A Hexokinase Associated with Catabolite Repression in Pachysolen tannophilus

Abstract

~ ~ ~~A hexokinase (ATP : D-hexose 6-phosphotransferase; EC 2.7.1.. 1) associated with catabolite repression was isolated and purified from the yeast Pachysolen tannophilus. The enzyme phosphorylated D-fructose at a rate 1.5 times greater than that for D-glUCOSe. The K , values for D-glucose and D-fructose were 0.36 and 2.28 mM, respectively. Neither xylose reductase nor xylitol dehydrogenase were subject to catabolite repression in mutants defective in this enzyme.Two of the enzymes associated with xylose catabolism, aldose (xylose) reductase and xylitol dehydrogenase, have been purified from Pachysolen tannophilus and examined (Ditzelmuller et al., 1984a(Ditzelmuller et al., , b, 1985 Verduyn et al., 1985;Bolen et al., 1986;Morimoto et al., 1986 Morimoto et al., , 1987. However, little is known about other enzymes in this yeast. We have recently demonstrated the existence of three hexose-phosphorylating enzymes in P . tannophilus; hexokinases A and B and a glucokinase specific for D-glucose (Wedlock et al., 1989; accompanying paper). In this paper, we report the isolation and partial purification of a hexokinase (ATP : D-hexose 6-phosphotransferase; EC 2.7.1 , I), designated A, from a mutant which is defective in the glucokinase. The abcPg1ce of the glucokinase, which co-elutes with hexokinase A in the wild-type strain of P . tannophilus, aids the isolation of the hexokinase without contamination by the glucokinase. The hexokinase PI1 enzyme of Saccharomyces cerevisiae has been implicated in catabolite repression (Kopetzki & Entian, 1985;Hong & Botstein, 1986) as has a hexokinase isolated from Schwanniomyces occidentalis (McCann et al., 1987). Xylose utilization by P. tannophilus is subject to hexose-sugar catabolite repression (Slininger et al., 1987; Bicho et al., 1988). The effect of the P . tannophilus hexokinase A on the activities of xylose reductase and xylitol dehydrogenase and on D-xylose utilization is described here. METHODSYeast strains and growth conditions. The Pachysolen tannophilus strains used in enzyme analyses and sugar utilization experiments were P444-3 the wild-type strain, P5 10-5A defective in a glucokinase enzyme (glul), P509-3C defective in hexokinase A (hxk2) and P509-1B defective in both hexokinase A and the glucokinase (hxk2 glul). Strain P510-5A was also used in the purification of hexokinase A.Cells were grown in 1 litre flasks containing 250 ml of YEP-glucose [ 1 %, w/v, yeast extract (Difco), 2%, w/v, peptone (Difco) and 2%, w/v, ~-glucose]. The culture was incubated for 42 h at 30 "C and 180 r.p.m. on a gyratory shaker. Cells grown for 48 h in YEP-glucose were used as the inoculum. For the measurement of xylose reductase and xylitol dehydrogenase activities, cells were grown for 16 h with either Dglucose, Dxylose, or D-glucose and Dxylose as carbon source.Growth and sugar utilization were followed in YNB [0.67% yeast nitrogen base (Difco), 2%, w/v, sugar] liquid media. A 48 h YNB-xylose culture (100 ml) was washed with deionized water and resuspended in water to an optical...

The early steps of glucose signalling in yeast

“…In contrast, in a similar experiment, Hxk1 from Y. lipolytica was fully effective (Petit & Gancedo, 1999) and there is preliminary evidence that Yl Hxk1 is involved in glucose repression of the LIP2 gene encoding an extracellular lipase in Y. lipolytica (Fickers et al , 2005). It has also been reported that in mutants of Schwanniomyces occidentalis or Pachysolen tannophilus , which lack a specific isoenzyme of hexokinase, glucose repression is defective (McCann et al , 1987; Wedlock & Thornton, 1989).…”

Yeast Carbon Catabolite Repression

“…The situation in other yeasts is not uniform. While it has been reported that mutants of Schwanniomyces occidentalis or Pachysolen tannophilus lacking a hexokinase isoenzyme have a defect in catabolite repression (226,371), in Candida utilis a strong decrease in hexokinase activity does not suppress glucose repression of ␣-glucosidase (103), and in Aspergillus nidulans, too, hexokinase is not involved in glucose repression (295).…”

Cloning and Biochemical Characterisation of an Aspergillus Niger Glucokinase

“…Hexokinases and glucokinases from several fungi have been purified and characterised including those from Saccharomyces cerevisiae (Maitra, 1970;Easterby and Rosemeyer, 1972), Aspergillus oryzae (Ruiz-Amil and Sols, 1961), and Pachysolen tunnophilus (Wedlock and Thornton, 1989). Steinbock et al (1994) reported the existence of a single hexokinase in A. niger which was purified and characterised.…”