Re‐Investigation of Hydration Potential of Rhodococcus Whole‐Cell Biocatalysts towards Michael Acceptors

Abstract

The implementation of a stereoselective Michael addition with water as substrate is still a major challenge by classical, chemical means. Inspired by nature's ability to carry out this attractive reaction with both high selectivity and efficiency, the interest in hydratases (EC 4.2.1.x) to accomplish a selective water addition is steadily rising. The gram‐positive bacterial genus Rhodococcus is known as biocatalytic powerhouse and has been reported to hydrate various Michael acceptors leading to chiral alcohols. This study aimed at the in‐depth re‐investigation of the hydration potential of Rhodococcus whole‐cells towards Michael acceptors. Here, two concurrent effects responsible for the hydration reaction were found: while the majority of substrates was hydrated in an oxygen‐independent manner by amino‐acid catalysis, an enzyme‐catalysed water addition to (E)‐4‐hydroxy‐3‐methylbut‐2‐enoic acid was proven to be oxygen‐dependent. 18O2‐labelling studies showed that no 18O2 was incorporated in the product. Therefore, a novel O2‐dependent hydratase distinct from all characterised hydratases so far was found.

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

“…In the course of identifying novel hydratases with interesting properties in the genus Rhodococcus, an Orthologous Matrix (OMA) algorithm approach was used combining a large number of Rhodococcus strains from different families. The strain R. pyridinivorans DSM 20415 showed interesting behaviour towards α,β-unsaturated Michael acceptors in previous studies (Busch et al 2020). As no genomic sequence data was available, it was decided to sequence the whole genome to subsequently incorporate this interesting strain into the generated Orthologous Matrix algorithm to further investigate whether this strains also produces an Ohy.…”

“…Genome analysis showed sequence diversity among Ohys that can be exploited for the discovery of novel features and will broaden future applications. Rhodococcus cells host a large number of interesting catabolic enzymes and are known for their extraordinary potential for bioremediation (Kim et al 2018;Busch et al 2020;Busch et al 2019). The genus Rhodococcus was investigated in detail for the presence of oleate hydratases using an Orthologous Matrix algorithm.…”

Rhodococcus as a Versatile Biocatalyst in Organic Synthesis

“…Upon water addition, an internal nucleophilic attack leads to the formation of useful synthon ( R )-4-hydroxy-4-methyldihydrofuran-2(3H)-one. The stereochemical course of the reaction was investigated, showing that the water-addition takes place in syn- fashion [225,226]. Interestingly, this hydratase demonstrated unusual behavior as it requires oxygen for higher activities, but labeling studies with D 2 O and 18 O 2 proved a true water addition instead of an oxidative process [226].…”

“…The stereochemical course of the reaction was investigated, showing that the water-addition takes place in syn- fashion [225,226]. Interestingly, this hydratase demonstrated unusual behavior as it requires oxygen for higher activities, but labeling studies with D 2 O and 18 O 2 proved a true water addition instead of an oxidative process [226]. Unfortunately, this membrane-associated enzyme has not been isolated and the coding gene remains elusive up to this point.…”

Novel oleate hydratases and potential biotechnological applications

“…Several promising microbial activities for novel hydratases have been described in literature. These enzymes have proven to be hard to isolate and characterise (Busch et al 2020a ).…”