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

Abstract

Enzymatic conversion of fatty acids (FAs) by fatty acid hydratases (FAHs) presents a green and efficient route for high‐value hydroxy fatty acid (HFA) production. However, limited diversity was achieved among HFAs, to date, with respect to chain length and hydroxy position. In this study, two highly similar FAHs from Lactobacillus acidophilus were compared: FA‐HY2 has a narrow substrate scope and strict regioselectivity, whereas FA‐HY1 utilizes longer chain substrates and hydrates various double‐bond positions. It is revealed that three active‐site residues play a remarkable role in directing substrate specificity and regioselectivity of hydration. If these residues on FA‐HY2 are mutated to the corresponding ones in FA‐HY1, a significant expansion of substrate scope and a distinct enhancement in hydration of double bonds towards the ω‐end of FAs is observed. A three‐residue mutant of FA‐HY2 (TM‐FA‐HY2) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting the ratio of the HFA regioisomers (10‐OH/13‐OH) from 99:1 to 12:88. Notable changes in regioselectivity were also observed for arachidonic acid and for C18 polyunsaturated fatty acid substrates. In addition, TM‐FA‐HY2 converted eicosapentaenoic acid into its 12‐hydroxy product with high conversion at the preparative scale. Furthermore, it is demonstrated that microalgae are a source of diverse FAs for HFA production. This study paves the way for tailor‐made FAH design to enable the production of diverse HFAs for various applications from the polymer industry to medical fields.

Exploring the abundance of oleate hydratases in the genus Rhodococcus—discovery of novel enzymes with complementary substrate scope

“…This shows a clear preference for the 10-position over the 13-position for both Rhodococcus Ohys which can be explained by the two amino acids V393 and H395 (ReOhy (PR4)) and T390 and N392 (RpOhy) (corresponding to T436 and N438 in Em-OAH1) following the results from recent studies (Eser et al 2020). In FA-HY1, these positions are occupied by two smaller serine amino acids meaning that the substrate is entering the substrate tunnel further leading to the water addition in 13-position (Eser et al 2020). In our example, however, histidine and asparagine are bulkier thereby directing a water addition into 10-position.…”

“…Up to now, only two other Ohys from Lactobacillus acidophilus (NTV001 (FA-HY1) and LMG 11470) were shown to convert pinolenic acid whereby both catalyse the water addition in 13-position (Engleder et al 2015;Hirata et al 2015;Kim et al 2015). This shows a clear preference for the 10-position over the 13-position for both Rhodococcus Ohys which can be explained by the two amino acids V393 and H395 (ReOhy (PR4)) and T390 and N392 (RpOhy) (corresponding to T436 and N438 in Em-OAH1) following the results from recent studies (Eser et al 2020). In FA-HY1, these positions are occupied by two smaller serine amino acids meaning that the substrate is entering the substrate tunnel further leading to the water addition in 13-position (Eser et al 2020).…”

“…A recent study investigated the impact of these two amino acids on the final regio-and stereoselectivity and showed that selective mutations can lead to a complete reversal of selectivity with the example of the two fatty acid hydratases from Lactobacillus acidophilus (FA-HY1 and FA-HY2). Here, less bulky substituents in these two positions enabled the substrate to go deeper into the carboxylate end of the substrate channel (Eser et al 2020).…”

A Bi‐Enzymatic Cascade Pathway towards Optically Pure FAHFAs**

“…As we reported previously, FA‐HY1 and FA‐HY2 are highly stereospecific enzymes that generate a single stereoisomer product with an enantiomeric excess of over 95 % [14] . Therefore, several pairs of FAH‐substrates that exhibited high conversion rates in our previous study, [14a] were chosen for FAHFA preparation. By using either FA‐HY1 or FA‐HY2, oleic acid (OA), LA, cis ‐vaccenic acid (CVA) and α‐linolenic acid (ALA) were converted to corresponding HFAs with different −OH position, followed by the esterification with PA catalyzed by CALA.…”

Fatty Acid Hydratases: Versatile Catalysts to Access Hydroxy Fatty Acids in Efficient Syntheses of Industrial Interest

“…Other confines are the substrate scope, which is limited by the seven carbon atoms between carboxylic residue and double bond, as well as the length of the fatty acids and the carboxylic acid itself. These problems have already been addressed in some publications, like the hydration of unactivated olefins by using short chain acids as decoy molecules [77], the rational of engineering a fatty acid hydratase to enhance the substrate scope [78] and the hydration of oleic acid derivatives without a free carboxylate moiety [79]. Thus, the current status represents a highly promising basis for further studies in this field with the perspective of a range of industrial applications for a sustainable manufacture of industrial chemicals for various commercial segments, based on the utilization of biorenewable raw materials.…”