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 |
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Erscheinungsjahr: |
2022 |
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Erschienen: |
2022 |
Enthalten in: |
Zur Gesamtaufnahme - volume:23 |
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Enthalten in: |
Chembiochem : a European journal of chemical biology - 23(2022), 4 vom: 16. Feb., Seite e202100606 |
Sprache: |
Englisch |
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Beteiligte Personen: |
Zhang, Yan [VerfasserIn] |
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Links: |
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Themen: |
Biocatalysis |
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Anmerkungen: |
Date Completed 08.03.2022 Date Revised 08.03.2022 published: Print-Electronic Citation Status MEDLINE |
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doi: |
10.1002/cbic.202100606 |
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funding: |
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Förderinstitution / Projekttitel: |
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PPN (Katalog-ID): |
NLM334650941 |
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520 | |a 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 | ||
650 | 4 | |a Journal Article | |
650 | 4 | |a Research Support, Non-U.S. Gov't | |
650 | 4 | |a Biocatalysis | |
650 | 4 | |a enzyme engineering | |
650 | 4 | |a fatty acid hydratases | |
650 | 4 | |a hydroxy fatty acids | |
650 | 4 | |a regioselectivity | |
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650 | 7 | |a Hydrolases |2 NLM | |
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700 | 1 | |a Breum, Niels Mikkel Dyrby |e verfasserin |4 aut | |
700 | 1 | |a Schubert, Sune |e verfasserin |4 aut | |
700 | 1 | |a Hashemi, Negin |e verfasserin |4 aut | |
700 | 1 | |a Kyhnau, Rikke |e verfasserin |4 aut | |
700 | 1 | |a Knauf, Marius Sandholt |e verfasserin |4 aut | |
700 | 1 | |a Mathialakan, Masuthan |e verfasserin |4 aut | |
700 | 1 | |a Takeuchi, Michiki |e verfasserin |4 aut | |
700 | 1 | |a Kishino, Shigenobu |e verfasserin |4 aut | |
700 | 1 | |a Ogawa, Jun |e verfasserin |4 aut | |
700 | 1 | |a Kristensen, Peter |e verfasserin |4 aut | |
700 | 1 | |a Guo, Zheng |e verfasserin |4 aut | |
700 | 1 | |a Eser, Bekir Engin |e verfasserin |4 aut | |
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