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Rational Engineering of Hydratase from Lactobacillus Acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

preprint
submitted on 02.06.2019 and posted on 03.06.2019 by Bekir Engin Eser, Michal Poborsky, Rongrong Dai, Shigenobu Kishino, Anita Ljubic, Michiki Takeuchi, Charlotte Jacobsen, Jun Ogawa, Peter Kristensen, Zheng Guo
Enzymatic conversion of abundant fatty acids (FAs) through fatty acid hydratases (FAHs) presents an environment-friendly and efficient route for production of high-value hydroxy fatty acids (HFAs). However, a limited diversity was achieved among HFAs to date with respect to chain length and hydroxy group position, due to high substrate- and regio-selectivity of hydratases. In this study, we compared two highly similar FAHs from Lactobacillus acidophilus: FA-HY2 has narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilize longer chain substrates and hydrate various double bond positions. We reveal three active-site residues that play remarkable role in directing substrate specificity and regioselectivity of hydration. When these residues on FA-HY2 are mutated to the corresponding residues in FA-HY1, we observe a significant expansion of substrate scope and distinct shift and enhancement in hydration of double bonds towards -end of FAs. A three-residue mutant of FA-HY2 (TM-FA-HY2; T391S/H393S/I378P) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting ratio of the HFA product regioisomers (10-OH:13-OH) from 99:1 to 12:88. Although kcat values are still low in comparison to wild-type FA-HY1, TM-FA-HY2 exhibited about 60-fold increase in catalytic efficiency (kcat/Km) compared to wild-type FA-HY2. Important changes in regioselectivity were also observed with mutant enzymes for arachidonic acid and C18 PUFAs. In addition, TM-FA-HY2 variant exhibited high conversion rates for cis-5, cis-8, cis-11, cis-14, cis-17-eicosapentaenoic acid (EPA) and cis-8, cis-11, cis-14-eicosatrienoic acid (ETA) at preparative scale and enabled isolation of 12-hydroxy products with moderate yields. Furthermore, we demonstrated the potential of microalgae as a source of diverse FAs for HFA production. Our study paves the way for tailor-made FAH design and for efficient conversion of FA sources into diverse range of HFAs with high potential for various applications from polymer industry to medical field.

Funding

Novo Nordisk Foundation (grant NNF16OC0021740)

AUFF-NOVA from Aarhus Universitets Forskningsfond (AUFF-E-2015-FLS-9-12)

History

Email Address of Submitting Author

bekireser@eng.au.dk

Institution

Aarhus University, Department of Engineering

Country

Denmark

ORCID For Submitting Author

0000-0002-8836-1251

Declaration of Conflict of Interest

The authors declare no conflict of interest

Version Notes

Version 1.0

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