Effects of the engineering of a single binding pocket residue on specificity and regioselectivity of hydratases from Lactobacillus Acidophilus
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Effects of the engineering of a single binding pocket residue on specificity and regioselectivity of hydratases from Lactobacillus Acidophilus. / Zhang, Yan; Eser, Bekir Engin; Kougioumtzoglou, Georgios; Eser, Zekiye; Poborsky, Michal; Kishino, Shigenobu; Takeuchi, Michiki; Ogawa, Jun; Kristensen, Peter; Guo, Zheng.
In: Biochemical Engineering Journal, Vol. 171, 108006, 2021.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Effects of the engineering of a single binding pocket residue on specificity and regioselectivity of hydratases from Lactobacillus Acidophilus
AU - Zhang, Yan
AU - Eser, Bekir Engin
AU - Kougioumtzoglou, Georgios
AU - Eser, Zekiye
AU - Poborsky, Michal
AU - Kishino, Shigenobu
AU - Takeuchi, Michiki
AU - Ogawa, Jun
AU - Kristensen, Peter
AU - Guo, Zheng
N1 - Publisher Copyright: © 2021 Elsevier B.V.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - Enzyme engineering
KW - Fatty acid hydratase
KW - Hydroxy fatty acid
KW - Regioselectivity
KW - Site saturation mutagenesis
U2 - 10.1016/j.bej.2021.108006
DO - 10.1016/j.bej.2021.108006
M3 - Journal article
AN - SCOPUS:85103708005
VL - 171
JO - Biochemical Engineering Journal
JF - Biochemical Engineering Journal
SN - 1369-703X
M1 - 108006
ER -
ID: 272123120