Effects of the engineering of a single binding pocket residue on specificity and regioselectivity of hydratases from Lactobacillus Acidophilus
Research output: Contribution to journal › Journal article › Research › peer-review
Fatty acid hydratase (FAH) mediated hydroxy fatty acid (HFA) production is a promising enzymatic route that demands diversification of hydration position to access a broader range of high-value HFAs. FA-HY1 is a promiscuous FAH from Lactobacillus Acidophilus, whereas its homolog from the same organism, FA-HY2, is strict in substrate scope and regioselectivity. Our earlier work demonstrated that three amino acid mutations at the carboxylate end of the substrate (T391/H393/I378 in FA-HY2) shift regioselectivity of FA-HY2 towards that of FA-HY1. Here, we explore alanine 216 of FA-HY2 as a hot-spot residue at the omega end of the substrate. A quadruple mutant (T391S/H393S/I378 P/A216S) demonstrates further shift in regioselectivity towards FA-HY1. Moreover, site-saturation mutagenesis of this residue in FA-HY1 (S218) led to novel variants exhibiting significant changes in regioselectivity for EPA (eicosapentaenoic acid) as substrate, where, unlike wild-type enzyme, 15-OH product was the dominant product (63:37 for wild-type vs. 26:74 for S218I mutant; 12-OH:15-OH). Alterations in conversion levels that indicate pronounced correlation to the exchanged residue type were also detected. A likely explanation for the observed differences is provided based on structural, statistical and kinetic analysis.
Original language | English |
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Article number | 108006 |
Journal | Biochemical Engineering Journal |
Volume | 171 |
ISSN | 1369-703X |
DOIs | |
Publication status | Published - 2021 |
Externally published | Yes |
Bibliographical note
Publisher Copyright:
© 2021 Elsevier B.V.
- Enzyme engineering, Fatty acid hydratase, Hydroxy fatty acid, Regioselectivity, Site saturation mutagenesis
Research areas
ID: 272123120