A low-molecular-mass protein from Methylococcus capsulatus (Bath) is responsible for the regulation of formaldehyde dehydrogenase activity in vitro

An 8.6 kDa protein, which the authors call a modifin, has been purified fro m Methylococcus capsulatus (Bath) and has been shown to alter the substrate specificity and kinetics of NAD(+)-linked formaldehyde dehydrogenase (FDH) isolated from the same organism. Purification methods for both the modifin and FDH are presented which reliably produced pure protein for further ana lysis. Analysis of the molecular mass and N-terminal sequence of both FDH a nd the modifin indicate that they are unique proteins and show no similarit y to alcohol or aldehyde dehydrogenase enzymes isolated from methylotrophic bacteria. Substrate specificity studies demonstrated that FDH oxidized for maldehyde exclusively in the presence of the modifin; a diverse range of al dehydes and alcohols were oxidized by FDH in the absence of the modifin. No formaldehyde oxidation was detected in the absence of the modifin. Attempt s to replace the modifin with glutathione or high concentrations of methano l to stimulate formaldehyde oxidation failed. With acetaldehyde as substrat e, FDH showed standard Michaelis-Menten kinetics; interaction of FDH with t he modifin using formaldehyde as substrate altered the kinetics of the reac tion to sigmoidal. Kinetic analysis during turnover experiments indicated t hat the FDH may be associated with bound formaldehyde following enzyme isol ation and that NAD may also be associated with the enzyme but in a form tha t is less tightly bound than found with the methanol dehydrogenase from Bac illus methanolicus. Data are presented which indicate that the modifin may play an important role in regulating formaldehyde concentration in vivo.