Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression.

Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.     

The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.     

Physiological properties of Saccharomyces cerevisiae from which hexokinase II has been deleted

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H(+)-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.     

The hexokinase isoenzyme PII of Saccharomyces cerevisiae ia a protein kinase

The HXK2 gene product has an important role in controlling carbon catabolite repression in Saccharomyces cerevisiae. We have raised specific antibodies against the hexokinase PII protein and have demonstrated that it is a 58 kDa phosphoprotein with protein kinase activity. The predicted amino acid sequence of the HXK2 gene product has significant homology to the conserved catalytic domain of mammalian and yeast protein kinases. Protein kinase activity was located in a different domain of the protein from the hexose-phosphorylating activity. The hexokinase PII protein level remained unchanged in P2T22D mutant cells (hxk1 HXK2 glk1) growing in a complex medium with glucose. The protein kinase activity of hexokinase PII is regulated by the glucose concentration of the culture medium. Exit from the carbon catabolite repression phase and entry into derepression phase may be controlled, in part, by modulation of the 58 kDa protein kinase activity by changes in cyclic AMP concentration.     

Genetics of yeast hexokinase

Two independent isolates of Saccharomyces cerevisiae lacking hexokinase activity (EC 2.7.1.1) are described. Both mutant strains grow on glucose but are unable to grow on fructose, and contain two mutant genes hxk1 and hxk2 each. The mutations are recessive and noncomplementing. Genetic analysis suggests that these two unlinked genes hxk1 and hxk2 determine, independently of each other, the synthesis of hexokinase isozymes P1 and P2, respectively. hxk1 is located on chromosome VIR distal to met10, and hxk2 is on chromosome IIIR distal to MAL2. Of four hexokinase-positive spontaneous reversions, one is very tightly linked to hxk1 and the other three to the hxk2 locus. The reverted enzymes are considerably more thermolabile than the respective wild-type enzymes, and in one case show altered immunological properties. Data are presented which suggest that the hxk1 and hxk2 mutations are missense mutations in the structural genes of hexokinase P1 and hexokinase P2, respectively. These are presumably the only enzymes that allow S. cerevisiae to grow on fructose.     

Physiological Properties of Saccharomyces cerevisiae from Which Hexokinase II Has Been Deleted

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H⁺-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.     

Kinetic and proteomic analyses of <Emphasis Type="Italic">S</Emphasis>-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism

Summary. Mammalian hexokinase (HXK) is found at the outer mitochondrial membrane, exposed to mitochondrial oxygen- and nitrogen-radicals. Given the important role of this enzyme in metabolic pathways and diseases, the effect of S-nitrosoglutathione (GSNO) on HXK A structure and activity was studied. To focus on the catalytic domain, yeast HXK A was used because it has a significant homology to the mammalian domain that contains both the regulatory and catalytic sites. Biologically relevant [GSNO]/[HXK] caused a significant decrease in V_max with glucose (but not with fructose), along with oxidation of 5 Met and nitration of 4 Tyr. Preincubation of HXK with glucose abrogated the effect of GSNO whereas fructose was ineffective. These results are interpreted by considering the tight binding of glucose to the enzyme as opposed to that of fructose. The segment comprised from amino acids 304 to 306 contained the most modifications. Given that this sequence is highly conserved in HXK from various species, a decline in activity is expected when a high-affinity substrate is presented. Considering that changes in primary structure are envisioned at high [GSNO]/[HXK] ratios, like those present under normal conditions, it could be hypothesized that the high concentration of hexokinase present in fast growing tumors may serve not only to sustain high glycolysis rates, but also to minimize protein damage that might result in activity decline, compromising energy metabolism.     

Distinct modulations of the hexokinase1-mediated glucose response and hexokinase1-independent processes by HYS1/CPR5 in Arabidopsis

The Arabidopsis mutant hypersenescence 1 (hys1), that is allelic to constitutive expresser of pathogenesis-related genes 5 (cpr5), displays phenotypes related to glucose signalling and defence responses. In the present study, it is shown that the hys1 mutation boosts the inhibitory effects of glucose upon the greening of seedlings and reduces the antagonistic activities of ethylene and cytokinin toward this inhibition. Neither the glucose content nor the sensitivities to ethylene, cytokinin, and abscisic acid were found to differ between wild-type and hys1 seedlings. However, disruption of the gene encoding hexokinase1 (HXK1), which acts as a glucose sensor, partially suppressed the glucose hypersensitive phenotype of the hys1 mutant. These results thus suggest that the hys1 mutation promotes a process associated with the HXK1-mediated glucose response during greening. By contrast, additional hys1 phenotypes, including an increase in salicylic acid (SA), production of abnormal trichomes, and early senescence, were not suppressed by the loss of HXK1. Surprisingly, the hxk1 and hys1 mutations acted synergistically towards an increased SA accumulation. Hence, HYS1/CPR5 appears to be a versatile protein that modulates both the HXK1-mediated glucose response and various HXK1-indepndent processes that are involved in growth control. A possible role for HYS1/CPR5 as a component of the networks that regulate growth control is discussed.     

Mixed and diverse metabolic and gene-expression regulation of the glycolytic and fermentative pathways in response to a HXK2 deletion in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the HXK2 gene, which encodes the glycolytic enzyme hexokinase II, is involved in the regulatory mechanism known as 'glucose repression'. Its deletion leads to fully respiratory growth at high glucose concentrations where the wild type ferments profusely. Here we describe that deletion of the HXK2 gene resulted in a 75% reduction in fermentative capacity. Using regulation analysis we found that the fluxes through most glycolytic and fermentative enzymes were regulated cooperatively by changes in their capacities (Vmax) and by changes in the way they interacted with the rest of the metabolism. Glucose transport and phosphofructokinase were regulated purely at the metabolic level. The reduction of fermentative capacity in the mutant was accompanied by a remarkable resilience of the remaining capacity to nutrient starvation. After starvation, the fermentative capacity of the hxk2Delta mutant was similar to that of the wild type. Based on our results and previous reports, we suggest an inverse correlation between glucose repression and the resilience of fermentative capacity towards nutrient starvation. Only a limited number of glycolytic enzyme activities changed upon starvation of the hxk2Delta mutant and we discuss to what extent this could explain the stability of the fermentative capacity.     

Genetics of Yeast Glucokinase

Mutants of Saccharomyces cerevisiae lacking glucokinase (EC 2.7.1.2) have no discernible phenotypic difference from the wild-type strain; in a hexokinaseless background, however, they are unable to grow on any sugar except galactose. Reversion studies with glucokinase mutants indicate that the yeast S. cerevisiae has no other enzyme for phosphorylating glucose except the two hexokinases, P1 and P2, and glucokinase. Spontaneous revertants of hxk1 hxk2 glk1 strains collected on glucose regain any one of these three enzymes. The majority of glucokinase revertants synthesize species of enzyme activity that are kinetically or otherwise indistinguishable from the wild-type enzyme. In a few cases the reverted enzyme is very perceptibly altered in properties with a Km for glucose two orders of magnitude higher than that of the enzyme from the wild-type parent. These recessive, noncomplementing mutants, thus, define a single structural gene GLK1 of glucokinase. Yeast diploids lacking all of the three enzymes for glucose phosphorylation fail to sporulate. Heterozygosity of either of the hexokinase genes HXK1 or HXK2, but not GLK1, restores sporulation. The location of GLK1 on chromosome III was indicated by loss of this chromosome when hexokinaseless diploids heterozygous for glk1 were selected for resistance to 2-deoxyglucose; the homologue of chromosome III carrying GLK1, the mating-type allele and other nutritional markers on this chromosome was lost. Meiotic mapping of glucokinase executed with heterozygosity of one of the hexokinases indicated that the gene GLK1 defining the structure of glucokinase protein is located on the left arm of chromosome III 24 cM to the left of his4 in the order: leu2--his4--glk1. --Only two of 206 independent glucokinase mutants are nonsense ochre, both of which map at one end of the gene. In hxk1 only one of 130 isolates is a nonsense mutation, whereas in hxk2 none has been found among 220 independent mutants. These results raise the possibility that the protein products of these genes have some other essential function. --An earlier mapping result for hxk2 has been corrected. The new location is on the left arm of chromosome VII, 17 cM distal to ade5 in the order: lys5--ade5--hxk2.     

The 15 N-terminal amino acids of hexokinase II are not required for in vivo function: analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae

The function of the N-terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form). This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short form protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild-type protein dimerizes. Kinetic studies show that the enzymatic activities are very similar to wild-type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form of the enzyme is similar to the wild-type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N-terminal amino acids of hexokinase II are not required in vivo either for phosphorylation of hexoses or for glucose repression.     

Saccharomyces Cerevisiae Null Mutants in Glucose Phosphorylation: Metabolism and Invertase Expression

A congenic series of Saccharomyces cerevisiae strains has been constructed which carry, in all combinations, null mutations in the three genes for glucose phosphorylation: HXK1, HXK2 and GLK1, coding hexokinase 1 (also called PI or A), hexokinase 2 (PII or B), and glucokinase, respectively: i.e., eight strains, all of which grow on glucose except for the triple mutant. All or several of the strains were characterized in their steady state batch growth with 0.2% or 2% glucose, in aerobic as well as respiration-inhibited conditions, with respect to growth rate, yield, and ethanol formation. Glucose flux values were generally similar for different strains and conditions, provided they contained either hexokinase 1 or hexokinase 2. And their aerobic growth, as known for wild type, was largely fermentative with ca. 1.5 mol ethanol made per mol glucose used. The strain lacking both hexokinases and containing glucokinase was an exception in having reduced flux, a result fitting with its maximal rate of glucose phosphorylation in vitro. Aerobic growth of even the latter strain was largely fermentative (ca. 1 mol ethanol per mol glucose). Invertase expression was determined for a variety of media. All strains with HXK2 showed repression in growth on glucose and the others did not. Derepression in the wild-type strain occurred at ca. 1 mM glucose. The metabolic data do not support- or disprove-a model with HXK2 having only a secondary role in catabolite repression related to more rapid metabolism.     

31P NMR and 13C NMR studies of recombinant Saccharomyces cerevisiae with altered glucose phosphorylation activities

Manipulation of cellular metabolism to maximize the yield and rate of formation of desired products may be achieved through genetic modification. Batch fermentations utilizing glucose as a carbon source were performed for three recombinant strains of Saccharomyces cerevisiae in which the glucose phosphorylation step was altered by mutation and genetic engineering. The host strain (hxk1 hxk2 glk) is unable to grow on glucose or fructose; the three plasmids investigated expressed hexokinase PI, hexokinase PII, or glucokinase, respectively, enabling more rapid glucose and fructose phosphorylation in vivo than that provided by wild-type yeast.Intracellular metabolic state variables were determined by ³¹P NMR measurements of in vivo fermentations under nongrowth conditions for high cell density suspensions. Glucose consumption, ethanol and glycerol production, and polysaccharide formation were determined by ¹³C NMR measurements under the same experimental conditions as used in the ³¹P NMR measurements. The trends observed in ethanol yields for the strains under growth conditions were mimicked in the nongrowth NMR conditions.Only the strain with hexokinase PI had higher rates of glucose consumption and ethanol production in comparison to healthy diploid strains in the literature. The hexokinase PII strain drastically underutilized its glucose-phosphorylating capacity. A regulation difference in the use of magnesium-free ATP for this strain could be a possible explanation. Differences in ATP levels and cytoplasmic pH values among the strains were observed that could not have been foreseen. However, cytoplasmic pH values do not account for the differences observed among in vivo and in vitro glucose phosphorylation activities of the three recombinant strains.     

Construction of a flocculating yeast for fructose production from inulin

Construction of flocculating yeast lacking for fructose utilisation was realised by integration of the FLO1 flocculation gene in the ribosomal DNA of an hexokinase deficient (hxk1, hxk2) Saccharomyces cerevisiae strain (ATCC36859). Simultaneous production of ethanol and fructose was obtained from glucose/fructose mixtures or from hydrolysed Jerusalem artichoke extracts using the transformed yeast in batch fermentations and in a continuous reactor with internal biomass recycle. This allowed the production of 5 g ethanol/L and 48 g sugars/L containing up to 99 % fructose from diluted hydrolysed Jerusalem artichoke extracts containing 60 g sugars/L. © Rapid Science Ltd. 1998     

Complete nucleotide sequence of the hexokinase PI gene (HXK1) of Saccharomyces cerevisiae

The nucleotide sequence of the yeast glycolytic hexokinase isoenzyme PI-gene, HXK1, has been determined by sequencing the yeast DNA insert of the previously isolated plasmid HXK1 clone [Entian et al., Mol. Gen. Genet. 198 (1984) 50-54]. The structural gene sequence included 1452 bp coding for 484 amino acid (aa) residues corresponding to the Mr of 153 605 for the HXK1 monomer. Several initiation regions and termination points were located using nuclease S1 mapping. The HXK1 sequence was 76% homologous with that of HXK2, which is responsible for triggering glucose repression in yeasts. Since HXK1 is not involved in this regulatory system, the regulatory function of HXK2 must correspond to one or more of the differences between both isoenzymes. Most changes in the amino acid sequence were statistically distributed; however, four clustered regions with more than five altered aa residues were identified.