Reversal of Regioselectivity in Zinc‐Dependent Medium‐Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives

Abstract

The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/ mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.

Stairway to Stereoisomers: Engineering Short‐ and Medium‐Chain Ketoreductases To Produce Chiral Alcohols

“…Thermoanaerobacter ethanolicus MDR multiple substrates ABY93890 [108] ADH1 Pyrococcus furiosus DSM 3638 SDR low promiscuity AAC25556 [109] BtADH Bacillus thermoleovorans B23 MDR low promiscuity AB040809 [110] ApADH Aeropyrum pernix K1 MDR multiple substrates APE2239 [111] LbADH Lactobacillus brevis ATCC 14869 SDR multiple substrates Q84EX5 [112] ReADH Rhodococcus erythropolis MDR multiple substrates AAN73270 [113] Kacr1 Kluyveromyces aestuarii SDR low promiscuity BAD01116 [114,115] Cscr1…”

Rhodococcus strains as a good biotool for neutralizing pharmaceutical pollutants and obtaining therapeutically valuable products: Through the past into the future

“…10.3389/fmicb.2022 Frontiers in Microbiology 07 frontiersin.org Thus, 2-hydroxyacetophenone was successfully converted to (R)-(-)-1-phenyl-1,2-ethanediol (99% ee), an important pharmaceutical precursor (Han et al, 2021). The zinc-dependent alcohol dehydrogenase from R. erythropolis DSM 43297 (ReADH) converted ethyl 2-oxo-4-phenylbutyrate to ethyl 2-hydroxy-4-phenylbutanoate, an important intermediate for anti-hypertension drugs such as enalaprilat and lisinopril (Dhoke et al, 2020).…”