Biomedical subjects

G J Ruijter

Publications and source records attributed to G J Ruijter.

13 recordsLinked to original sources

PubMed2001

The Aspergillus niger D-xylulose kinase gene is co-expressed with genes encoding arabinan degrading enzymes, and is essential for growth on D-xylose and L-arabinose.

The Aspergillus niger D-xylulose kinase encoding gene has been cloned by complementation of a strain deficient in D-xylulose kinase activity. Expression of xkiA was observed in the presence of L-arabinose, L-arabitol and D-xylose. Expression of xkiA is not mediated by XLNR, the xylose-dependent positively-acting xylanolytic regulator. Although the expression of xkiA is subject to carbon catabolite repression, the wide domain regulator CREA is not directly involved. The A. niger D-xylulose kinase was purified to homogeneity, and the molecular mass determined using electrospray ionization mass spectrometry agreed with the calculated molecular mass of 62816.6 Da. The activity of XKIA is highly specific for D-xylulose. Kinetic parameters were determined as Km(D-xylulose) = 0.76 mM and Km(ATP) = 0.061 mM. Increased transcript levels of the genes encoding arabinan and xylan degrading enzymes, observed in the xylulose kinase deficient strain, correlate with increased accumulation of L-arabitol and xylitol, respectively. This result supports the suggestion that L-arabitol may be the specific low molecular mass inducer of the genes involved in arabinan degradation. It also suggests a possible role for xylitol in the induction of xylanolytic genes. Conversely, overproduction of XKIA did not reduce the size of the intracellular arabitol and xylitol pools, and therefore had no effect on expression of genes encoding xylan and arabinan degrading enzymes nor on the activity of the enzymes of the catabolic pathway.

Adenosine Diphosphate↗

PubMed2000

Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production.

Using a combination of dye adsorption and affinity elution we purified Aspergillus niger citrate synthase to homogeneity using a single column and characterised the enzyme. An A. niger citrate synthase cDNA was isolated by immunological screening and used to clone the corresponding citA gene. The deduced amino acid sequence showed high similarity to other fungal citrate synthases. After processing upon mitochondrial import, the calculated M(r) of A. niger citrate synthase is 48501, which agrees well with the estimated molecular mass of the purified protein (48 kDa). In addition to an N-terminal mitochondrial import signal, a peroxisomal target sequence (AKL) was found at the C-terminus of the protein. Whether both signals are functional in vivo is not clear. Strains overexpressing citA were made by transformation and cultured under citric acid-producing conditions. Up to 11-fold overproduction of citrate synthase did not increase the rate of citric acid production by the fungus, suggesting that citrate synthase contributes little to flux control in the pathway involved in citric acid biosynthesis by a non-commercial strain.

Acetyl Coenzyme A↗

PubMed2000

Measurement of intracellular (compartmental) pH by 31P NMR in Aspergillus niger.

31P nuclear magnetic resonance (31P NMR) was used to monitor cytoplasmic and vacuolar pH values in the filamentous fungus Aspergillus niger. To obtain a homogeneous cell sample and to be able to perform long term in vivo NMR measurements A. niger mycelium was kept in a setup that allows perfusion of the cell plug within the NMR tube. Mycelial samples, however, became rapidly clogged during perfusion leading to (partial) anaerobiosis of the plug with subsequent acidification of the cytoplasm. As a result, only short-term NMR measurements (5-10 min) were possible using free mycelium. To increase and to prolong perfusion, A. niger was immobilized in Ca(2+)-alginate beads. Deteriorated spectra recorded under hypoxia could be completely restored in the presence of oxygen. With this system perfusion in the presence of citrate could be maintained for at least 18 h at much higher rates (15 ml min-1 compared with 4 ml min-1 for free mycelium). During this period 31P NMR spectra were highly invariable, indicating approximate steady-state intracellular conditions during long term measurements. Perfusion in the presence of glucose resulted in complete depletion of the vacuolar inorganic phosphate pool within 45 min and yielded a higher pH gradient over the tonoplast than when citrate was used (delta pH = 1.6 and 1.4, respectively).

Alginates↗

PubMed1999

Characterization of Aspergillus niger phosphoglucose isomerase. Use for quantitative determination of erythrose 4-phosphate.

Phosphoglucose isomerase (PGI) was purified from Aspergillus niger and the in vitro kinetic properties of the enzyme were related to its functioning in vivo. A new assay method was developed to study the forward reaction making use of mannitol 1-P dehydrogenase as the coupling enzyme. In this simple assay system mannitol 1-P dehydrogenase converts fructose 6-P and NADH to mannitol 1-P and NAD+, respectively. At pH 7.5 the Km for glucose 6-P was 0.48 mM, whereas the Km for fructose 6-P was 0.32 mM. The pentose phosphate pathway intermediates 6-phosphogluconate and erythrose 4-P (E4P) were competitive inhibitors of PGI with Ki values of approximately 0.2 mM and 1 microM respectively. In citric acid producing A. niger mycelium inhibition by 6-phosphogluconate is of minor physiological significance (10% inhibition). Since E4P could not be detected by an existing procedure, a novel assay was developed based on the strong inhibition of PGI by E4P. Although the new assay is very sensitive (detection limit 25 pmol), E4P could still not be detected in metabolite extracts indicating that a very low level of E4P is present in the cells. Using in vitro kinetics and concentrations of intracellular metabolites the in vivo activity of PGI was calculated and closely matched the steady state glycolytic flux observed during citric acid production.

Aspergillus niger↗

PubMed1998

Cloning and biochemical characterisation of Aspergillus niger hexokinase--the enzyme is strongly inhibited by physiological concentrations of trehalose 6-phosphate.

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 54090, 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 D-galactose, L-sorbose, methyl alpha-D-glucoside and the pentoses L-arabinose and D-xylose are not. The enzyme has high affinity for glucose (Km = 0.35 mM at pH 7.5) and for fructose (Km = 2.0 mM at pH 7.5) and is inhibited by ADP. The enzyme is strongly inhibited by physiological concentrations (0.1-0.2 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 (Ki = 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 3-4 h after depletion of the carbon source.

Amino Acid Sequence↗

PubMed1997

Carbon repression in Aspergilli.

Many microorganisms prefer easily metabolizable carbon sources over alternative, less readily metabolized carbon sources. One of the mechanisms to achieve this is repression of the synthesis of enzymes related to catabolism of the alternative carbon sources, i.e. carbon repression. It is now clear that in Aspergillus nidulans and Aspergillus niger the repressor protein CREA plays a major role in carbon repression. CREA inhibits transcription of many target genes by binding to specific sequences in the promoter of these genes. Unfortunately there is little information on other components of the signalling pathway that triggers repression by CREA. In this review we summarize the current understanding of carbon repression in Aspergilli.

Aspergillus↗

PubMed1997

Overexpression of phosphofructokinase and pyruvate kinase in citric acid-producing Aspergillus niger.

Phosphofructokinase and pyruvate kinase were overexpressed in the filamentous fungus Aspergillus niger. Moderate overexpression of these glycolytic enzymes in A. niger N400 (3-5-fold the wild-type level), either individually or simultaneously, did not increase citric acid production by the fungus significantly. Thus, phosphofructokinase and pyruvate kinase do not seem to contribute in a major way to flux control of the metabolism involved in the conversion of glucose to citric acid. Overexpression of phosphofructokinase and pyruvate kinase did not influence the activities of other enzymes in the pathway, nor did it change intermediary metabolite levels. However, in strains overexpressing phosphofructokinase, the level of fructose 2,6-bisphosphate, a positive allosteric effector of phosphofructokinase, was reduced almost 2-fold compared to the wild-type strain. Measurements with purified phosphofructokinase, using substrate, product and effector concentrations found intracellularly, showed that such a reduction in the fructose-2,6-bisphosphate level could decrease the specific activity of phosphofructokinase in the cell significantly. Thus, the fungus seems to adapt to overexpression of phosphofructokinase by decreasing the specific activity of the enzyme through a reduction in the level of fructose 2,6-bisphosphate.

Aspergillus niger↗

PubMed1996

Cloning and biochemical characterisation of an Aspergillus niger glucokinase. Evidence for the presence of separate glucokinase and hexokinase enzymes.

The Aspergillus niger glucokinase gene glkA has been cloned using a probe generated by polymerase chain reaction with degenerate oligonucleotides. The DNA sequence of the gene was determined, and the deduced amino acid sequence shows significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to the Saccharomyces cerevisiae glucokinase protein. The encoded protein was purified from a multicopy glkA transformant, and extensively characterised. The protein has a molecular mass of 54536 Da and a pI of 5.2. The enzyme has high affinity for glucose (K(m) 0.063 mM at pH 7.5) and a relatively low affinity for fructose (K(m) 120 mM at pH 7.5), and in vivo fructose phosphorylation by glucokinase is consequently negligible. The configurations at C1 and C4 of the substrate appear to be essential for substrate specificity. The A. niger glucokinase shows non-competitive inhibition by ADP towards ATP and uncompetitive inhibition by ADP towards glucose. The kcal (turnover number) decreases rapidly below pH 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. In addition, proof is provided for the presence of a second hexosephosphorylating enzyme in A. niger. This enzyme is probably a hexokinase, since unlike glucokinase, this activity is inhibited by trehalose 6-phosphate.

Adenosine Diphosphate↗

PubMed1996

Characterisation of the Aspergillus nidulans frA1 mutant: hexose phosphorylation and apparent lack of involvement of hexokinase in glucose repression.

Hexose phosphorylation was studied in Aspergillus nidulans wild-type and in a fructose non-utilising mutant (frA). The data indicate the presence of at least one hexokinase and one glucokinase in wild-type A. nidulans, while the frA1 mutant lacks hexokinase activity. The A. nidulans gene encoding hexokinase was isolated by complementation of the frA1 mutation. The absence of hexokinase activity in the frA1 mutant did not interfere with glucose repression of the enzymes involved in alcohol and L-arabinose catabolism. This suggest that, unlike the situation in yeast where mutation of hexokinase PII abolishes glucose repression, the A. nidulans hexokinase might not be involved in glucose repression.

Amino Acid Sequence↗

PubMed1995

An extreme creA mutation in Aspergillus nidulans has severe effects on D-glucose utilization.

Aspergillus nidulans wild-type and the extreme carbon catabolite derepressed mutant creAd-30 were characterized with respect to enzyme activities, metabolite concentrations and polyol pools all related to glycolysis, after growth on D-glucose. In the creAd-30 strain the enzymes hexokinase and fructose-6-phosphate reductase showed a two- and threefold increase in activity, respectively, whereas phosphofructokinase and pyruvate kinase activity decreased two- and threefold, respectively, in comparison with the wild-type strain. The most notable changes in metabolite concentrations were that fructose 2,6-bisphosphate and fructose 1,6-bisphosphate showed a 2.5-fold increase, whereas both pyruvate and citrate decreased in the creAd-30. Striking differences were found for the polyol concentrations measured for the two strains tested. Intracellular glycerol and arabitol concentrations were 10-fold higher and erythritol fivefold higher in creAd-30, whereas intracellular trehalose and mannitol were both decreased. The total internal polyol concentration appears to be constant at approximately 700 mumol (g dry wt)-1. All polyols were also detected in high amounts in the culture filtrate of the creAd-30 mutant strain but no extracellular trehalose was found. The overall production of polyols in this strain was therefore much higher than in the wild-type. The high level of polyols produced and the changes in metabolite concentrations in the creAd-30 strain suggest that the differences in enzyme activities result in an altered flow through glycolysis leading to a more rapid formation of polyols which are subsequently secreted.

Aspergillus nidulans↗

PubMed1992

Analysis of mutations that uncouple transport from phosphorylation in enzyme IIGlc of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.

Mutations that uncouple glucose transport from phosphorylation were isolated in plasmid-encoded Escherichia coli enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The uncoupled enzymes IIGlc were able to transport glucose in the absence of the general phosphoryl-carrying proteins of the PTS, enzyme I and HPr, although with relatively low affinity. Km values of the uncoupled enzymes IIGlc for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal IIGlc. Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for IIGlc), indicating that transport and phosphorylation are separable functions of the enzyme. Some of the uncoupled enzymes IIGlc transported glucose with a higher rate and lower apparent Km in a pts+ strain than in a delta ptsHI strain lacking the general proteins enzyme I and HPr. Since the properties of these uncoupled enzymes IIGlc in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type IIGlc, these mutants appeared to be conditionally uncoupled. Sequencing of the mutated ptsG genes revealed that all amino acid substitutions occurred in a hydrophilic segment within the hydrophobic N-terminal part of IIGlc. These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate.

Base Sequence↗

PubMed1991

Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc.

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme IIGlc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat- and batch cultures were compared. Strain PP799 transported and phosphorylated glucose via an intact PTS, while strain PP952 took up glucose exclusively via uncoupled enzyme IIGlc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP799 and PP952 exhibited similar YGlc values. Assuming equal YATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme IIGlc.

Anaerobiosis↗

PubMed1990

Adaptation of Salmonella typhimurium mutants containing uncoupled enzyme IIGlc to glucose-limited conditions.

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP):glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. As a result of the ptsG mutation, the apparent Km of the system for glucose transport is increased about 1,000-fold (approximately 18 mM) compared with wild-type PTS-mediated glucose transport. An S. typhimurium mutant containing uncoupled enzyme IIGlc as the sole system for glucose uptake was grown in glucose-limited chemostat cultures. Selective pressure during growth in the chemostat resulted in adaptation to the glucose-limiting conditions in two different ways. At first, mutations appeared that led to a decrease in Km value of uncoupled enzyme IIGlc. These results suggested that uncoupled enzyme IIGlc had significant control on the growth rate under glucose-limiting conditions. More efficient glucose uptake enabled a mutant to outgrow its parent and caused a decrease in the steady-state glucose concentration in the chemostat. At very low glucose concentrations (10 microM), mutants arose that contained a constitutively synthesized methyl-beta-galactoside permease. Apparently, further changes in the uncoupled enzyme IIGlc did not lead to a substantial increase in growth rate at very low glucose concentrations.

Biological Transport↗