Functional identification of sll1383 from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp PCC 6803 as L-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K _m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V _max (∼0.033 μmol Pi/min/mg protein) was low. Li⁺ was found to be an inhibitor (IC₅₀ 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na⁺, K⁺ NH₄⁺) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg²⁺ dependent, which can be substituted partially by Mn²⁺. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.     

L-<Emphasis Type="Italic">myo</Emphasis>-inositol-1-phosphate synthase: partial purification and characterisation from <Emphasis Type="Italic">Gleichenia glauca</Emphasis>

A screening for the enzyme L-myo-inositol-1-phosphate synthase [EC 5.5.1.4] has been made first time in both vegetative and reproductive parts of the representative members of pteridophytes: Lycopodium, Selaginella, Equisetum, Polypodium, Dryopteris, and Gleichenia. The enzyme has been partially purified following low-speed centrifugation, streptomycin sulphate precipitation, ammonium sulphate fractionation, chromatography on DEAE-cellulose and gel-filtration through Sephadex G-200, and characterised from the reproductive pinnules of Gleichenia glauca Smith. The enzyme has a pH optimum at 7.5. The K_m for glucose-6-P and NAD⁺ were 0.922 × 10^–3 M and 0.9 × 10^–4 M, respectively. A basal activity of the enzyme has been recorded in absence of exogenous NAD⁺. The enzyme activity was augmented with NH₄Cl, but heavy metals like Hg²⁺, Cu²⁺ and Zn²⁺ inactivated it.     

A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay—the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg²⁺ and is not enhanced by other divalent metal ions (Zn²⁺ and Mn²⁺), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors.     

sll1722, an unassigned open reading frame of<Emphasis Type="Italic"> Synechocystis</Emphasis> PCC 6803, codes for <Emphasis Type="SmallCaps">l</Emphasis>-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate synthase

l-myo-Inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes conversion of glucose 6-phosphate to l-myo-inositol 1-phosphate, the first and the rate-limiting step in the production of inositol, and has been reported from evolutionarily diverse organisms. Two forms of the enzyme have been characterized from higher plants, viz. cytosolic and chloroplastic, and the presence of MIPS has been earlier reported from the cyanobacteria (e.g. Spirulina sp.), the presumed chloroplast progenitors. The present study demonstrates possible multiple forms of MIPS and identifies the gene for one of them in the cyanobacterium Synechocystis sp. PCC 6803. Following detection of at least two immunologically cross-reactive MIPS forms, we have been able to identify from the fully sequenced Synechocystis genome an as yet unassigned open reading frame (ORF), sll1722, coding for the approx. 50-kDa MIPS protein, by using biochemical, molecular and bioinformatics tools. The DNA fragment corresponding to sll1722 was PCR-amplified and functional identity of the gene was confirmed by a complementation assay in Saccharomyces cerevisiae mutants containing a disrupted INO1 gene for the yeast MIPS. The sll1722 PCR product was cloned in Escherichia coli expression vector pET20b and the isopropyl -d-thiogalactopyranoside (IPTG)-induced overexpressed protein product was characterized following complete purification. Comparison of the sll1722 sequences with other MIPS sequences and its phylogenetic analysis revealed that the Synechocystis MIPS gene is quite divergent from the others.Abbreviations CBB Coomassie Brilliant Blue - EST Expressed sequence tag - G6P d-Glucose 6-phosphate - IPTG Isopropyl -d-thiogalactopyranoside - MIPS l–myo-Inositol 1-phosphate synthase - ORF Open reading frame     

Characterization of a mutation that causes overproduction of inositol in Neurospora crassa

Summary Slow-growing (inl ^+/-) spontaneous mutants have been isolated from an inositol requiring (inl) strain of Neurospora crassa that produces defective myo-inositol-1-phosphate synthase (MIPS), the enzyme responsible for the production of inositol-1-phosphate from glucose-6-phosphate. The defective enzyme has some residual activity. In the inl ^+/- strain the synthesis of the defective enzyme is enhanced, which enables the strain to grow slowly on minimal medium. The mutation (opi1) responsible for the partial inositol independence segregates independently from the inositol locus, and suppresses the inositolless character by overproduction of defective MIPS. opi1 acting upon the wild type (inl ⁺) allele increases MIPS production and causes inositol excretion.     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Purification and characterisation of NAD+-dependent xylitol dehydrogenase from Fusarium oxysporum

An NAD⁺-dependent xylitol dehydrogenase (XDH) from Fusarium oxysporum, a key enzyme in the conversion of xylose to ethanol, was purified to homogeneity and characterised. It was homodimeric with a subunit of M _r 48 000, and pI 3.6. It was optimally active at 45 °C and pH 9–10. It was fully stable at pH 6–7 for 24 h and 30 °C. K _m values for d-xylitol and NAD⁺ were 94 mM and 0.14 mM, respectively. Mn²⁺ at 10 mM increased XDH activity 2-fold and Cu²⁺ at 10 mM inhibited activity completely.     

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

l-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of d-glucose 6-phosphate to l-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989–998, 2004). One of the forms with a ~50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as a ~65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified ~1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive ~65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.Anirban Chatterjee and Krishnarup Ghosh Dastidar contributed equally.     

NAD kinase from Bacillus licheniformis: inhibition by NADP and other properties

NAD kinase was purified 180-fold from Bacillus licheniformis to determine the role it plays in NADP turnover in this organism. The enzyme was found to have a pH optimum of 6.8 and an apparent K _m for NAD of 2.7 mM. The ATP saturation curve was not hyperbolic; 5.5 mM ATP was required to reach half maximal activity. Both Mn²⁺ and Ca²⁺ could be substituted for Mg²⁺. Several compounds including nicotinic acid, nicotinamide, nicotinamide mononucleotide, quinolinic acid, NADPH, ADP, AMP and cyclic AMP did not affect NAD kinase activity. In contrast, the enzyme was inhibited by NADP at concentrations typically found in logarithmic cells of B. licheniformis. This inhibition was competitive with NAD and had a K _i of 0.13 mM. It is suggested that in vivo NAD kinase activity is highly dependent on the concentrations of NAD and ATP and the proportion of oxidized and reduced NADP.This paper is dedicated to Sydney C. Rittenberg on the occassion of his retirement, with respect and much affection, in appreciation for his friendship and years of distinguished service as a teacher and scientist     

Fructose-1,6-bisphosphatase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>: expression and deletion of the<Emphasis Type="Italic"> fbp</Emphasis> gene and biochemical characterization of the enzyme

The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K_m value of about 14 µM and a V_max of about 5.4 µmol min^–1 mg^–1 and k_cat of about 3.2 s^–1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg²⁺ or Mn²⁺ and was inhibited by the monovalent cation Li⁺ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.     

NAD+ mediated differential thermotolerance between chloroplastic and cytosolic L-myo-inositol-1-phosphate synthase from Diplopterygium glaucum (Thunb.) Nakai

Relative thermotolerance of the enzyme, L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4), from the chloroplastic and cytosolic sources of Diplopterygium glaucum was studied. The purification involved streptomycin sulphate precipitation, ammonium sulphate fractionation, ion-exchange chromatography, and molecular sieve chromatography. After the final chromatography, 16.62% of chloroplastic and 13.47% of cytosolic MIPS could be recovered. Between 15 degrees C and 55 degrees C, the two forms of MIPS exhibited differential thermal stability, which is related to the presence of the MIPS co-factor, NAD+. Added NAD+ increased the lower thermotolerance of the chloroplastic MIPS and the removal of 'built-in' NAD+ decreased the higher thermal stability of the cytosolic MIPS.     

Essential sulfhydryl group of malic enzyme fromEscherichia coli

The activity of malic enzyme fromEscherichia coli was unaffected by the monovalent cations Na⁺ or Li⁺ at 10 mM. At 100 mM, Li⁺ or Na⁺ inhibited the enzyme activity by 88% and 83%, respectively. However, the enzyme activity was stimulated by 40–80-fold with 10 mM K⁺, Rb⁺, Cs⁺, or NH ₄ ⁺ . Less stimulation was observed with 100 mM of these stimulating cations. The stimulatory effect was lost after the enzyme was dialyzed against Tris-Cl buffer, but was regained after incubating the dialyzed enzyme with dithiothreitol. The regenerated enzyme was inactivated by 5,5-dithiobis(2-nitrobenzoic acid). The resulting inactive thionitrobenzoyl enzyme could be regenerated to the active thiol-enzyme by eithiothreitol or converted to the inactive thiocyanoylated enzyme by KCN. The thiocyanoylated enzyme was insensitive to K⁺ stimulation, which suggested the essentiality of the sulfhydryl groups of theE. coli malic enzyme.     

Expression of sucrose: fructan 6-fructosyltransferase (6-SFT) and myo -inositol 1-phosphate synthase (MIPS) genes in barley (Hordeum vulgare) leaves

Fructan is an important class of non-structural carbohydrates present in cool-season grasses. Sucrose: fructan 6-fructosyltransferase (6-SFT, EC 2.4.1.10), one of the enzymes thought to be involved in grass fructan biosynthesis, catalyzes the initiation and extension of 2,6-linked fructans.Myo-inositol is a central component in several metabolic pathways in higher plants.Myo-inositol 1-phosphate synthase (MIPS) (EC 5.5.1.4), the first enzyme in inositolde novo biosynthesis, catalyzes the formation ofmyo-inositol 1-phosphate (MIP) from glucose-6-phosphate. The expression of 6-SFT and MIPS genes is compared in barley (Hordeum vulgare L.) leaves under various conditions. In cool temperature treatments, both 6-SFT and MIPS mRNAs accumulate within two days and then decline after four days. Under warm temperatures and continuous illumination, the amount of 6-SFT and MIPS mRNA gradually accumulated in detached leaves and increased significantly by 8 h. In contrast, we observed no significant changes over time in attached (control) leaves. Treating detached leaves with glucose or sucrose in the dark resulted in accumulations of both 6-SFT and MIPS mRNA. Homologous expression patterns for 6-SFT and MIPS genes suggest that they may be similarly regulated in barley leaves. Although sucrose and glucose may play important roles in the expression of 6-SFT and MIPS genes, regulation likely involves multiple factors.     

Reverse Reaction of Malic Enzyme for HCO3 − Fixation into Pyruvic Acid to Synthesize L-Malic Acid with Enzymatic Coenzyme Regeneration

Malic enzyme [L-malate: NAD(P)⁺ oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)⁺). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO₃ ^? fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD⁺, and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO₃ ^? and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 °C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD⁺.     

Purification and characterization of a NAD+-dependent secondary alcohol dehydrogenase from propane-grown Rhodococcus rhodochrous PNKb1

NAD⁺-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD⁺-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C₂–C₈) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K _m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.Abbreviations HPLC high performance liquid chromatography - DEAE diethyl amino ethyl - IEF isoelectrofocusing - NTG nitrosoguanidine - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - pI isoelectric point     

Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrificans grown on glucose/nitrate

Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ l-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD⁺-and NADP⁺-linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg²⁺ requirement. Kinetics for both NAD(P)⁺ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD⁺-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP⁺-linked activity. Neither of the two activities are inhibited by 100 M NADH but both are inhibited by NADPH. The NAD⁺-linked activity is far more sensitive to inhibition by NADPH than the NADP⁺-linked activity.     

Purification and characterization of threonine dehydrogenase from Clostridium sticklandii

Threonine dehydrogenase from Clostridium sticklandii has been purified 76-fold from cells grown in a defined medium to a homogeneous preparation of 234 units · mg^-1 protein. Purification was obtained by chromatography on Q-Sepharose fast flow and Reactive green 19-Agarose. The native enzyme had a molecular mass of 67 kDa and consisted of two identical subunits (33 kDa each). The optimum pH for catalytic activity was 9.0. Only l-threo-threo-nine, dl--hydroxynorvaline and acetoin were substrates; only NAD was used as the natural electron acceptor. The apparent K _m values for l-threonine and NAD were 18 mM and 0.1 mM, respectively. Zn²⁺, Co²⁺ and Cu²⁺ ions (0.9 mM) inhibited enzyme activity. The N-terminal amino acid sequence revealed similarities to the class of non-metal short-chain alcohol dehydrogenases, whereas the threonine dehydrogenase from Escherichia coli belongs to the class of medium chain, zinc-containing alcohol dehydrogenases.Abbreviations PMSF phenylmethylsulfonyl fluoride - Dea diethanolamine - Tris tris-(hydroxy-methyl)-aminomethane - Nbs ₂ 5,5-dithiobis-(2-nitrobenzoic acid) - ApADN 3-acetylpyridine adenine diucleotide - thio-NAD thionicotinamide adenine dinucleotide - NBT nitro blue tetrazolium chloride     

Effect of Osmolality and myo-Inositol Deprivation on the Transport Properties of myo-Inositol in Primary Astrocyte Cultures

myo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na⁺ with a Km of 13–18 M and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 M with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na⁺-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.     

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     

Purification and Characterization of the NAD-Dependent Glutamate Dehydrogenase in the Ectomycorrhizal FungusLaccaria bicolor(Maire) Orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) fromLaccaria bicolorwas purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE–Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate–polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at −75°C, 4 days at 4°C, and 1 h at 50°C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at −75°C. NAD-GDH activity was stimulated by Ca²⁺and Mg²⁺but strongly inhibited by Cu²⁺and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 μM, 89 μM, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and itsK_mvalue increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid.