Dihydroxyacetone Synthase: a Special Transketolase for Formaldehyde Fixation from the Methylotrophic Yeast Candida boidinii CBS 5777

Abstract

The transketolase synthesized by Candida boidinii CBS 5777 during growth on non-C, substrates has been purified and shown to have a molecular weight of approximately 135 000 and to consist of two subunits of approximate molecular weight 68000. The enzyme is active with xylulose 5phosphate as glycolyl donor and ribose 5-phosphate as acceptor; under the conditions of assay formaldehyde was inactive as acceptor. Candida boidinii CBS 5777 also synthesizes a second transketolase during growth on methanol. This enzyme, which is unstable, has been purified to homogeneity. It has a wide substrate specificity towards glycolyl donors and acceptors; formaldehyde is a good acceptor and in terms of its physiological role during growth on methanol the enzyme can be described as a dihydroxyacetone synthase. This enzyme has a molecular weight of 105000-1 10000, with two subunits of molecular weight 62000-67000. Its properties have been compared with analogous enzymes purified elsewhere from methanolgrown Candida boidinii KD1 and Candida boidinii (Kloeckera sp.) 2201.

Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha

“…After washing twice with buffer A plus 3 M sorbitol to remove the mercaptoethanol, the cells were resuspended in the same buffer (0.06 g wet weight/ml). Then the cell wall digesting enzyme Zymolyase 20,000 was added to a final concentration of 1 mg/ml and the mixture was incubated for 3 h at 35~ All further steps were carried out in 5 mM MES-buffer pH 5.5 containing 1 mM MgClz, 1 mM EDTA, 1 mM DTT and 0.5 mM TPP (buffer B) in order to stabilize DHAS activity (Waites and Quayle 1983). After cell wall digestion, the protoplasts were sedimented (10 rain 10,000 • g) and washed twice with buffer B plus 3 M sorbitol, then dialysed against buffer B plus 1 M sorbitol for I h at 4~ and finally homogenized in a Potter Elvehjem homogenizer (Potter 1955) by applying 10 strokes at 200 rpm.…”

Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803

“…The classical transketolase found in cells grown on C 1 compounds as well as on multicarbon substrates, however, is known to exhibit strong activity on aldose phosphates such as (12,37). C. boidinii KD1, however, was found to have DHAS which uses formaldehyde as the only glycoaldehyde group acceptor among those aldehydes and aldose phosphates tested (5), indicating that the DHAS of this bacterium is more specific than those of the other two C. boidinii strains.…”

“…The DHAS present in cells grown only on methanol is a special transketolase able to use formaldehyde as the acceptor for the glycoaldehyde group from the donor substrates (5,12,37). The classical transketolase found in cells grown on C 1 compounds as well as on multicarbon substrates, however, is known to exhibit strong activity on aldose phosphates such as (12,37).…”

“…Erythrose 4-phosphate was found to be the best acceptor for Acinetobacter sp. strain JC1 enzyme, while glycolaldehyde was preferred for the two C. boidinii enzymes (12,37).…”

“…DHAS activity was assayed, depending on the purpose of the experiment, by one of three methods described previously (12,37), with several modifications. For routine assay and to test the effects of glycoaldehyde acceptors on DHAS activity, the activity was measured by a modification of the method of Kato et al (12) (method A).…”