PURIFICATION AND PROPERTIES OF METHYL FORMATE SYNTHASE, A MITOCHONDRIAL ALCOHOL-DEHYDROGENASE, PARTICIPATING IN FORMALDEHYDE OXIDATION IN METHYLOTROPHIC YEASTS

Methyl formate synthase, which catalyzes methyl formate formation duri ng the growth of methylotrophic yeasts, was purified to homogeneity fr om methanol-grown Candida boidinii and Pichia methanolica cells. Both purified enzymes were tetrameric, with identical subunits with molecul ar masses of 42 to 45 kDa, containing two atoms of zinc per subunit. T he enzymes catalyze NAD(+)-linked dehydrogenation of the hydroxyl grou p of the hemiacetal adduct [CH2(OH)OCH3] of methanol and formaldehyde, leading to the formation of a stoichiometric amount of methyl formate . Although neither methanol nor formaldehyde alone acted as a substrat e for the enzymes, they showed simple NAD(+)-linked alcohol dehydrogen ase activity toward aliphatic long-chain alcohols such as octanol, sho wing that they belong to the class III alcohol dehydrogenase family. T he methyl formate synthase activity of C. boidinii was found in the mi tochondrial fraction in subcellular fractionation experiments, suggest ing that methyl formate synthase is a homolog of Saccharomyces cerevis iae Adh3p. These results indicate that formaldehyde could be oxidized in a glutathione-independent manner by methyl formate synthase in meth ylotrophic yeasts. The significance of methyl formate synthase in both formaldehyde resistance and energy metabolism is also discussed.