Coenzyme Acceptance in Ene‐Reductases From Herbaspirillum seropedicae

Ene‐reductases (EREDs) are commonly employed for the asymmetric synthesis of important industrial chemicals. In this research, we characterise two EREDs (HSI and HSII) belonging to the ‘thermophilic‐like’ Old Yellow Enzyme (OYE) family from the beta‐proteobacterium Herbaspirillum seropedicae . Both HSI and HSII show promiscuity towards the coenzymes NADH and NADPH. Despite high sequence identities of 46% and 76% with NADH/NADPH‐dependent xenobiotic reductase A (XenA) from Pseudomonas putida , HSI prefers NADPH, whereas HSII shows equal preference for both coenzymes. To study coenzyme specificity, ancestral sequence reconstruction was performed. Two ancestral enzymes (N29 and N38) displayed a strong preference for NADH over NADPH, with N29 exhibiting the highest specific activity with NADH (12 U mg −1 ). We conclude that oligomerisation and a conserved ‘arginine finger’ contribute to NADPH specificity, and demonstrate the practical utility of HSI and HSII by using them as coenzyme regeneration catalysts for 5‐hydroxymethylfurfural oxidation, resulting in yields of 34.5% and 25.0%.