Identification and engineering on the nonconserved residues of metallo-ß-lactamase-type thioesterase to improve the enzymatic activity.
Biotechnol Bioeng
; 118(12): 4623-4634, 2021 12.
Article
en En
| MEDLINE
| ID: mdl-34427915
ABSTRACT
The standalone metallo-ß-lactamase-type thioesterase (MßL-TE), belongs to the group V nonreducing polyketide synthase agene cluster, catalyzes the rate-limiting step of product releasing. Our work first investigated on the orthologous MßL-TEs from different origins to determine which nonconserved amino acid residues are important to the hydrolysis efficiency. A series of chimeric MßL-TEs were constructed by fragment swapping and site-directed mutagenesis, in vivo enzymatic assay showed that two nonconserved residues A19 and E75 (numbering in HyTE) were critical to the catalytic performance. Protein structure modeling suggested that these two residues are located in different areas of HyTE. A19 is on the entrance to the active sites, whereas E75 resides in the linker between the two ß strands which hold the metal-binding sites. Combining with computational simulations and comparative enzymatic assay, different screening criteria were set up for selecting the variants on the two noncatalytic and nonconserved key residues to improve the catalytic activity. The rational design on A19 and E75 gave five candidates in total, two (A19F and E75Q) of which were thus found significantly improved the enzymatic performance of HyTE. The double-point mutant was constructed to further improve the activity, which was increased by 28.4-fold on product accumulation comparing to the wild-type HyTE. This study provides a novel approach for engineering on nonconserved residues to optimize enzymatic performance.
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1
Banco de datos:
MEDLINE
Asunto principal:
Tioléster Hidrolasas
/
Beta-Lactamasas
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Sitios de Unión
/
Mutagénesis Sitio-Dirigida
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Idioma:
En
Año:
2021
Tipo del documento:
Article