Dihydroxyacetone Kinases in Saccharomyces cerevisiaeAre Involved in Detoxification of Dihydroxyacetone

Abstract

The genes YML070W/DAK1 and YFL053W/DAK2 in the yeast Saccharomyces cerevisiae were characterized by a combined genetic and biochemical approach that firmly functionally classified their encoded proteins as dihydroxyacetone kinases (DAKs), an enzyme present in most organisms. The kinetic properties of the two isoforms were similar, exhibiting K m(DHA) of 22 and 5 M and K m(ATP) of 0.5 and 0.1 mM for Dak1p and Dak2p, respectively. We furthermore show that their substrate, dihydroxyacetone (DHA), is toxic to yeast cells and that the detoxification is dependent on functional DAK. The importance of DAK was clearly apparent for cells where both isogenes were deleted (dak1⌬dak2⌬), since this strain was highly sensitive to DHA. In the opposite case, overexpression of either DAK1 or DAK2 made the dak1⌬dak2⌬ highly resistant to DHA. In fact, overexpression of either DAK provided cells with the capacity to grow efficiently on DHA as the only carbon and energy source, with a generation time of about 5 h. The DHA toxicity was shown to be strongly dependent on the carbon and energy source utilized, since glucose efficiently suppresses the lethality, whereas galactose or ethanol did so to a much lesser extent. However, this suppression was found not to be explained by differences in DHA uptake, since uptake kinetics revealed a simple diffusion mechanism with similar capacity independent of carbon source. Salt addition strongly aggravated the DHA toxicity, independent of carbon source. Furthermore, the DHA toxicity was not linked to the presence of oxygen or to the known harmful agents methylglyoxal and formaldehyde. It is proposed that detoxification of DHA may be a vital part of the physiological response during diverse stress conditions in many species.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…However, as a consequence of the expected decrease in phosphoryl group flux through DhaM, DhaL will primarily be present with bound ADP, and due to the presence of dihydroxyacetone, DhaK will primarily be present with bound substrate. Both signals favor dhaKLM expression, and this unusual regulation mode was therefore thought to ensure a constant dihydroxyacetone turnover rate and detoxification, which might be necessary if dihydroxyacetone accumulates owing to intracellular metabolic processes (567).…”

Cloning, Heterologous Expression, and Characterization of Three Aquaglyceroporins from Trypanosoma brucei

“…Interestingly, a concentration of 0.2 M DHA is toxic for yeast cells (30). In glucose-containing medium, 0.2 M DHA led to a slightly decreased growth rate of the fps1⌬ deletion strain, whereas growth of TbAQP-transformed fps1⌬ was drastically reduced (Fig.…”

“…Moreover, trypanosomes incubated in buffer containing different DHA concentrations as the sole energy source did not survive (data not shown). In literature, DHA has been related to anaerobic glycerol metabolism and osmoregulation, but cell toxicity has also been described (30,34,35).…”

A mechanism of covalent substrate binding in the x-ray structure of subunit K of the Escherichia coli dihydroxyacetone kinase

“…We propose that formation of the hemiaminal is the chemical basis for efficient discrimination between short-chain ketoses and aldoses on one hand and the structurally similar polyalcohols on the other. The former are chemically reactive and potentially toxic, even at low concentrations (31,32). The latter are compatible solutes, which accumulate to high concentrations as osmoprotectants, for instance in yeast (33).…”