ω‐Hydroxyacid Dehydrogenases From Clostridium viride : Iron‐Containing Biocatalysts With Biotechnological Potential

ω ‐Hydroxy and oxo acids are versatile platform chemicals, accessible via enzymes of the iron‐containing alcohol dehydrogenase (FeADH) superfamily. Compared with well‐characterized zinc‐dependent ADHs, FeADHs producing such compounds remain poorly defined regarding assignment to genetic locus, biochemical and biophysical properties, and metal specificity. Here, we uncover two FeADHs from Clostridium viride : a highly selective 4‐hydroxybutyrate dehydrogenase and a broad‐spectrum 5‐hydroxyvalerate dehydrogenase highly active on C5–C12 substrates, identified by purification from the native organism. Chemical synthesis of 5‐oxovalerate enabled determination of kinetics for the physiological direction ( V max = 0.47 ± 0.05 kU/mg, K m = 0.047 ± 0.011 mM). These values compare favorably with those of the oxidation ( V max = 0.13 ± 0.02 kU/mg, K m = 0.42 ± 0.08 mM). We report the first crystal structure of an FeADH in complex with both substrate and NADH at 1.73 Å resolution. This enables identification of active‐site iron and catalytic residues, providing insight into substrate recognition. High iron occupancy in the nonreconstituted enzymes, together with Mössbauer and electron paramagnetic resonance (EPR) spectroscopic identification of high‐spin Fe 2+ , establishes iron as the native cofactor. These findings highlight the potential of C. viride FeADHs as attractive biocatalysts for accessing sustainable solvents, polymers, and fine chemicals via ω ‐functionalized acids.