Semi-rational Engineering of a Promiscuous Fatty Acid Hydratase for Alteration of Regioselectivity

© 2021 Wiley-VCH GmbH..

Fatty acid hydratases (FAHs) catalyze regio- and stereo-selective hydration of unsaturated fatty acids to produce hydroxy fatty acids. Fatty acid hydratase-1 (FA-HY1) from Lactobacillus Acidophilus is the most promiscuous and regiodiverse FAH identified so far. Here, we engineered binding site residues of FA-HY1 (S393, S395, S218 and P380) by semi-rational protein engineering to alter regioselectivity. Although it was not possible to obtain a completely new type of regioselectivity with our mutant libraries, a significant shift of regioselectivity was observed towards cis-5, cis-8, cis-11, cis-14, cis-17-eicosapentaenoic acid (EPA). We identified mutants (S393/S395 mutants) with excellent regioselectivity, generating a single hydroxy fatty acid product from EPA (15-OH product), which is advantageous from application perspective. This result is impressive given that wild-type FA-HY1 produces a mixture of 12-OH and 15-OH products at 63 : 37 ratio (12-OH : 15-OH). Moreover, our results indicate that native FA-HY1 is at its limit in terms of promiscuity and regiospecificity, thus it may not be possible to diversify its product portfolio with active site engineering. This behavior of FA-HY1 is unlike its orthologue, fatty acid hydratase-2 (FA-HY2; 58 % sequence identity to FA-HY1), which has been shown earlier to exhibit significant promiscuity and regioselectivity changes by a few active site mutations. Our reverse engineering from FA-HY1 to FA-HY2 further demonstrates this conclusion.

Medienart:

E-Artikel

Erscheinungsjahr:

2022

Erschienen:

2022

Enthalten in:

Zur Gesamtaufnahme - volume:23

Enthalten in:

Chembiochem : a European journal of chemical biology - 23(2022), 4 vom: 16. Feb., Seite e202100606

Sprache:

Englisch

Beteiligte Personen:

Zhang, Yan [VerfasserIn]
Breum, Niels Mikkel Dyrby [VerfasserIn]
Schubert, Sune [VerfasserIn]
Hashemi, Negin [VerfasserIn]
Kyhnau, Rikke [VerfasserIn]
Knauf, Marius Sandholt [VerfasserIn]
Mathialakan, Masuthan [VerfasserIn]
Takeuchi, Michiki [VerfasserIn]
Kishino, Shigenobu [VerfasserIn]
Ogawa, Jun [VerfasserIn]
Kristensen, Peter [VerfasserIn]
Guo, Zheng [VerfasserIn]
Eser, Bekir Engin [VerfasserIn]

Links:

Volltext

Themen:

Biocatalysis
EC 3.-
Enzyme engineering
Fatty Acids
Fatty acid hydratases
Hydrolases
Hydroxy fatty acids
Journal Article
Regioselectivity
Research Support, Non-U.S. Gov't

Anmerkungen:

Date Completed 08.03.2022

Date Revised 08.03.2022

published: Print-Electronic

Citation Status MEDLINE

doi:

10.1002/cbic.202100606

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

NLM334650941