Enzymes of mannitol metabolism in the human pathogenic fungus Aspergillus fumigatus--kinetic properties of mannitol-1-phosphate 5-dehydrogenase and mannitol 2-dehydrogenase, and their physiological implications

The human pathogenic fungus Aspergillus fumigatus accumulates large amounts of intracellular mannitol to enhance its resistance against defense strategies of the infected host. To explore their currently unknown roles in mannitol metabolism, we studied A. fumigatus mannitol-1-phosphate 5-dehydrogenase (AfM1PDH) and mannitol 2-dehydrogenase (AfM2DH), each recombinantly produced in Escherichia coli, and performed a detailed steady-state kinetic characterization of the two enzymes at 25 °C and pH 7.1. Primary kinetic isotope effects resulting from deuteration of alcohol substrate or NADH showed that, for AfM1PDH, binding of D-mannitol 1-phosphate and NAD(+) is random, whereas D-fructose 6-phosphate binds only after NADH has bound to the enzyme. Binding of substrate and NAD(H) by AfM2DH is random for both D-mannitol oxidation and D-fructose reduction. Hydride transfer is rate-determining for D-mannitol 1-phosphate oxidation by AfM1PDH (k(cat) = 10.6 s(-1)) as well as D-fructose reduction by AfM2DH (k(cat) = 94 s(-1)). Product release steps control the maximum rates in the other direction of the two enzymatic reactions. Free energy profiles for the enzymatic reaction under physiological boundary conditions suggest that AfM1PDH primarily functions as a D-fructose-6-phosphate reductase, whereas AfM2DH acts in D-mannitol oxidation, thus establishing distinct routes for production and mobilization of mannitol in A. fumigatus. ATP, ADP and AMP do not affect the activity of AfM1PDH, suggesting the absence of flux control by cellular energy charge at the level of D-fructose 6-phosphate reduction. AfM1PDH is remarkably resistant to inactivation by heat (half-life at 40 °C of 20 h), consistent with the idea that formation of mannitol is an essential component of the temperature stress response of A. fumigatus. Inhibition of AfM1PDH might be a useful target for therapy of A. fumigatus infections.     

Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK _m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD⁺. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK _m value of 1.2 mM and a high requirement of Mg²⁺ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK _m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD⁺. For the non-specific HK, only theK _m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase     

Mannitol-1-phosphate dehydrogenase activity in <Emphasis Type="Italic">Ectocarpus siliculosus</Emphasis>, a key role for mannitol synthesis in brown algae

Mannitol represents a major end product of photosynthesis in brown algae (Phaeophyceae), and is, with the β-1,3-glucan laminarin, the main form of carbon storage for these organisms. Despite its importance, little is known about the genes and enzymes responsible for the metabolism of mannitol in these seaweeds. Taking benefit of the sequencing of the Ectocarpus siliculosus genome, we focussed our attention on the first step of the synthesis of mannitol (reduction of the photo-assimilate fructose-6-phosphate), catalysed by the mannitol-1-phosphate dehydrogenase (M1PDH). This activity was measured in algal extracts, and was shown to be regulated by NaCl concentration in the reaction medium. Genomic analysis revealed the presence of three putative M1PDH genes (named EsM1PHD1, EsM1PDH2 and EsM1PDH3). Sequence comparison with orthologs demonstrates the modular architecture of EsM1PHD1 and EsM1PDH2, with an additional N-terminal domain of unknown function. In addition, gene expression experiments carried out on samples harvested through the diurnal cycle, and after several short-term saline and oxidative stress treatments, showed that EsM1PDH1 is the most highly expressed of these genes, whatever the conditions tested. In order to assess the activity of the corresponding protein, this gene was expressed in Escherichia coli. Cell-free extracts prepared from bacteria containing EsM1PDH1 displayed higher M1PDH activity than bacteria transformed with an empty plasmid. Further characterisation of recombinant EsM1PDH1 activity revealed its very narrow substrate specificity, salt regulation, and sensitivity towards an inhibitor of SH-enzymes.     

Nitrophenide (Megasul) blocks Eimeria tenella development by inhibiting the mannitol cycle enzyme mannitol-1-phosphate dehydrogenase.

Unsporulated oocysts of the protozoan parasite Eimeria tenella contain high levels of mannitol, which is thought to be the principal energy source for the process of sporulation. Biosynthesis and utilization of this sugar alcohol occurs via a metabolic pathway known as the mannitol cycle. Here, results are presented that suggest that 3-nitrophenyl disulfide (nitrophenide, Megasul), an anticoccidial drug commercially used in the 1950s, inhibits mannitol-1-phosphate dehydrogenase (M1PDH), which catalyzes the committed enzymatic step in the mannitol cycle. Treatment of E. tenella-infected chickens with nitrophenide resulted in a 90% reduction in oocyst shedding. The remaining oocysts displayed significant morphological abnormalities and were largely incapable of further development. Nitrophenide treatment did not affect parasite asexual reproduction, suggesting specificity for the sexual stage of the life cycle. Isolated oocysts from chickens treated with nitrophenide exhibited a dose-dependent reduction in mannitol, suggesting in vivo inhibition of parasite mannitol biosynthesis. Nitrophenide-mediated inhibition of MIPDH was observed in vitro using purified native enzyme. Moreover, MIPDH activity immunoprecipitated from E. tenella-infected cecal tissues was significantly lower in nitrophenide-treated compared with untreated chickens. Western blot analysis and immunohistochemistry showed that parasites from nitrophenide-treated and untreated chickens contained similar enzyme levels. These data suggest that nitrophenide blocks parasite development at the sexual stages by targeting M1PDH. Thus, targeting of the mannitol cycle with drugs could provide an avenue for controlling the spread of E. tenella in commercial production facilities by preventing oocyst shedding.     

D-mannitol metabolism by Aspergillus candidus

Pathways of mannitol biosynthesis and utilization in Aspergillus candidus NRRL 305 were studied in cell-free extracts of washed mycelia prepared by sonic and French pressure cell treatments. A nicotinamide adenine dinucleotide-linked mannitol-1-phosphate (M1P) dehydrogenase was found in French pressure cell extracts of d-glucose-grown cells, whereas a specific mannitol-1-phosphatase was present in extracts prepared by both methods. The existence of these two enzymes indicated that mannitol may be synthesized in this organism by the reduction of fructose-6-phosphate. A specific nicotinamide adenine dinucleotide phosphate-linked mannitol dehydrogenase was also identified in both extracts. This enzyme may have been involved in mannitol utilization. However, the level of the mannitol dehydrogenase appeared to be substantially reduced in extracts from mannitol-grown cells, whereas the level of M1P dehydrogenase was increased. A hexokinase has been identified in this organism. Fructose-6-phosphatase, glucose isomerase, and mannitol kinase could not be demonstrated.     

Mannitol Synthesis in Higher Plants : Evidence for the Role and Characterization of a NADPH-Dependent Mannose 6-Phosphate Reductase

Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase ¹⁴C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent K_m (M6P) was 15.8 millimolar, but the apparent K_m (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg²⁺, Ca²⁺, Zn²⁺, and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPH-dependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.     

The mannitol cycle in Pleurotus ostreatus (Florida)

In mushroom, presence of the mannitol cycle has not been reported so far although the polyol is supposed to be generated by the reduction of fructose by mannitol dehydrogenase. This study submits evidence for the presence of the mannitol cycle in Pleurotus ostreatus. The key enzyme of the cycle, mannitol-1-phosphate dehydrogenase (M1PDH), was present appreciably in all the developmental stages of the mushroom. However, the enzyme level dropped significantly at the onset of sporulation. The presence of M1DPH was confirmed by isozyme analysis and RT-PCR mediated amplification of a approximately 400 bp DNA fragment.     

Mannitol in six autotrophic stramenopiles and Micromonas

Mannitol plays a central role in brown algal physiology since it represents an important pathway used to store photoassimilate. Several specific enzymes are directly involved in the synthesis and recycling of mannitol, altogether forming the mannitol cycle. The recent analysis of algal genomes has allowed tracing back the origin of this cycle in brown seaweeds to a horizontal gene transfer from bacteria, and furthermore suggested a subsequent transfer to the green micro-alga Micromonas. Interestingly, genes of the mannitol cycle were not found in any of the currently sequenced diatoms, but were recently discovered in pelagophytes and dictyochophytes. In this study, we quantified the mannitol content in a number of ochrophytes (autotrophic stramenopiles) from different classes, as well as in Micromonas. Our results show that, in accordance with recent observations from EST libraries and genome analyses, this polyol is produced by most ochrophytes, as well as the green alga tested, although it was found at a wide range of concentrations. Thus, the mannitol cycle was probably acquired by a common ancestor of most ochrophytes, possibly after the separation from diatoms, and may play different physiological roles in different classes.Key words: algae, stramenopiles, mannitol cycle, primary metabolism, osmotic stress, evolutionBrown algae produce mannitol directly from the photoassimilate fructose-6-phosphate. Its metabolism occurs through the mannitol cycle, which involves four enzymatic reactions: (1) the reduction of fructose-6-phosphate (F6P) to mannitol-1-phosphate (M1P) via the activity of an M1P dehydrogenase (M1PDH); (2) the production of mannitol from M1P via an M1P phosphatase (M1Pase); (3) the oxidation of mannitol via the activity of a mannitol-2-dehydrogenase (M2DH) yielding fructose; and (4) the phosphorylation of fructose yielding F6P and involving a hexokinase (HK).^1,2 The first completed draft of a brown algal genome enabled the identification of candidate genes for each of these steps.³ As these genes were not found in the genomes of the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum, mannitol metabolism in stramenopiles was considered a trait typical for brown algae. The corresponding genes were thought to have been acquired horizontally from bacteria and subsequently transferred to some green algae.⁴ Recently, however, homologs of several genes of the cycle were also found in the genome of the pelagophyte Aureococcus anophagefferens⁵ and an EST library produced for the dictyochophyte Pseudochattonella farcimen (Dittami et al. personal communication). These observations prompted us to examine the presence of mannitol in a range of strains covering different classes of autotrophic stramenopiles (ochrophytes). In addition, because of the identification of genes encoding enzymes for the production of mannitol through the mannitol cycle in the green alga Micromonas, one strain of this genus was also included in our analysis.     

Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis.

One step in de novo pyridoxine (vitamin B6) and pyridoxal 5'-phosphate biosynthesis was predicted to be an oxidation catalyzed by an unidentified D-erythrose-4-phosphate dehydrogenase (E4PDH). To help identify this E4PDH, we purified the Escherichia coli K-12 gapA- and gapB-encoded dehydrogenases to homogeneity and tested whether either uses D-erythrose-4-phosphate (E4P) as a substrate. gapA (gap1) encodes the major D-glyceraldehyde-3-phosphate dehydrogenase (GA3PDH). The function of gapB (gap2) is unknown, although it was suggested that gapB encodes a second form of GA3PDH or is a cryptic gene. We found that the gapB-encoded enzyme is indeed an E4PDH and not a second GA3PDH, whereas gapA-encoded GA3PDH used E4P poorly, if at all, as a substrate under the in vitro reaction conditions used in this study. The amino terminus of purified E4PDH matched the sequence predicted from the gapB DNA sequence. Purified E4PDH was a heat-stable tetramer with a native molecular mass of 132 kDa. E4PDH had an apparent Km value for E4P [Kmapp(E4P)] of 0.96 mM, an apparent kcat catalytic constant for E4P [kcatapp(E4P)] of 200 s-1, Kmapp(NAD+) of 0.074 mM, and kcatapp(NAD+) of 169 s-1 in steady-state reactions in which NADH formation was determined. From specific activities in crude extracts, we estimated that there are at least 940 E4PDH tetramer molecules per bacterium growing in minimal salts medium plus glucose at 37 degrees C. Thin-layer chromatography confirmed that the product of the E4PDH reaction was likely the aldonic acid 4-phosphoerythronate. To establish a possible role of E4PDH in pyridoxal 5'-phosphate biosynthesis, we showed that 4-phosphoerythronate is a likely substrate for the 2-hydroxy-acid dehydrogenase encoded by the pdxB gene. Implications of these findings in the evolution of GA3PDHs are also discussed. On the basis of these results, we propose renaming gapB as epd (for D-erythrose-4-phosphate dehydrogenase).     

Comparative activity of rat liver dihydrofolate reductase with 7,8-dihydrofolate and other 7,8-dihydropteridines

The various interactions of rat liver dihydrofolate reductase with two unconjugated 7,8-dihydropteridines, 7,8-dihydrobiopterin and 6-methyl-7,8-dihydropteridine, have been compared with those of 7,8-dihydrofolate and folate. Of particular interest was the reactivity demonstrated by 7,8-dihydrobiopterin because of the potential physiological significance of this reaction both in the regeneration of tetrahydrobiopterin, a cofactor for various biological hydroxylations, and as a step in the biosynthesis of this compound from GTP. Kinetic experiments gave Km values of 0.17, 6.42, and 10.2 microM for 7,8-dihydrofolate, 7,8-dihydrobiopterin, and 6-methyl-7,8-dihydropteridine, respectively, with Vmax = 6.22, 2.39, and 1.54 mumol min-1 mg-1. With folate the enzyme showed high affinity (Km = 0.88 microM) but low Vmax (0.20 mumol min-1 mg-1). The natural cofactor was NADPH and a Km of approximately 0.7 microM was measured with each substrate. The enzyme was activated by both p-hydroxymercuribenzoate and urea when assayed with 7,8-dihydrofolate but was inhibited when 7,8-dihydrobiopterin was the substrate. The pH optimum for dihydrofolate reduction was 4 with enhancement at pH greater than or equal to 5.5 in the presence of 1 M NaCl. Peak activity with 7,8-dihydrobiopterin occurred at pH 4.8; this was shifted to pH 5.3 but was not enhanced by 1 M NaCl. Inhibition with methotrexate was similar whether the enzyme was assayed with either the conjugated or unconjugated 7,8-dihydro derivatives. The rat liver enzyme, highly unstable after purification, was stabilized in the presence of the nonionic detergent, Tween-20 (0.1%); however, the comparative properties toward the conjugated and unconjugated substrates were not altered by this treatment.     

Mannitol‐1‐phosphate dehydrogenases/phosphatases: a family of novel bifunctional enzymes for bacterial adaptation to osmotic stress

The nutritionally versatile soil bacterium Acinetobacter baylyi ADP1 copes with salt stress by the accumulation of compatible solutes, a strategy that is widespread in nature. This bacterium synthesizes the sugar alcohol mannitol de novo in response to osmotic stress. In a previous study, we identified MtlD, a mannitol‐1‐phosphate dehydrogenase, which is essential for mannitol biosynthesis and which catalyses the first step in mannitol biosynthesis, the reduction of fructose‐6‐phosphate (F‐6‐P) to the intermediate mannitol‐1‐phosphate (Mtl‐1‐P). Until now, the identity of the second enzyme, the phosphatase that catalyses the dephosphorylation of Mtl‐1‐P to mannitol, was elusive. Here we show that MtlD has a unique sequence among known mannitol‐1‐phosphate dehydrogenases with a haloacid dehalogenase (HAD)‐like phosphatase domain at the N‐terminus. This domain is indeed shown to have a phosphatase activity. Phosphatase activity is strictly Mg²⁺ dependent. Nuclear magnetic resonance analysis revealed that purified MtlD catalyses not only reduction of F‐6‐P but also dephosphorylation of Mtl‐1‐P. MtlD of A. baylyi is the first bifunctional enzyme of mannitol biosynthesis that combines Mtl‐1‐P dehydrogenase and phosphatase activities in a single polypeptide chain. Bioinformatic analysis revealed that the bifunctional enzyme is widespread among Acinetobacter strains but only rarely present in other phylogenetic tribes.     

Ectopic expression of sorbitol-6-phosphate 2-dehydrogenase gene from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis> enhances salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

d-Sorbitol-6-phosphate 2-dehydrogenase (S6PDH, E.C. 1.1.1.140) catalyzes the NADH-dependent conversion of d-fructose 6-phosphate (F6P) to d-sorbitol 6-phosphate (S6P). In this work, recombination and characterization of Haloarcula marismortui d-sorbitol-6-phosphate 2-dehydrogenase are reported. Haloarcula marismortui d-sorbitol-6-phosphate 2-dehydrogenase was expressed in P. pastoris and Arabidopsis thaliana. Enzyme assay indicated that HmS6PDH catalyzes the reduction of d-fructose 6-phosphate to d-sorbitol 6-phosphate and HmS6PDH activity was enhanced by NaCl. Furthermore, transgenic A. thaliana ectopic expressing HmS6PDH accumulate more sorbitol under salt stress. These results suggest that the ectopic expression of HmS6PDH in plants can facilitate future studies regarding the engineering and breeding of salt-tolerant crops.     

Biochemical characterization of mannitol metabolism in the unicellular red alga Dixoniella grisea (Rhodellophyceae)

The mannitol cycle has been verified in a unicellular red alga (Rhodellophyceae) for the first time. All four enzymes involved in the cycle (mannitol-1-phosphate dehydrogenase, Mt1PDH: EC 1.1.1.17; mannitol-1-phosphatase, Mt1Pase: EC 3.1.3.22; mannitol dehydrogenase, MtDH: 1.1.1.67; hexokinase, HK: 2.7.1.1.) were detected and characterized in crude algal extracts from Dixoniella grisea. These enzymes, with the exception of Mt1Pase, were specific to their corresponding substrates and nucleotides. The activities of enzymes in the anabolic pathway (fructose-6-P reduction by Mt1PDH and mannitol-6-P reduction by Mt1Pase) were at least 2- to 4-fold greater than those of the catabolic pathway (mannitol oxidation by MtDH and fructose oxidation by HK). There appears to be, therefore, a net carbon flow in D. grisea towards a high intracellular mannitol pool. The mannitol cycle guarantees a rapid accumulation or degradation of mannitol within algal cells in response to changing salinity in natural habitats. Moreover, the demonstration of the mannitol cycle within the Rhodellophyceae provides evidence that this metabolic pathway is of ancient origin in the red algal lineage.     

Salt-Regulated Mannitol Metabolism in Algae

Mannitol, one of the most widely occurring sugar alcohol compounds, is found in bacteria, fungi, algae, and plants. In these organisms the compound acts as a compatible solute and has multiple functions, including osmoregulation, storage, and regeneration of reducing power, and scavenging of active oxygen species. Because of the diverse functions of mannitol, introducing the ability to accumulate it has been a hallmark of attempts to generate highly salt-tolerant transgenic plants. However, transgenic plants have not yet improved significantly in their salt tolerance. Recently, we purified and characterized 2 enzymes that biosynthesize mannitol, mannitol-1-phosphate dehydrogenase (M1PDH) and mannitol-1-phosphate-specific phosphatase, from the marine red alga Caloglossa continua, which grows in estuarine areas where tide levels fluctuate frequently. The activation of Caloglossa M1PDH is unique in that it is regulated by salt concentration at enzyme level. In this review we focus on the metabolism of mannitol, mainly in marine photosynthetic organisms, and suggest how this might be applied to producing salt-tolerant transgenic plants.     

Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide.

The algC gene from Pseudomonas aeruginosa has been shown to encode phosphomannomutase (PMM), an essential enzyme for biosynthesis of alginate and lipopolysaccharide (LPS). This gene was overexpressed under control of the tac promoter, and the enzyme was purified and its substrate specificity and metal ion effects were characterized. The enzyme was determined to be a monomer with a molecular mass of 50 kDa. The enzyme catalyzed the interconversion of mannose 1-phosphate (M1P) and mannose 6-phosphate, as well as that of glucose 1-phosphate (G1P) and glucose 6-phosphate. The apparent Km values for M1P and G1P were 17 and 22 microM, respectively. On the basis of Kcat/Km ratio, the catalytic efficiency for G1P was about twofold higher than that for M1P. PMM also catalyzed the conversion of ribose 1-phosphate and 2-deoxyglucose 6-phosphate to their corresponding isomers, although activities were much lower. Purified PMM/phosphoglucomutase (PGM) required Mg2+ for maximum activity; Mn2+ was the only other divalent metal that showed some activation. The presence of other divalent metals in addition to Mg2+ in the reaction inhibited the enzymatic activity. PMM and PGM activities could not be detected in nonmucoid algC mutant strain 8858 and in LPS-rough algC mutant strain AK1012, while they were present in the wild-type strains as well as in algC-complemented mutant strains. This evidence suggests that AlgC functions as PMM and PGM in vivo, converting phosphomannose and phosphoglucose in the biosynthesis of both alginate and LPS.     

Biosynthesis and catabolism of mannitol is developmentally regulated in the protozoan parasite Eimeria tenella

The mannitol cycle is a metabolic branch of the glycolytic pathway found in Eimeria tenella. In this paper, we describe the biosynthesis and consumption of mannitol during parasite development. Low micromolar levels of mannitol were detected in all of the asexual stages and mannitol production increased sharply during the sexual phase of the life cycle. Unsporulated oocysts had high mannitol content (300 mM or 25% of the oocyst mass). Mannitol-1-phosphate dehydrogenase (M1PDH), the first committed step of the mannitol cycle, was also elevated in sexual stages and this coincides with mannitol levels. Approximately 90% of the mannitol present in unsporulated oocysts was consumed in the first 15 hr of sporulation, and levels continued to drop until the sporulation process was complete at approximately 35 hr. Thus, mannitol appears to be the "fuel" for sporulation during the vegetative stage of the parasite life cycle. Evaluation of oocyst extracts from 6 additional Eimeria species for mannitol content and the presence of M1PDH indicated that the mannitol cycle was broadly present in this genus. This finding combined with the lack of mannitol metabolism in higher eukaryotes makes this pathway an attractive chemotherapeutic target.     

Polyol-specific long-chain dehydrogenases/reductases of mannitol metabolism in Aspergillus fumigatus: biochemical characterization and pH studies of mannitol 2-dehydrogenase and mannitol-1-phosphate 5-dehydrogenase

Functional genomics data suggests that the metabolism of mannitol in the human pathogen Aspergillus fumigatus involves the action of two polyol-specific long-chain dehydrogenases/reductases, mannitol-1-phosphate 5-dehydrogenase (M1PDH) and mannitol 2-dehydrogenase (M2DH). The gene encoding the putative M2DH was expressed in Escherichia coli, and the purified recombinant protein was characterized biochemically. The predicted enzymatic function of a NAD(+)-dependent M2DH was confirmed. The enzyme is a monomer of 58kDa in solution and does not require metals for activity. pH profiles for M2DH and the previously isolated M1PDH were recorded in the pH range 6.0-10.0 for the oxidative and reductive direction of the reactions under conditions where substrate was limiting (k(cat)/K) or saturating (k(cat)). The pH-dependence of logk(cat) was usually different from that of log(k(cat)/K), suggesting that more than one step of the enzymatic mechanism was affected by changes in pH. The greater complexity of the pH profiles of log(k(cat)/K) for the fungal enzymes as compared to the analogous pH profiles for M2DH from Pseudomonas fluorescens may reflect sequence changes in vicinity of the conserved catalytic lysine.     

31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida characterization of a new mannitol-producing metabolic pathway.

Glucose metabolism of Pasteurella multocida was examined in resting cells in vivo using 13C NMR spectroscopy, in cell-free extracts in vitro using 31P NMR spectroscopy and using enzyme assays. The NMR data indicate that glucose is converted by the Embden-Meyerhof and pentose phosphate pathways. The P. multocida fructose 6-phosphate phosphotransferase activity (the key enzyme of the Embden-Meyerhof pathway) was similar to that of Escherichia coli. Nevertheless, and in contrast to that of E. coli, its activity was inhibited by alpha glycerophosphate. This inhibition is consistent with the very low fructose 6-phosphate phosphotransferase activity found in cell-free extracts of P. multocida using a spectrophotometric method. The dominant end products of glucose metabolism were mannitol, acetate and succinate. Under anaerobic conditions, P. multocida was able to constitutively produce mannitol from glucose, mannose, fructose, sucrose, glucose 6-phosphate and fructose 6-phosphate. We propose a new metabolic pathway in P. multocida where fructose 6-phosphate is reduced to mannitol 1-phosphate by fructose 6-phosphate reductase. Mannitol 1-phosphate produced is then converted to mannitol by mannitol 1-phosphatase.     

Algal heme oxygenase from Cyanidium caldarium. Partial purification and fractionation into three required protein components

Enzymatic heme oxygenase activity has been partially purified from extracts of the unicellular red alga Cyanidium caldarium, and the macromolecular components have been separated into three protein fractions, referred to as Fractions I, II, and III, by serial column chromatography through DEAE-cellulose and Reactive Blue 2-Sepharose. Fraction I is retained by DEAE-cellulose at low salt concentration and eluted by 1 M NaCl. Fraction II is retained by Blue Sepharose at low salt concentration and eluted by 1 M NaCl. Fraction III is retained on 2',5'-ADP-agarose and eluted by 1 mM NADPH, while Fraction II is not retained on ADP-agarose. Fractions I-III, have Mr values of 22,000, 38,000, and 37,000, respectively (all +/- 2,000), as determined by Sephadex gel filtration chromatography. In vitro heme oxygenase activity requires the presence of all three fractions, plus substrate, O2, reduced pyridine nucleotide, and another reductant. Ascorbate, isoascorbate, and phenylenediamine serve equally well as the second reductant, but hydroquinone can also be used, with lower activity resulting. Fractions I-III are heat sensitive and inactive by Pronase digestion. Fraction I has a visible absorption spectrum similar to that of ferredoxin and is bleached by dithionite reduction or incubation with p-hydroxymercuribenzoate. Fraction I can be replaced by commercially available ferredoxin derived from the red alga Porphyra umbilicalis, and to a smaller extent, by spinach ferredoxin. Fraction III contains ferredoxin-linked cytochrome c reductase activity and can be partially replaced by spinach ferredoxin-NADP+ oxidoreductase. Reconstituted heme oxygenase and ferredoxin-linked cytochrome c reductase activities are both abolished if Fraction I or III is preincubated with 0.1 mM p-hydroxymercuribenzoate, but heme oxygenase activity is only slightly affected if Fraction II is preincubated with p-hydroxymercuribenzoate. Preincubation of Fraction II with 0.5 mM diethylpyrocarbonate inactivates heme oxygenase in the reconstituted system, and 10 microM mesohemin partially protects this Fraction against diethylpyrocarbonate inactivation. Algal heme oxygenase is inhibited 80% by 2 microM Sn-protoporphyrin even in the presence of 20 microM mesohemin. Fraction II is rate limiting in unfractionated and reconstituted incubation mixtures. None of the three cell fractions could be replaced by bovine spleen microsomal heme oxygenase or NADPH-cytochrome P450 reductase.     

Hysteretic behaviour of glucose-6-phosphate dehydrogenase of pea chloroplasts

Glucose-6-phosphate dehydrogenase (G6PDH) from pea chloroplasts has at least two interconvertible kinetic states which differ from one another in their catalytic activities (‘hyperactive’ and ‘hypoactive’ forms). Preincubation of chloroplast extracts with 10 mM glucose-6-phosphate (G6P) led to the accumulation of a ‘hyperactive’ G6PDH form which exhibited a burst of activity at the start of the assay; steady state was reached after a period of several minutes. Preincubation of the pea chloroplast extracts in the absence of G6P resulted in the formation of a ‘hypoactive’ enzyme from which exhibited a lag during the assay. Steady state was reached after several minutes. The enzyme activity in the steady state was the same for both forms. The length of the lag (τ) was inversely related to the concentration of G6DH and substrate concentration. These results show that the G6PDH of pea chloroplasts, like the enzyme of cyanobacteria, behaves as a hysteretic enzyme.