Dihydroxyacetone kinase of methanol‐assimilating yeasts II. Partial purification and some properties of dihydroxyacetone kinase from Candida methylica

Abstract

Dihydroxyacetone kinase (DHAII) from the cell-free extract of methanol-grown Candida rnethylica was partially purified about 100-fold by a procedure employing streptomycine sulfate fractionation, ammonium sulfate fractionation, negative absorption on Cibacron blue F3G-A sephadex G 200 and DEAE-cellulose column chromatography. The enzyme was stable in 50 mnf Tris-HC1 buffer p H 7.5 containing 60% glycerol at -18 "C.The p H optimum for the activity of DHAK from C. methylica was 7.5. The purified enzyme phosphorylated dihydroxyacetone four times faster than D,L-glyceraldehyde. The apparent MI-CHAELIS-MENTEN constants for dihydroxyacetone and D,L-glyceraldehyde were 0.011 mM and 0.024 mM. Other C, compounds including glycerol were not phosphorylated. I T P and U T P were used as phosphate donors with a reaction rate of 11% and 3.1%, respectively, in relation t o ATP, whereas the reaction rates of DHAK from C. methylica with CTP or GTP were much lower than 1%. The reaction of DHAK depends upon the presence of divalent cations in the assay. The highest activity was found with MgZt ions. The reaction rates with Co2+ or Caz+ ions were only 57.3% and 30.3%, respectively, in relation t o the assay with magnesium ions. Manganese chloride in the assay led t o a complete loss of activity.The phosphorylation of glyceraldehyde and dihydroxyacetone by means of triokinases plays an important role in the metabolism of glycerol via NAD+-and NADP+-linked glycerol dehydrogenases and glycerol oxidase in niicroorganisms (LIN 1976, TOM et al. 1978. Recently it was found that dihydroxyacetone is an intermediate in the assimilation of methanol by yeasts, and high activities of dihydroxyacetone kinase were measured in crude extracts from methanol-grown yeasts ( VAN DIJXEN et al. 1978, BABEL and LOFFIIAGEN 1979). However, the knowledge about the kinetic and regulatory properties of triokinases from microorganisms is very limited. Previously we have investigated the regulation of the dihydroxyacetone kinase from the methanol-grown yeast Candida methylica in situ (HOFMANN and BABEL 1980). The present paper will describe the procedure for purification, the substrate specificity and the kinetic properties of the enzyme from Candida methylica. Materials and methodsCarLdida methylica was obtained from Dr. Y. A. TROTSENKO, Pushchino, USSR, and was grown as reported previously (HOFMANN and BABEL 1980). For the preparation of cell-free extracts cells were harvested by centrifugation, washed twice with 20 miw Tris-HC1 buffer (pH 7.5) and resuspended in the same buffer. The cell suspension was passed three times through a FRENCH pressure cell (9.9 kN m-2) followed by centrifugation of the homogenate at 20,000 g for 20 min. Protein concentration was determined by the method of protein-dye binding (BRADFORD 1976) in which human serum albumin as a standard was used. The enzyme activity of dihydroxyacetone kinase was measured at 30 "C using a method based on that of HEINZ a.nd LAMPRECHT (1961). The assay mixtures consisted of 100 miv imidazole-HC1 buffe...

Purification and Properties of Dihydroxyacetone Kinase from Schizosaccharomyces pombe

“…However, the DHAK of K . pneumonia was also reported to be at least as active with Ca2+ as with Mg2+ (Johnson et al, 1984) though that from C. rnethyfica was only 30% as active with Ca2+ as with Mg2+ (Hofmann & Babel, 1981).…”

“…an ATP : dihydroxyacetone phosphotransferase) has been reported in the methanol-utilizing yeasts Hansenula polymorpha and Candida boidinii (van Dijken et al, 1978) and Candida methylica (Hofmann & Babel, 1981). It resembles the S .…”

Small substrate, big surprise: fold, function and phylogeny of dihydroxyacetone kinases

“…glycerol also phosphorylate Dha and glyceraldehyde [37][38][39][40]. The Dha-specific kinases have been characterized in bacteria [10,41] and in methylotrophic yeast (for a review see [42][43][44]). They have been purified from yeasts [16,17,42,43,[45][46][47][48][49], algae [50], tomato [51], rat brain [31], porcine kidney [52] and from bacteria, viz.…”

Bifunctional Homodimeric Triokinase/FMN Cyclase

“…Despite the relevance of the Hers pathway in liver (1)(2)(3)(4)(5) and the much heralded concerns on the effects of the high consumption of fructose in human health (5)(6)(7)(8)(9)(10)(11)(12), mammalian triokinase, contrary to fructokinase and aldolase B (13,14), is still without an established molecular identity, and it is not yet firmly associated to a specific gene. However, there are a few reports suggesting that mammalian triokinase could be a product of DAK (15,16), a gene named by homology to yeast and bacterial genes coding for ATP-dependent dihydroxyacetone (DHA) kinases (17,18), some of which are known to be active as GA kinases too (18,19).…”