Cobalt modulates methanol turnover of the alcohol dehydrogenase in Desulfofundulus kuznetsovii strain TPOSR

Desulfofundulus kuznetsovii strain 17T oxidizes methanol via a two-pathway system involving both alcohol dehydrogenases (ADH) and a cobalt-dependent methanol methyltransferase (MT). In contrast, D. kuznetsovii strain TPOSR lacks the MT pathway, relying solely on ADH for growth on methanol. Despite the absence of the MT pathway, cobalt starvation resulted in lower methanol uptake rates and reduced growth rates in strain TPOSR, suggesting a critical role of cobalt in methanol metabolism outside of its role in the MT system. Given the often-crucial role of metal cofactors such as iron, zinc, and other metals in the active site of ADHs, we hypothesized that cobalt could influence the catalytic activity of the TPOSR ADHs. The gene encoding for the most abundant ADH during growth on methanol, Adh1, was heterologously expressed in Escherichia coli, and the enzyme was purified for kinetic studies. Adh1 exhibited optimal activity at 55°C and is oxygen tolerant. The methanol turnover rate increased from 1.76 (95% Cl [1.56, 1.99]) s⁻¹ to 3.5 (95% Cl [3.3, 3.72]) s⁻¹ with the addition of 2 µM CoSO4, while higher cobalt concentrations (>5 µM) inhibited Adh1 activity. Similarly, NiSO4 addition (1–1000 µM) enhanced Adh1 activity, with a 75% improvement observed at an optimum concentration of 200 µM. Our findings suggest that the importance of cobalt for the methanol metabolism of sulfate-reducing organisms extends beyond its involvement in the MT system.