Cloning and biochemical characterisation of Aspergillus niger hexokinase

Abstract

The Aspergillus niger hexokinase gene hxkA has been cloned by heterologous hybridisation using the Aspergillus nidulans hexokinase gene as a probe. The DNA sequence of the gene was determined, and the deduced amino acid sequence showed significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to those of the budding yeasts. The encoded protein was purified from a multicopy hxkA transformant, and extensively characterised. The hexokinase protein has a molecular mass of 54 090, a pI of 4.9 and is a homodimer. D-Glucose, the glucose analogue 2-deoxy-D-glucose, D-fructose, D-mannose and D-glucosamine are phosphorylated by hexokinase, whereas the hexoses Dgalactose, L-sorbose, methyl A-D-glucoside and the pentoses L-arabinose and D-xylose are not. The enzyme has high affinity for glucose (K m ϭ 0.35 mM at pH 7.5) and for fructose (K m ϭ 2.0 mM at pH 7.5) and is inhibited by ADP. The enzyme is strongly inhibited by physiological concentrations (0.1Ϫ0.2 mM) of trehalose 6-phosphate, which may be of importance for in vivo regulation of the enzyme. Inhibition of A. niger hexokinase by trehalose 6-phosphate is competitive towards the sugar substrate (K i ϭ 0.01 mM). Based on the kinetic constants of hexokinase and glucokinase their relative contribution to in vivo glucose phosphorylation was calculated and found to be strongly dependent on intracellular pH and glucose concentration. At pH 7.5 glucokinase is predominant, whereas at pH 6.5 hexokinase is predominant at glucose concentrations higher than 0.5 mM. Expression of the hexokinase and the glucokinase gene requires active carbon metabolism. Also on carbon sources which are not substrates for hexokinase or glucokinase, clear expression is observed. The hexokinase and glucokinase enzymes are quite stable in vivo. Even in the absence of transcription, active glucokinase and hexokinase remain present in the cells at almost the same level for at least 3Ϫ4 h after depletion of the carbon source.Keywords : Aspergillus niger; hexokinase; glucokinase; purification; expression ; trehalose 6-phosphate.In a recent publication we described the cloning of the Aspergillus niger glucokinase gene and provided evidence for the existence of separate glucokinase and hexokinase enzymes in this industrially important microorganism .Both metabolic modelling (Torres, 1994a, b) and experimental studies (Schreferl-Kunar et al., 1989) indicate that hexose phosphorylation may be of major importance in the regulation of carbon flux in A. niger. Recently, Arisan-Atac et al. (1996) found increased rates of citric acid production in a strain in which the trehalose 6-phosphate synthase gene (ggs1) was disrupted. Saccharomyces cerevisiae hexokinases PI and PII are strongly inhibited by trehalose 6-phosphate, which is also a potent inhibitor of A. niger hexokinase (Arisan-Atac et al., 1996; this report). This inhibition may play an important role in the regulation of glycolytic flux in S. cerevisiae, although the precise mechanism by which trehalose 6-phosphate or ...

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

“…In A. niger, hexokinase HxkA and glucokinase GlkA were found to contribute similarly to glucose phosphorylation. However, this was strongly dependent on both the intracellular pH and the glucose concentration (Panneman et al, 1998).…”

“…In Aspergillus niger, the hexokinase and the glucokinase have been purified and biochemically characterized. Both enzymes seem to contribute similarly to the rate of glucose phosphorylation in vivo, dependent on the pH and the glucose concentration (Panneman et al, 1996(Panneman et al, , 1998). …”

“…Similar to yeast hexokinases, the A. niger hexokinase is strongly inhibited by trehalose 6-phosphate, which plays a role in the regulation of hexokinase activity also in vivo (Arisan-Atac et al, 1996;Panneman et al, 1998). In A. nidulans, the possible involvement of hexokinases in catabolite repression was indicated by the analysis of mutants defective in either hexo-or glucokinase or in both enzymes (Flipphi et al, 2003).…”

Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli

“…In the latter case, the glucokinase was suggested to regulate the blood sugar concentration with a high K m [47,48]. Compared with much lower K m values of glucokinase (0.063 mM) and hexokinase (0.35 mM) for glucose in A. niger [49,50], K m value of LG kinase at about 71 mM for levoglucosan indicated that A. niger cells might accumulate levoglucosan in vivo at a rate higher than that of glucose to produce enough amount of glucose 6-phosphate and then convert it into high yield of citric acid. Besides this, some enzymatic properties of LG kinase different from those of other general hexose kinases may also contribute to the metabolism of levoglucosan in A. niger: Firstly, the activity of glucokinase in A. niger decreases rapidly at pHs below 7.5 ( 56% at pH 7.0 and 17% at pH 6.5) and this may have important implications for the in vivo regulation of activity [49], while LG kinase in A. niger keeps its activity in a wide range of pHs from 6.0 to 10.0.…”

“…LG kinase reaction product, glucose 6-phosphate also had no inhibition. On the contrary, hexokinase in A. niger can be strongly inhibited by physiological concentrations of the reaction product, such as trehalose 6-phosphate [50].…”