Chembiochem. 2026 Aug 27;27(16):e70511. doi: 10.1002/cbic.70511.
ABSTRACT
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 (Vmax = 0.47 ± 0.05 kU/mg, Km = 0.047 ± 0.011 mM). These values compare favorably with those of the oxidation (Vmax = 0.13 ± 0.02 kU/mg, Km = 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 Fe2+, 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.
PMID:42626997 | DOI:10.1002/cbic.70511