Molecular cloning and characterization of the geneHXK1 encoding the hexokinase fromYarrowia lipolytica

Abstract

We have cloned the gene HXK1 from the dimorphic yeast Yarrowia lipolytica that encodes the unique hexokinase of this yeast. The gene has an intron located 39 base pairs after the A of the first ATG. The putative protein contains a sequence of 40 amino acids which is absent from other known hexokinase sequences. Y. lipolytica strains devoid of hexokinase grew in glucose slower than wild‐type. This growth was due to the existence of a glucokinase. The hexokinase from Y. lipolytica substituted effectively for hexokinase II from S. cerevisiae in catabolite repression of invertase. The hexokinases from Schizosaccharomyces pombe or Kluyveromyces lactis were much less effective in this role. The Km for glucose and fructose of hexokinase was 0·38 mM and 3·56 mM , respectively. The Km of glucokinase for glucose was 0·17 mM . While the hexokinase was strongly inhibited by trehalose‐6‐phosphate (Ki=3·6 µM), glucokinase was not affected by this compound. Copyright © 1999 John Wiley & Sons, Ltd.

The Role of Hexokinase and Hexose Transporters in Preferential Use of Glucose over Fructose and Downstream Metabolic Pathways in the Yeast Yarrowia lipolytica

“…We hypothesized that the molecular mechanism controlling preferential uptake of glucose over fructose in Y. lipolytica might hold in the affinity toward glucose and fructose of hexose transporters and/or hexokinase. YlHxk1 has over 9-fold higher affinity for glucose than for fructose [21], which could be one of the reasons for the yeasts' strict glucose preference. As mentioned above, some yeast species, such as S. pombe, have fructophilic hexokinase with a higher affinity for fructose than for glucose [25].…”

“…Furthermore, SpHxk1 might not interact with native proteins in the same manner as YlHxk1 does, therefore, allowing for uninterrupted fructose import. Structurally, YlHxk1 has a unique feature, namely a 37-amino acid loop not found in other known hexokinases, including SpHxk1 (Figure S5; [21]), which has been shown to be important in inhibitor binding and gene expression regulation [22]. This structure could, therefore, interact with YHT3 in effect decelerating sugar intake and defending the cell from the carbon overflow in the central carbon metabolism (Figure 5) and/or increased intracellular osmotic pressure.…”

“…Two enzymes with hexokinase activity have been identified in Y. lipolytica. One of them is a glucokinase, with glucose as the only substrate; the other one phosphorylates glucose, fructose and mannose [21] and is encoded by the YlHXK1 (YALI0B22308g) gene. The primary structure of YlHxk1 has quaint characteristics absent in other known hexokinases.…”

“…The primary structure of YlHxk1 has quaint characteristics absent in other known hexokinases. In addition to conserved domains allowing for the action on hexoses, YlHxk1 contains an atypical 37-amino acid loop spanning between glucose-and ATP-binding domains [21], which indirectly influences enzyme activity [22]. The overexpression of YlHXK1 enhanced fructose utilization in the Y. lipolytica strains showing tedious growth on this monosaccharide, such as the wild strain W29, and more generally improved hexose utilization, increased carbon flux through glycolysis, changed the preferential use of glycerol to glucose when both substrates were present and reduced filamentation [20,22,23].…”

“…The overexpression had, however, no effect on preferential uptake of glucose over fructose. The preference might in turn result from the kinetic properties of YlHxk1 as the enzyme has higher affinity for glucose than for fructose with a Km of 0.38 mM and 3.56 mM, respectively [21], even more pronounced than for the ScHxk2 hexokinase of S. cerevisiae (0.25 mM for glucose; 1.5 mM for fructose). In contrast, hexokinase from Schizosaccharomyces pombe (SpHxk1) is fructophilic with a Km of 8.4 mM for glucose and 1.5 mM for fructose [25].…”