Sugar kinases reveal a multilayered control system governing glycolytic entry in a fungal pathogen

The opportunistic fungal pathogen Nakaseomyces glabratus, formerly known as Candida glabrata, survives in some of the most nutritionally unpredictable corners of the human body, from the sugar-sparse bloodstream to the transiently fed oral and gastrointestinal tracts. A new study published in the journal Fungal Biology and Biotechnology maps, in unusual detail, how this fungus decides when and how fast to let sugar into glycolysis, the central pathway that converts hexose sugars into energy and building blocks for biosynthesis. The answer, the researchers report, is not a single enzyme or a single switch but a layered control architecture built from three specialized sugar kinases, feedback inhibition by a trehalose metabolite, condition-dependent gene expression and a direct coupling between phosphorylation and sugar import.

Homology searches against well-characterized kinases from the baker’s yeast Saccharomyces cerevisiae and the pathogen Candida albicans turned up five candidates: Hxk1, Hxk2, Hxk2b, Glk1 and Glk1b. A maximum-likelihood phylogenetic tree built from these protein sequences, together with known kinases from the two comparison species, separated the enzymes into two major clades corresponding to hexokinases and glucokinases. Pairwise amino acid identities among the five N. glabratus proteins ranged from 14.85 percent to 85.39 percent, with the closest pair being Hxk2 and Hxk2b at 78 percent identity and the most distant pair Hxk1 and Glk1b.

Sequence inspection already hinted that not all five enzymes would work. Hxk2, Hxk2b and Glk1 conserved the canonical glucose-binding and ATP-binding residues seen in characterized fungal kinases, while Hxk1 and Glk1b diverged sharply at the ATP-binding sites and lacked key glucose-binding residues, with Glk1b missing all of them. Motif and domain analyses reinforced the split: Glk1b carries only a single hexokinase-2 domain, whereas the other four enzymes possess both hexokinase-1 and hexokinase-2 domains. Experimental confirmation followed. When the researchers deleted HXK2, HXK2b and GLK1 together, all detectable phosphorylation of glucose, fructose and mannose disappeared from cell extracts, proving that Hxk1 and Glk1b contribute nothing to hexose phosphorylation under the tested conditions. The triple deletion strain also failed to grow on every fermentable carbon source tested, confirming that the fungus relies exclusively on the three active kinases for glycolytic entry.

Kinetic profiling of strains expressing only a single functional kinase revealed a clear division of labor. The two hexokinases, Hxk2 and Hxk2b, behaved as high-capacity, lower-affinity enzymes, achieving high maximal phosphorylation rates that suit sugar-abundant conditions. Glk1, by contrast, showed the lowest apparent Km values for glucose and mannose but consistently the lowest maximal rates, the signature of a high-affinity, low-capacity scavenger optimized for scarce sugar. Substrate chemistry shaped the pattern across all enzymes: mannose, an aldose like glucose, was bound most tightly, fructose, a ketose, was the poorest substrate with the highest apparent Km values, and Glk1 showed no fructose phosphorylation at all, leaving fructose metabolism entirely to the hexokinases. Apparent catalytic efficiencies were comparable for glucose and mannose but markedly lower for fructose, where Hxk2 outperformed Hxk2b by more than two-fold.

Yet intrinsic kinetics did not predict physiological importance. Although Hxk2 and Hxk2b displayed broadly similar catalytic properties, deleting HXK2b alone cut total phosphorylation by 49 percent for glucose, 61 percent for fructose and 43 percent for mannose, whereas removing HXK2 or GLK1 individually had no significant effect on overall activity. Hxk2b alone was sufficient to sustain wild-type phosphorylation levels on glucose and fructose, marking it as the dominant hexose-phosphorylating enzyme under the conditions tested. The authors attribute this dominance not to catalytic superiority but to differential regulation, an organizational principle they note is echoed in S. cerevisiae and C. albicans, where Hxk2 likewise leads hexose phosphorylation.

A second regulatory layer came from trehalose-6-phosphate, or T6P, a central intermediate of trehalose metabolism derived from glucose-6-phosphate and a known inhibitor of fungal hexokinases. Using equimolar hexose and T6P in enzyme assays, the researchers found that T6P strongly inhibited both hexokinases while leaving Glk1 untouched. Hxk2 was the more sensitive of the two, losing 60 percent of its glucose phosphorylation, 66 percent on fructose and 35 percent on mannose, whereas Hxk2b lost 29, 55 and 21 percent respectively. The authors argue that this split makes functional sense: feedback inhibition reins in the high-capacity hexokinases when sugar is plentiful and uncontrolled phosphorylation could deplete ATP and trigger metabolic imbalance, while the T6P-insensitive Glk1 keeps operating during scarcity, when strong feedback would be counterproductive. They point out that a glucose pulse in S. cerevisiae diverts roughly 30 percent of incoming sugar into trehalose metabolism, and that T6P-insensitive hexokinases exist in the fission yeast Schizosaccharomyces pombe and in most mammalian cells, underscoring how flexible this control mechanism is across evolution.

Transcriptional tuning added a third layer. When cells grown on glycerol were pulsed with sugar for an hour, HXK2 expression was consistently higher at 2 percent sugar than at 0.1 percent, regardless of which hexose was offered. HXK2b moved the opposite way, rising under low-sugar conditions significantly in fructose- and mannose-containing media, and GLK1 was strongly induced by sugar limitation, with transcripts at 0.1 percent sugar more than doubling relative to 2 percent. Notably, GLK1 responded to fructose availability even though Glk1 cannot phosphorylate fructose, which the authors interpret as regulation by overall carbon status rather than direct substrate use. The low-sugar regime matters physiologically: blood glucose is held near 3 to 5 millimolar, roughly 0.06 to 0.1 percent, vaginal glucose often falls below 0.05 percent, and fructose and mannose circulate at concentrations orders of magnitude lower. Deleting individual kinase genes, meanwhile, did not induce compensatory upregulation of the remaining kinases, indicating that N. glabratus relies on pre-established specialization rather than dynamic rebalancing, a contrast with the compensatory network described in S. cerevisiae.

A fourth layer emerged from subcellular localization. All three enzymes were detected throughout the cell, but their nuclear-to-cytoplasmic distribution shifted with glucose availability: Hxk2 and Hxk2b became about 10 percent more nuclear-enriched upon glucose starvation, while Glk1, already less nuclear than cytoplasmic under high glucose, dropped a further 10 percent. Western blots confirmed only full-length fusion proteins were present, ruling out cleaved free mCherry as an artifact. The pattern is notable because it runs opposite to the classical model in S. cerevisiae and C. albicans, where nuclear accumulation of hexokinases accompanies glucose abundance and glucose repression. The authors conclude that nuclear localization in N. glabratus is probably not tightly coupled to glucose repression and may reflect broader metabolic or regulatory functions whose consequences remain unclear.

The final and perhaps most striking layer connects phosphorylation to import itself. Using radiolabeled glucose and fructose, the team showed that uptake of each sugar was nearly abolished in strains lacking the enzymes needed to phosphorylate it, supporting the idea that kinases maintain a low intracellular free sugar pool and thereby sustain the concentration gradient that drives transport. But the uptake phenotypes were not explained by kinase activity alone. Deleting GLK1 reduced glucose import despite the mutant having wild-type glucose kinase activity, while deleting both hexokinases, which left only about 20 percent residual activity, unexpectedly increased glucose uptake. Manipulating the trehalose pathway clarified part of the puzzle: deleting TPS1, which prevents T6P production, increased sugar import, whereas deleting TPS2, which causes T6P to accumulate, reduced it, nearly abolishing uptake in strains dependent on T6P-sensitive hexokinases. Intriguingly, TPS1 and TPS2 deletions still modulated glucose uptake even in the hexokinase-double-deletion background, and the authors suggest trehalose metabolism may act through downstream glycolytic flux, transporter interactions or transporter-kinase complexes, mechanisms they did not directly assess.

Taken together, the study portrays glycolytic entry in N. glabratus as an integrated control point rather than a simple gate. Enzyme specialization, transcriptional tuning, T6P-mediated feedback and phosphorylation-coupled uptake combine to keep carbon flowing across sugar concentrations that swing from feast to famine in different host niches, a flexibility the authors link to the fungus’s ability to grow robustly in fluctuating and often sugar-limited environments. The work also carries context beyond basic metabolism: glycolytic enzymes have been proposed as potential antifungal drug targets, and glycolytic activity is upregulated during specific infection stages in Candida albicans. The authors note their analysis was limited to mRNA levels, so post-transcriptional compensation cannot be excluded, and that condition-dependent changes in kinase oligomerization, not examined here, could further refine the physiological roles of the three active enzymes.

Subject of Research: Multilayered regulation of hexose phosphorylation and uptake by specialized sugar kinases in the pathogenic fungus Nakaseomyces glabratus

Article Title: Multilayered control of hexose uptake and phosphorylation through specialized sugar kinases in Nakaseomyces glabratus

Article References: Vreys, J., Schraepen, E., Peterson, T., Wijnants, S., & Van Dijck, P. (2026). Multilayered control of hexose uptake and phosphorylation through specialized sugar kinases in Nakaseomyces glabratus. Fungal Biology and Biotechnology, 13(1), Article 10. https://doi.org/10.1186/s40694-026-00216-0

Image Credits: AI Generated

DOI: 10.1186/s40694-026-00216-0

Keywords: Nakaseomyces glabratus, sugar kinases, hexokinase, glucokinase, glycolysis, trehalose-6-phosphate, hexose uptake, enzyme kinetics, glucose repression, fungal metabolism, opportunistic pathogen, gene expression