnl

Related information

Gene_locus hevbr-hnl
Structure hevbr-hnl    8EUO

Citations formats

Casadevall G, Pierce C, Guan B, Iglesias-Fernandez J, Lim HY, Greenberg LR, Walsh ME, Shi K, Gordon W, Aihara H, Evans RL, Kazlauskas R, Osuna S (2023)
Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase
Biorxiv :

Casadevall G, Pierce C, Guan B, Iglesias-Fernandez J, Lim HY, Greenberg LR, Walsh ME, Shi K, Gordon W, Aihara H, Evans RL, Kazlauskas R, Osuna S (2023)
Biorxiv :