Revealing Additional Stereocomplementary Pairs of Old Yellow Enzymes by Rational Transfer of Engineered Residues

Abstract

Every year numerous protein engineering and directed evolution studies are published, increasing the knowledge that could be used by protein engineers. Here we test a protein engineering strategy that allows quick access to improved biocatalysts with very little screening effort. Conceptually it is assumed that engineered residues previously identified by rational and random methods induce similar improvements when transferred to family members. In an application to ene-reductases from the Old Yellow Enzyme (OYE) family, the newly created variants were tested with three compounds, revealing more stereocomplementary OYE pairs with potent turnover frequencies (up to 660 h ) and excellent stereoselectivities (up to >99 %). Although systematic prediction of absolute enantioselectivity of OYE variants remains a challenge, "scaffold sampling" was confirmed as a promising addition to protein engineers' collection of strategies.

Metagenomic ene-reductases for the bioreduction of sterically challenging enones

“…Under these reaction conditions turnover frequencies (TOFs) were high; pQR1907 achieved a high TOF of 722 h À1 while pQR1445 reached 590 h À1 molecules per hour, better than many reported ERs and close to the best reported TOF for (S)-carvone (z1000 h À1 ), by LacER. 37,38 The bioreduction of S-5 with NCR provided the same selectivity in 78% yield and with a TOF of 645 h À1 in accordance with the literature. 38 With the ER from pQR1907 and NCR, isomer R-5 was reduced in 66% and 63% respectively, giving exclusively (2R,5R)-7.…”

Catalytic Performance of a Class III Old Yellow Enzyme and Its Cysteine Variants

“…Because C25G is highly active with N -methylmaleimide, but poorly active with cyclic ketones, it appears that residue 25 is critical for determining the substrate specificity of OYERo2 catalysis. This effect was shown with C25G variants of Rm OYE and Ts OYE where lower conversions were found for R -carvone with respect to WT (Nett et al, 2017 ).…”

Stereodivergent Synthesis of Carveol and Dihydrocarveol through Ketoreductases/Ene‐Reductases Catalyzed Asymmetric Reduction

“…The biocatalytic reduction of activated C=C bond of carvone ( 1 ) to give dihydrocarvone ( 3 ) has been well studied by several ene‐reductases ,,,. For example, (1 R ,4 R )‐ 3 and (1 R ,4 S )‐ 3 could be obtained through asymmetric reduction of corresponding (4 R )‐ 1 and (4 S )‐ 1 by various ene‐reductases, such as OYE1,, PETNR, and so on . However, only a few ene‐reductase variants, such as OYEW116A/V and TsER C25G/I67T, displayed switched stereoselectivity to yield dihydrocarvone with S configuration at C‐1 position.…”

“…For example, (1 R ,4 R )‐ 3 and (1 R ,4 S )‐ 3 could be obtained through asymmetric reduction of corresponding (4 R )‐ 1 and (4 S )‐ 1 by various ene‐reductases, such as OYE1,, PETNR, and so on . However, only a few ene‐reductase variants, such as OYEW116A/V and TsER C25G/I67T, displayed switched stereoselectivity to yield dihydrocarvone with S configuration at C‐1 position. In addition, the observed (1 S ,4 R )‐ 3 by OYEW116A/V variants had medium de values (about 50 %), which was not sufficient for the next reduction by ketoreductases.…”