Regulation of gluconate and ketogluconate production in Gluconobacter oxydans ATCC 621-H

Gluconobacter spp. possess the enzymic potential for two pathways of direct glucose oxidation. It has been proposed that the major part of glucose is oxidized to gluconate via NADP-dependent glucose dehydrogenase and that reoxidation of NADPH under these conditions proceeds via recycling of gluconate through ketogluconates. This hypothesis was tested in experiments in which Gluconobacter oxydans ATCC 621-H was grown in glucose-yeast extract medium containing [¹⁴C]2-ketogluconate. As expected, glucose was almost quantitatively oxidized to gluconate, without further accumulation of 2- and 5-ketogluconate. Interestingly, the total amount of neither [¹⁴C]2-ketogluconate nor [¹⁴C]gluconate did change significantly during this oxidation phase, indicating that recycling of gluconate through ketogluconates did not occur. An analysis of enzyme activities in cell-free extracts of glucose-grown cells of G. oxydans ATCC 621-H showed that the membrane-bound glucose dehydrogenase was far more active than the NADP-linked glucose dehydrogenase. The activity of the latter enzyme constituted only 10–15% of that of quinoprotein glucose dehydrogenase and was far too low to match the in vivo rates of gluconate production in batch cultures of G. oxydans. It is concluded that under these conditions glucose is mainly oxidized to gluconate via the membrane-bound glucose dehydrogenase. Implications of these results for the regulation of ketogluconate formation are discussed.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - PQQ pyrrolo-quinoline quinone     

The role of magnesium and calcium ions in the glucose dehydrogenase activity of Klebsiella pneumoniae NCTC 418

Magnesium-limited chemostat cultures of Klebsiella pneumoniae NCTC 418 with 20 M CaCl₂ in the medium showed a low rate of gluconate plus 2-ketogluconate production relative to potassium- or phosphate-limited cultures. However, when the medium concentration of CaCl₂ was increased to 1 mM, the glucose dehydrogenase (GDH) activities also increased and became similar to those observed in potassium- or phosphate limited cultures. It is concluded that this is due to Mg²⁺ and Ca²⁺ ions being involved in the binding of pyrroloquinoline quinone (PQQ) to the GDH apoenzyme. There seems to be an absolute requirement of divalent cations for proper enzyme functioning and in this respect Ca²⁺ ions could replace Mg²⁺ ions. The high GDH activity which has been found in cells grown under Mg^2–-limited conditions in the presence of higher concentrations of Ca²⁺ ions, is compatible with the earlier proposal that GDH functions as an auxiliary energy generating system involved in the maintenance of high transmembrane ion gradients.Abbreviations PQQ pyrroloquinoline quinone - GDH glucose dehydrogenase (EC 1.1.99.17) - GaDH gluconate dehydrogenase (EC 1.1.99.3) - CAP chloramphenicol - WB Wurster's Blue [1,4-bis-(dimethylamino)-benzene perchlorate]     

The separate roles of PQQ and apo-enzyme syntheses in the regulation of glucose dehydrogenase activity in Klebsiella pneumoniae NCTC 418

No holoenzyme pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and only very low apoenzyme levels could be detected in cells of Klebsiella pneumoniae, growing anaerobically, or carrying out a fumarate or nitrate respiration. Low glucose dehydrogenase activity in some aerobic glucose-excess cultures of K. pneumoniae (ammonia or sulphate limitation) was increased significantly by addition of PQQ, whereas in cells already possessing a high glucose dehydrogenase activity (phosphate or potassium limitation) extra PQQ had almost no effect. These observations indicate that the glucose dehydrogenase activity in K. pneumoniae is modulated by both PQQ synthesis and synthesis of the glucose dehydrogenase apo-enzyme.Abbreviations PQQ 2, 7, 9-tricarboxy-1H-pyrrolo-(2,3-f)quinoline-4,5-dione - WB Wurster's Blue (1,4-bis-(dimethylamino)-benzene perchlorate)     

Gluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture

The metabolism of gluconate by Klebsiella pneumoniae NCTC 418 was studied in continuous culture. Under all gluconate-excess conditions at low culture pH values (pH 4.5–5.5) the majority (70–90%) of the gluconate metabolized was converted to 2-oxogluconate via gluconate dehydrogenase (GADH), although specific 2-oxogluconate production rates under potassium-limited conditions were significantly lower than under other gluconate-excess conditions. At high culture pH values, metabolism shifted towards production of acetate. Levels of GADH were highest at low culture pH values and synthesis was stimulated by the presence of (high concentrations of) gluconate. An increase in activity of the tricarboxylic acid cycle was accompanied by a decrease in GADH activity in vivo and in vitro, suggesting that the GADH serves a role as an alternative energy-generating system. Anaerobic 2-oxogluconate production was found to be possible in the presence of nitrate as electron acceptor. Levels of gluconate kinase were highest when K. pneumoniae was grown under gluconate-limited conditions. Under carbon-excess conditions, levels of this enzyme correlated with the intracellular catabolic flux.Abbreviations GADH gluconate dehydrogenase (EC 1.1.99.3) - GAK gluconate kinase (EC 2.7.1.12) - GDH glucose dehydrogenase (EC 1.1.99.17) - PQQ pyrroloquinoline quinone [2,7,9-tricarboxy-1-H-pyrrolo (2,3-f) quinoline-4,5-dione] - TCA trichloroacetic acid     

Quantitative aspects of glucose metabolism by Escherichia coli B/r,grown in the presence of pyrroloquinoline quinone

Escherichia coli B/r was grown in chemostat cultures under various limitations with glucose as carbon source. Since E. coli only synthesized the glucose dehydrogenase (GDH) apo-enzyme and not the appropriate cofactor, pyrroloquinoline quinone (PQQ), no gluconate production could be observed. However, when cell-saturating amounts of PQQ (nmol to mol range) were pulsed into steady state glucose-excess cultures of E. coli, the organisms responded with an instantaneous formation of gluconate and an increased oxygen consumption rate. This showed that reconstitution of GDH in situ was possible.Hence, in order to examine the influence on glucose metabolism of an active GDH, E. coli was grown aerobically in chemostat cultures under various limitations in the presence of PQQ. It was found that the presence of PQQ indeed had a sizable effect: at pH 5.5 under phosphate- or sulphate- limited conditions more than 60% of the glucose consumed was converted to gluconate, which resulted in steady state gluconate concentrations up to 80 mmol/l. The specific rate of gluconate production (0.3–7.6 mmol·h^-1·(g dry wt cells)^-1) was dependent on the growth rate and the nature of the limitation. The production rate of other overflow metabolites such as acetate, pyruvate, and 2-oxoglutarate, was only slightly altered in the presence of PQQ. The fact that the cells were now able to use an active GDH apparently did not affect apo-enzyme synthesis.Abbreviations HEPES N-2-hydroxy-ethylpiperazine-N-2-ethane sulphonic acid - MES 2-morpholinoethane sulphonic acid - PQQ pyrroloquinoline quinone (systematic name: 2,7,9-tricarboxy-1H-pyrrolo-(2,3-f)-quinoline-4,5-dione) - WB Wurster's Blue (systematic name: 1,4-bis-(dimethylamino)-benzene perchlorate     

Regulation of the glucolytic enzymes inPseudomonas putida

Summary The synthesis of glucose catabolizing enzymes is under inductive control inPseudomonas putida. Glucose, gluconate and 2-ketogluconate are the best nutritional inducers of these enzymes. Mutants unable to catabolize gluconate or 2-ketogluconate synthesized relatively high levels of glucose dehydrogenase and gluconate-6P dehydrase activities when grown in the presence of these substrates. This identifies both compounds as true inducers of these enzymes. KDGP aldolase is induced by its substrate, as evidenced by the inability of mutant cells unable to form KDGP to produce this enzyme at levels above the basal one. A 3-carbon compound appears to be the inducer of glyceraldehyde-3P dehydrogenase. This pattern of regulation suggests that there is a low degree of coordinate control in the synthesis of the glucolytic enzymes byP. putida. This is also supported by the lack of proportionality found in the levels of two enzymes governed by the same inducers, glucose dehydrogenase and gluconate-6P dehydrase, in cells grown on different conditions.Abbrevitions P phosphate - KDGP 2-Keto-3-deoxygluconate-6-phosphate - GDH glucose dehydrogenase - GNDH gluconate dehydrogenase - GK glucokinase - GNK gluconokinase - KGK ketogluconokinase - KGR 2-Ketogluconate-6-phosphate reductase - GPDH glucose-6-phosphate dehydrogenase - GNPD gluconate-6-phosphate dehydrase - KDGPA 2-Keto-3-deoxygluconate-6-phosphate aldolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase     

The production of soluble pyrroloquinoline quinone glucose dehydrogenase by Klebsiella pneumoniae, the alternative host of PQQ enzymes

Klebsiella pneumoniae, which produces PQQ and is available for use with a conventional expression vector system, was selected as the host strain for soluble PQQ glucose dehydrogenase (PQQGDH-B) production. The recombinant K. pneumoniaeexpressed PQQGDH-B in its holo-form at about 18000 U l^–1, equal to that achieved in recombinant Escherichia coli. The signal sequence of recombinant PQQGDH-B produced by K. pneumoniaewas correctly processed. K. pneumoniaecan become an alternative host microorganism not only for PQQGDH-B production but also for recombinant PQQ enzymes production.     

The functional significance of glucose dehydrogenase in Klebsiella aerogenes

In order to assess the functional significance of the quinoprotein glucose dehydrogenase recently found to be present in K⁺-limited Klebsiella aerogenes, a broad study was made of the influence of specific environmental conditions on the cellular content of this enzyme. Whereas high activities were manifest in cells from glucose containing chemostat cultures that were either potassium- or phosphate-limited, only low activities were apparent in cells from similar cultures that were either glucose-, sulphate- or ammonia-limited. With these latter two cultures, a marked increase in glucose dehydrogenase activity was observed when 2,4-dinitrophenol (1 mM end concentration) was added to the growth medium. These results suggested that the synthesis of glucose dehydrogenase is not regulated by the level of glucose in the growth medium, but possibly by conditions that imposed an energetic stress upon the cells. This conclusion was further supported by a subsequent finding that K⁺-limited cells that were growing on glycerol also synthesized substantial amounts of glucose dehydrogenase.The enzyme was found to be membrane associated, and preliminary evidence has been obtained that it is located on the periplasmic side of the cytoplasmic membrane and functionally linked to the respiratory chain. This structural and functional orientation is consistent with glucose dehydrogenase serving as a low impedance energy generating system.Abbreviations D dilution rate - DNP 2,4-dinitrophenol - PQQ 2,7,9-tricarboxy-1H-pyrrolo(2,3-f)quinoline-4,5-dione - PTS phosphoenolpyruvate: glucose phosphotransferase - WB Wurster's Blue     

The uptake of 2-ketogluconate by Pseudomonas putida

The uptake of 2-ketogluconate is inducible in Pseudomonas putida: 2-ketogluconate, glucose, gluconate, glycerol and glycerate were each good nutritional inducers of this ability. 2-Ketogluconate uptake obeyed saturation kinetics (apparent K _min 2-ketogluconate-grown cells was 0.4 mM). 2-Ketogluconate was transported against a concentration gradient, apparently in an unchanged state, and the process required metabolic energy, all of which indicate an active transport system.A number of independently isolated mutants with deranged activity of a common glucose-gluconate uptake system were found to be also defective in 2-ketogluconate transport. Strains unable to transport 2-ketogluconate which grew readily on glucose and gluconate were also isolated. These results suggest that 2-ketogluconate transport is governed by at least two genetic elements: one which is also required to take up glucose and gluconate and another which appears to be specific for 2-ketogluconate transport. Similarly glucose and gluconate transport appears to require at least one factor which is not necessary for 2-ketogluconate transport, as suggested by the lack of induction of the common glucose-gluconate uptake system by glycerol and glycerate, substrates which are good inducers of 2-ketogluconate uptake.Abbreviations CCCP carbonyl-cyanide-m-chlorophenyl-hydrazone - cpm radioactivity counts per minute - GGU glucose-gluconate uptake - PFU plaque forming units - U.V. ultraviolet Dedicated to Prof. Roger Y. Stainer on the occasion of his 60th birthday     

高浓度2-KLG对氧化葡萄糖酸杆菌WSH-003中2-KLG合成途径关键基因表达的影响

【目的】研究高浓度的2-KLG对其生产菌株氧化葡萄糖酸杆菌生产过程中关键的脱氢酶合成基因、辅因子合成基因及其转运蛋白编码基因的影响。【方法】测定高浓度梯度2-KLG下氧化葡萄糖酸杆菌的生长情况,确定合适的添加浓度对氧化葡萄糖酸杆菌进行胁迫。使用实时定量PCR技术检测2-KLG合成中关键山梨醇脱氢酶基因sld AB、关键辅因子PQQ合成基因pqq ABCDE及5个潜在转运蛋白合成基因的变化。【结果】根据氧化葡萄糖酸杆菌在2-KLG高浓度梯度下生长测定实验结果,选定40、80和120 g/L 2-KLG作为添加浓度。实时定量PCR结果显示,在高浓度的2-KLG压力下,PQQ合成基因pqq ABCDE未受到显著影响,山梨醇脱氢酶基因sld AB以及部分PQQ潜在转运蛋白编码基因的表达均显著下调。【结论】高浓度2-KLG会抑制氧化葡萄糖酸杆菌中山梨醇脱氢酶基因的表达,有可能会影响辅酶PQQ的转运,但不会显著影响辅酶PQQ的合成。     

The consequence of stimulating glucose dehydrogenase activity by the addition of PQQ on metabolite production by Agrobacterium radiobacter NCIB 11883

Summary Agrobacterium radiobacter NCIB 11 883 does not produce gluconate under conditions of glucose excess in batch or continuous culture. However, the addition of micromolar concentrations of pyrrolo quinoline quinone (PQQ) to fermentation media resulted in rapid excretion of gluconate by batch and continuous cultures. This rapid dehydrogenation of glucose was found in cells grown under carbon and nitrogen limitation and is constitutive which suggests that the only reason why this activity is not normally expressed is due to the inability of the organism to synthesize the prosthetic group (PQQ) of the glucose dehydrogenase enzyme.Although the addition of PQQ to batch and continuous cultures caused a very rapid specific rate of gluconate production (0.6–1.1 g gluconate g^-1 dry wt. h^-1) the rate of exopolysaccharide production remained unaltered. Indeed, when the rates of substrate and oxygen uptake are corrected for the rate of gluconate production in the presence of PQQ there appears to be little physiological consequence as a result of this oxidation.     

Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger

Batch cultures of Aspergillus niger grown from conidia on a medium with high C/N ratio accumulated gluconate from glucose with a yield of 57%. During almost the whole time of accumulation there was no net synthesis of total protein in the mycelium but the activity per flask and the specific activity of glucose oxidase (EC 1.1.3.4) in mycelial extracts increased whereas both values decreased for glucose dehydrogenase (EC 1.1.99.10) gluconate 6-phosphatase (cf. EC 3.1.3.1, 3.1.3.2), gluconokinase (EC 2.7.1.12), glucose 6-phosphate and phosphogluconate dehydrogenases (EC 1.1.1.49, EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), and most enzymes of the Embden-Meyerhof pathway and the tricarboxylic acid cycle. Gluconate dehydratase (EC 4.2.1.39), gluconate dehydrogenase (EC 1.1.99.3) and enzymes of the Entner-Doudoroff pathway could not be detected. By cycloheximide the increase of glucose oxidase activity was inhibited. It is concluded that the high yield of gluconate was due mainly to the net (de novo) synthesis of glucose oxidase which occurred during protein turnover after the exhaustion of the nitrogen source, and which was not accompanied by a net synthesis of the other enzymes investigated. Some gluconate may also have been formed by hydrolytic cleavage of gluconate 6-phosphate.Abbreviations GOD glucose oxidase - GD glucose dehydrogenase - PP pentose phosphate - EM Embden-Meyerhof - TCA tricarboxylic acid     

Growth yields and glucose metabolism of N<Subscript>2</Subscript>-fixing <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> at different culture pH values

Gluconacetobacter diazotrophicus was grown in chemostat under N₂-fixing conditions at different culture pH values (from 2.5 to 7.5) with glucose as the C-source. Maximum glucose and oxygen utilization yields were observed at pH values between 5.0 and 6.5. Yields, although lower, were not severely affected at acidic (2.5–4.5) and moderate alkaline (7.5) pH values. But, at pH values just over 7.5, cultures became unstable and washed out. Maximum biomass yields coincided with optimal activity (and minimal synthesis) of pyrroloquinoline quinone (PQQ)-linked glucose dehydrogenase (PQQ-GDH). At external pH values of 7.0 and above, whereas PQQ-GDH was actively synthesized, a very low in situ activity could be detected. The lack of PQQ-GDH activity at moderate alkaline pH values seems to be the cause of lack of growth of this organism under these conditions.     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent K _m values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - MDH methanol dehydrogenase - ADH acohol dehydrogenase - PQQ pyrroloquinoline, quinone - DTT dithiothreitol - NBT nitrobluetetrazolium - PMS phenazine methosulphate - DCPIP dichlorophenol indophenol     

Cooning of the genes encoding the two different glucose dehydrogenases fromAcinetobacter calcoaceticus

Glucose dehydrogenase (GDH) is a PQQ dependent bacterial enzyme which converts aldoses to their corresponding acids.A. calcoaceticus contains two different PQQ dependent glucose dehydrogenases designated GDH-A which is activein vivo and GDH-B of which onlyin vitro activity can be shown. We cloned the genes coding for the two GDH enzymes. The DNA sequences of bothgdh genes were determined. There is no obvious homology betweengdhA andgdhB. Both GDH enzymes oxidize D-glucosein vitro but disaccharides are specific GDH-B substrates and 2-deoxyglucose is specifically oxidized by GDH-A.     

Glucose Metabolism in Batch and Continuous Cultures of Gluconacetobacter diazotrophicus PAL 3

Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]–linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N₂ or NH₃). Its synthesis was stimulated by conditions of high energetic demand (i.e., N₂-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N₂-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)–linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.     

Bacterial NAD(P)-independent quinate dehydrogenase is a quinoprotein

Acinetobacter calcoaceticus LMD 79.41 produced significant amounts of pyrrolo-quinoline quinone (PQQ) in its culture medium when grown on quinic acid or shikimic acid. Studies with LMD 79.41 and PQQ^--mutants of this strain demonstrated that this organism contains an NAD(P)-independent quinate dehydrogenase (QDH) (EC 1.1.99.-), catalyzing the first degradation step of these compounds, and that the enzyme contains PQQ as a cofactor, i.e. is a quinoprotein. Synthesis of QDH was induced by protocatechuate and the enzyme appeared to be particle-bound. Acinetobacter lwoffi RAG-1 produced a quinoprotein QDH apoenzyme since growth on quinic acid only occurred in the presence of PQQ. The results obtained with the PQQ^--mutants of strain LMD 79.41 also provided some insight into the regulation of PQQ biosynthesis and assemblage of quinoprotein enzymes in the periplasmic space. Since two species of Pseudomonas also contained a quinoprotein QDH, it is assumed that bacterial NAD(P)-independent quinate dehydrogenase is a quinoprotein.Abbreviations DCPIP 2,6-dichlorophenolindophenol     

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