The distribution of the NADPH regenerating mannitol cycle among fungal species

Hult, Karl; Veide, Andres; Gatenbeck, Sten

doi:10.1007/bf00406168

Cited by 86 publications

(52 citation statements)

References 15 publications

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“…Nevertheless, we cannot rule out that there are situations in which Glk1 is important in addition to Hxk1. With regard to the Dhxk1 mutant, it was expected not to grow on fructose or on mannitol, which is metabolized via the mannitol cycle, involving the phosphorylation of fructose (Hult et al, 1980;Velez et al, 2007). However, the sensitivity of the Dhxk1 mutant to fructose was not expected.…”

Section: Discussionmentioning

confidence: 99%

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

Rui

Hahn

2007

Microbiology

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Hexose kinases play a central role in the initiation of sugar metabolism of living organisms and have also been implicated in carbon catabolite repression in yeasts and plants. In this study, the genes encoding glucokinase (Glk1) and hexokinase (Hxk1) from the plant-pathogenic ascomycete Botrytis cinerea were isolated and functionally characterized. Glk1-deficient mutants were indistinguishable from the wild-type in all growth parameters tested. In contrast, Dhxk1 mutants lacking Hxk1 showed a pleiotropic growth defect. On artificial media, vegetative growth was retarded, and conidia formation strongly reduced. No or only marginal growth of Dhxk1 mutants was observed when fructose, galactose, sucrose or sorbitol were used as carbon sources, and fructose inhibited growth of the mutant in the presence of other carbon sources. B. cinerea mutants containing hxk1 alleles with point mutations leading to enzymically inactive enzymes showed phenotypes similar to the Dhxk1 disruption mutant, indicating that loss of hexose phosphorylation activity of Hxk1 is solely responsible for the pleiotropic growth defect. Virulence of the Dhxk1 mutants was dependent on the plant tissue: on leaves, lesion formation was only slightly retarded compared to the wild-type, whereas only small lesions were formed on apples, strawberries and tomatoes. The low virulence of Dhxk1 mutants on fruits was correlated with their high contents of sugars, in particular fructose. Heterologous expression of Hxk1 and Glk1 in yeast allowed their enzymic characterization, revealing kinetic properties similar to other fungal hexokinases and glucokinases. Both Dglk1 and Dhxk1 mutants showed normal glucose repression of secreted lipase 1 activity, indicating that, in contrast to yeast, B. cinerea hexose kinases are not involved in carbon catabolite repression. INTRODUCTIONHexose kinases are crucial enzymes for most living cells, by catalysing the intracellular trapping and feeding into metabolism of the hexoses glucose and fructose after their uptake. In addition, hexokinases have been shown in yeast, plants and mammals to participate in glucose signalling (De Winde et al., 1996;Moore et al., 2003;Efrat et al., 1994). Saccharomyces cerevisiae has three enzymes catalysing hexose phosphorylation, namely hexokinases (EC 2 . 7 . 1 . 1) Hxk1 and Hxk2, and glucokinase (EC 2 . 7 . 1 . 2) Glk1. Each of these enzymes can support growth on glucose, while either of the two hexokinases is required for growth on fructose (Lobo & Maitra, 1977;Gancedo et al., 1977). Hxk2, in addition, is mainly required for catabolite repression, which prevents the expression of genes involved in catabolism of less preferred carbon sources in the presence of a more favourable nutrient such as glucose (Entian, 1980). In the presence of glucose, Hxk2 interacts with the carbon catabolite repressor Mig1 and thereby is translocated into the nucleus. In the nucleus, the Hxk2-Mig1 complex seems to form a repressor complex that binds to the promoters of carbon-catabolite-repressible genes (Ahuatzi ...

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Section: Discussionmentioning

confidence: 99%

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

Rui

Hahn

2007

Microbiology

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“…In nonphotosynthetic microorganisms, the oxidation of glucose 6-phosphate to ribulose 5-phosphate is usually considered to be the main pathway for NADPH generation. Hult and Gatenbeck (18) have proposed the involvement of the mannitol cycle in NADPH generation in the fungus A. alternata and have suggested that this cyclic pathway is also important for NADPH production in other fungi (19). If the mannitol cycle is required for NADPH production, one might expect that the expression of the mannitol cycle genes would be increased under conditions that require relatively large quantities of NADPH.…”

Section: Discussionmentioning

confidence: 99%

“…In A. niger conidiospores, D-mannitol is the predominant carbon-containing compound and makes up 10 to 15% of the dry weight (41). The metabolic pathway for mannitol biosynthesis in ascomycete and deuteromycete fungi most likely consists of two steps (19) (Fig. 1).…”

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confidence: 99%

“…The net result of mannitol cycle operation is transhydrogenase activity (NADH ϩ NADP ϩ 3 NAD ϩ ϩ NADPH) at the expense of ATP. The enzymes of the mannitol cycle are present in many fungi (19), but there is no evidence that mannitol turnover is required for NADPH production in any fungus. Singh et al (34) did not find any correlation between the activities of the mannitol cycle enzymes and the need for NADPH in A. nidulans.…”

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Mannitol Is Required for Stress Tolerance inAspergillus nigerConidiospores

et al. 2003

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D-Mannitol is the predominant carbon compound in conidiospores of the filamentous fungus Aspergillus niger and makes up 10 to 15% of the dry weight. A number of physiological functions have been ascribed to mannitol, including serving as a reserve carbon source, as an antioxidant, and to store reducing power. In this study, we cloned and characterized the A. niger mpdA gene, which encodes mannitol 1-phosphate dehydrogenase (MPD), the first enzyme in the mannitol biosynthesis pathway. The mpdA promoter contains putative binding sites for the development-specific transcription factors BRLA and ABAA. Furthermore, increased expression of mpdA in sporulating mycelium suggests that mannitol biosynthesis is, to a certain extent, developmentally regulated in A. niger. Inactivation of mpdA abolished mannitol biosynthesis in growing mycelium and reduced the mannitol level in conidiospores to 30% that in the wild type, indicating that MPD and mannitol 1-phosphate phosphatase form the major metabolic pathway for mannitol biosynthesis in A. niger. The viability of spores after prolonged storage and germination kinetics were normal in an mpdA null mutant, indicating that mannitol does not play an essential role as a reserve carbon source in A. niger conidia. However, conidiospores of a ⌬mpdA strain were extremely sensitive to a variety of stress conditions, including high temperature, oxidative stress and, to a lesser extent, freezing and lyophilization. Since mannitol supplied in the medium during sporulation repaired this deficiency, mannitol appears to be essential for the protection of A. niger spores against cell damage under these stress conditions. Polyols or polyhydroxyalcohols are present in all organisms, from bacteria to animals. In particular, plants and fungi are known to accumulate high levels of polyols intracellularly, up to several hundred millimoles per liter. The filamentous fungus Aspergillus niger produces a number of different polyols, including glycerol, erythritol, and D-mannitol (41). The intracellular concentrations of the individual polyols in A. niger depend on growth conditions and developmental stage, suggesting that polyols have important functions in fungal physiology.The hexitol D-mannitol is accumulated by vascular plants (37) and many fungal species (21). In A. niger conidiospores, D-mannitol is the predominant carbon-containing compound and makes up 10 to 15% of the dry weight (41). The metabolic pathway for mannitol biosynthesis in ascomycete and deuteromycete fungi most likely consists of two steps (19) (Fig. 1). Fructose 6-phosphate is reduced to mannitol 1-phosphate by NAD ϩ -dependent mannitol 1-phosphate dehydrogenase (MPD), followed by the dephosphorylation of mannitol 1-phosphate, which is catalyzed by mannitol 1-phosphate phosphatase (MPP), yielding mannitol. The presence of MPD and MPP in several fungi, such as A. niger (22), Aspergillus nidulans (34), and Alternaria alternata (18), has been demonstrated, but these enzymes are also responsible for mannitol biosynthesis in protozoa...

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“…If C. neoformans synthesizes and catabolizes mannitol via the unidirectional mannitol cycle originally proposed by Hult et al (1980) and if MPD is a key component of the biosynthetic arm of this pathway, one would predict that MPD mRNA levels would be higher in glucosegrown C. neoformans cells than in mannitol-grown cells. We found, however, almost as much MPD mRNA in C. neoformans cells cultured in SC-mannitol as in cells cultured in SC-glucose, which was consistent with the observation by Niehaus & Flynn (1994) that mannitol-grown C. neoformans contained both MPD and M1Pase.…”

Section: Discussionmentioning

confidence: 99%

Mannitol-1-phosphate dehydrogenase from Cryptococcus neoformans is a zinc-containing long-chain alcohol/polyol dehydrogenase The GenBank accession numbers for the nucleotide sequences for the C. neoformans mannitol-1-phosphate dehydrogenase cDNA and gene are AF175685 and AF186474, respectively.

2000

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Cryptococcus neoformans, the causative agent of cryptococcosis, produces large amounts of mannitol in culture and in infected mammalian hosts. Although there is considerable indirect evidence that mannitol synthesis may be required for wild-type stress tolerance and virulence in C. neoformans, this hypothesis has not been tested directly. It has been proposed that mannitol-1-phosphate dehydrogenase (MPD) is required for fungal mannitol synthesis, but no MPD-deficient fungal mutants or cDNAs or genes encoding fungal MPDs have been described. Therefore, C. neoformans was purified from a 148 kDa homotetramer of 36 kDa subunits that catalysed the reaction mannitol 1-phosphate MNAD ZY fructose 6-phosphate MNADH. Partial peptide sequences were used to isolate the corresponding cDNA and gene, and the deduced MPD protein was found to be homologous to the zinc-containing long-chain alcohol/polyol dehydrogenases. Lysates of Saccharomyces cerevisiae transformed with the cDNA of interest (but not vector-transformed controls) contained MPD catalytic activity. Lastly, Northern analyses demonstrated MPD mRNA in glucose-and mannitol-grown C. neoformans cells. Thus, MPD has been purified and characterized from C. neoformans, and the corresponding cDNA and gene (MPD1) cloned and sequenced. Availability of C. neoformans MPD1 should permit direct testing of the hypotheses that (i) MPD is required for mannitol biosynthesis and (ii) the ability to synthesize mannitol is essential for wild-type stress tolerance and virulence.

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The distribution of the NADPH regenerating mannitol cycle among fungal species

Cited by 86 publications

References 15 publications

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

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

Mannitol Is Required for Stress Tolerance inAspergillus nigerConidiospores

Mannitol-1-phosphate dehydrogenase from Cryptococcus neoformans is a zinc-containing long-chain alcohol/polyol dehydrogenase The GenBank accession numbers for the nucleotide sequences for the C. neoformans mannitol-1-phosphate dehydrogenase cDNA and gene are AF175685 and AF186474, respectively.

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