Biosynthesis, isolation and properties of NAD‐dependent formate dehydrogenase from the yeast Candida methylica

Abstract

NAD-dependent formate dehydrogenase (EC 1.2.1.2), was isolated from the methanol-utilizing yeast Crrridida methylica. Two purification techniques for the enzyme from the crude yeast extract have been developed: a twostep procedure, involving a sequential application of DEAE-cellulose ion-exchange chromatography and Sephadex G-200 gel filtration, and a single-step procedure, preparative isoelectric focusing in a granulated gel layer. The enzyme proved to be electrophoretically homogeneous. I t consisted of two identical subunits with a relative molecular mass of 46000, each containing one -SH group related to manifestation of the catalytic activity. The Michaelis constant was 1 lop4 M for NAD and 1.3 .M for formate. Formate dehydrogenasc was inhibited with p-chlormercuribenzoate, iodoacetamide, dithionitrobenzoatc, cyanide and azide.NAD-dependent formate dehydrogenase, catalyzing the oxidation of formate to COz, has been discovered in many microorganisms utilizing methanol and other C1 compounds as carbon sources [I -41. The homogeneous prcparations of NAD-dependent formate dehydrogenase were obtained from the methylotrophic yeasts Kloeckevu sp. 2201 [5] and Candida hoidinii [6] and their physico-chemical properties studied.In view of elaboration of the cofactor regeneration system based on NAD-dependent dehydrogenases from methylotrophic microorganisms, we isolated and compared formate dehydrogenases from the methylotrophic bacterium Acl7ro-tnohartrr parvulus [7,8] and the methylotrophic yeast Cmdida methqdica [9].The present paper describes the conditions of biosynthesis, isolation and some properties of NAD-dependent formate dehydrogenase from Candida mrthylica.

Purification and properties of formate dehydrogenase from Moraxella sp. strain C-1

“…(9,17,18,24,30). Moraxella formate dehydrogenase is markedly different from the formate dehydrogenases of anaerobes and P. oxalaticus (21), which are chromophoric iron-sulfur enzymes and are similar to those from the pea (23), methylotrophic yeasts (3,11,12,15,25), and A. parvulus (7,8), which contain no prosthetic group. In Table 2, some enzymatic and physicochemical properties of formate dehydrogenase from Moraxella sp.…”

“…The activity of the formate dehydrogenase was strongly inhibited by heavy metal ions such as Ag+, Hg2+, 5,5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide, sodium azide, KCN, and hydroxylamine, suggesting that essential sulfhydryl and carbonyl groups exist in the active site. This observation and the high Km value for formate are the common characteristics of NAD+-dependent formate dehydrogenases of methylotrophic microorganisms (3,7,11,12,15,25), excluding the formate dehydrogenase of P. oxalaticus, which shows a low Km value (21). The formate dehydrogenase of Moraxella sp.…”

Characterization of crystalline formate dehydrogenase from Candida methanolica

“…Bacterial formate dehydrogenases are less stable than the yeast enzymes. Thus, the purified enzymes so far reported were mostly from yeasts [6][7][8][9][10]201. The physicochemical and emzymatic properties of the formate dehydrogenase from C. methanolica, which we purified, resembled those of the enzymes from other methylotrophic yeasts [6 -101: molecular mass, sedimentation and diffusion coefficients, subunit structure, absorption coefficient, amino acid composition, substrate and coenzyme specificities, and K, value for NAD'.…”

NAD+-dependent formate dehydrogenase

“…The enzymes from eukaryotic organisms [14][15][16][17][18][19][20][38][39][40][41][42], as well as from some methylotrophic bacteria [21][22][23][24], have molecular masses ranging from 70 to 100 kDa and are composed of two chemically identical subunits. They display relatively low specific activity, a low affinity for formate ion and a broad pH optimum for catalytic activity at neutral pH ( Table 1).…”