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

The opportunistic pathogenic fungus Nakaseomyces glabratus inhabits diverse host niches with fluctuating nutrient availability. Therefore, efficient control of glycolytic entry is essential, yet the regulatory principles governing hexose phosphorylation in this species remain incompletely understood. Here, we investigated the functional organization of sugar kinases in N. glabratus. Among five predicted sugar kinases, only three (Hxk2, Hxk2b and Glk1) catalyzed phosphorylation of glucose, fructose or mannose, whereas Hxk1 and Glk1b lacked detectable activity. Kinetic analyses revealed a functional specialization, with the hexokinases acting as high-capacity enzymes and Glk1 functioning as a high-affinity, low-capacity kinase optimized for low-sugar concentrations. Despite similar intrinsic kinetics among the hexokinases, Hxk2b emerged as the physiologically dominant enzyme, reflecting differential regulation rather than catalytic properties alone. Both hexokinases, but not Glk1, were strongly inhibited by trehalose-6-phosphate, linking glycolytic entry to trehalose metabolism. Consistent with this, perturbation of trehalose synthesis modulated hexose uptake, revealing that phosphorylation capacity is a major driver of sugar import, while metabolic feedback further constrains uptake. Nuclear localization of sugar kinases and condition-dependent expression patterns indicate additional regulatory layers. Together, our results demonstrate that N. glabratus controls glycolytic entry through a multilayered architecture integrating enzyme specialization, transcriptional tuning, trehalose-6-phosphate-mediated feedback and uptake coupling. This systems-level organization results in robust growth across fluctuating and often sugar-limited host environments.