Aarhus University Seal

Effects of the engineering of a single binding pocket residue on specificity and regioselectivity of hydratases from Lactobacillus Acidophilus

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Yan Zhang
  • ,
  • Bekir Engin Eser
  • Georgios Kougioumtzoglou
  • ,
  • Zekiye Eser
  • ,
  • Michal Poborsky
  • ,
  • Shigenobu Kishino, Kyoto University
  • ,
  • Michiki Takeuchi, Kyoto University
  • ,
  • Jun Ogawa, Kyoto University
  • ,
  • Peter Kristensen, Aalborg University
  • ,
  • Zheng Guo

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 languageEnglish
Article number108006
JournalBiochemical Engineering Journal
Number of pages8
Publication statusPublished - Jul 2021

    Research areas

  • Enzyme engineering, Fatty acid hydratase, Hydroxy fatty acid, Regioselectivity, Site saturation mutagenesis

See relations at Aarhus University Citationformats

ID: 214749549