Abstract
Desulfofundulus kuznetsovii strain 17(T) 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 CoSO(4), while higher cobalt concentrations (>5 µM) inhibited Adh1 activity. Similarly, NiSO(4) 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.IMPORTANCEMethanol is a ubiquitous compound in natural environments, where it is produced geothermally or from plant and microbial biomass. Its microbial metabolism is particularly important in low-nutrient, oxygen-free environments, such as the deep subsurface, where specialized microbes compete for methanol and play a crucial role in the global carbon cycle. Typically, microbes in these settings rely on a cobalt-dependent methanol methyltransferase (MT) pathway for methanol breakdown. However, Desulfofundulus kuznetsovii TPOSR deviates from this, lacking the MT pathway and instead relying solely on alcohol dehydrogenases (ADH) to oxidize methanol. Despite the absence of the cobalt-dependent MT system, our study shows that cobalt strongly stimulates the activity of the most abundant ADH, revealing an unexpected, yet significant role for cobalt in this alternative methanol metabolism. Understanding these interactions not only sheds new light on methanol metabolism in nature but also opens up possibilities for developing more efficient and sustainable technologies for methanol conversion in industry.