[Efficient biosynthesis of D-mannitol by coordinated expression of a two-enzyme cascade].

D-mannitol is widely used in the pharmaceutical and medical industries as an important precursor of antitumor drugs and immune stimulants. However, the cost of the current enzymatic process for D-mannitol synthesis is high, thus not suitable for commercialization. To address this issue, an efficient mannitol dehydrogenase LpGDH used for the conversion and a glucose dehydrogenase BaGDH used for NADH regeneration were screened, respectively. These two enzymes were co-expressed in Escherichia coli BL21(DE3) to construct a two-enzyme cascade catalytic reaction for the efficient synthesis of d-mannitol, with a conversion rate of 59.7% from D-fructose achieved. The regeneration of cofactor NADH was enhanced by increasing the copy number of Bagdh, and a recombinant strain E. coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh was constructed to address the imbalance between cofactor amount and key enzyme expression level in the two-enzyme cascade catalytic reaction. An optimized whole cell transformation process was conducted under 30 ℃, initial pH 6.5, cell mass (OD600) 30, 100 g/L D-fructose substrate and an equivalent molar concentration of glucose. The highest yield of D-mannitol was 81.9 g/L with a molar conversion rate of 81.9% in 5 L fermenter under the optimal conversion conditions. This study provides a green and efficient biotransformation method for future large-scale production of D-mannitol, which is also of great importance for the production of other sugar alcohols.

Identification of multiple proteins whose interaction with mannitol dehydrogenase is induced by salicylic acid: Implications for unconventional secretion.

Although protein secretion was previously believed to be solely via ER/Golgi pathways, Golgi-independent secretion has now been described in both animals and plants. Secretion of the mannitol catabolic enzyme mannitol dehydrogenase (MTD) in response to the endogenous pathogen response signal salicylic acid (SA) was one of the first reports of unconventional protein secretion in plants. To begin assessing potential secretion-associated MTD protein interactors, we present here high-quality databases describing changes in MTD-interacting proteins following SA treatment of Arabidopsis thaliana cells expressing MTD.

Archaeal hyperthermostable mannitol dehydrogenases: A promising industrial enzymes for d-mannitol synthesis.

Recently, the term healthy lifestyle connected to low-calorie diets, although it is not possible to get rid of added sugars as a source of energy, despite the close relation of added sugars to some diseases such as obesity, diabetes, etc. As a result, the sweetener market has flourished, which has led to increased demand for natural sweeteners such as polyols, including d-mannitol. Various methods have been developed to produce d-mannitol to achieve high productivity and low cost. In particular, metabolic engineering for d-mannitol considers one of the most promising approaches for d-mannitol production on the industrial scale. To date, the chemical process is not ideal for large-scale production because of its multistep mechanism involving hydrogenation and high cost. In this review, we highlight and present a comparative evaluation of the biochemical parameters that affecting d-mannitol synthesis from Thermotoga neapolitana and Thermotoga maritima mannitol dehydrogenase (MtDH) as a potential contribution for d-mannitol bio-synthesis. These species were selected because purified mannitol dehydrogenases from both strains have been reported to produce d-mannitol with no sorbitol formation under temperatures (90-120 °C).

Defense Response in Chickpea Pod Wall due to Simulated Herbivory Unfolds Differential Proteome Profile.

The pod wall of legumes is known to protect the developing seeds from pests and pathogens. However, the mechanism of conferring defense against insects has not yet been deciphered. Here, we have utilized 2-dimensional gel electrophoresis (2D-GE) coupled with mass spectrometry (MS/MS) to identify over expressed proteins in the pod wall of two different cultivars (commercial cultivar: JG 11 and tolerant cultivar: ICC 506-EB) of chickpea after 12 h of application of Helicoverpa armigera oral secretions (simulated herbivory). The assays were performed with a view that larvae are a voracious feeder and cause substantial damage to the pod within 12 h. A total of 600 reproducible protein spots were detected on gels, and the comparative analysis helped identify 35 (12 up-regulated, 23 down-regulated) and 20 (10 up-regulated, 10 down-regulated) differentially expressed proteins in JG 11 and ICC 506-EB, respectively. Functional classification of protein spots of each cultivar after MS/MS indicated that the differentially expressed proteins were associated with various metabolic activities. Also, stress-related proteins such as mannitol dehydrogenase (MADH), disease resistance-like protein-CSA1, serine/threonine kinase (D6PKL2), endoglucanase-19 etc. were up-regulated due to simulated herbivory. The proteins identified with a possible role in defense were further analyzed using the STRING database to advance our knowledge on their interacting partners. It decoded the involvement of several reactive oxygen species (ROS) scavengers and other proteins involved in cell wall reinforcement. The biochemical analysis also confirmed the active role of ROS scavengers during simulated herbivory. Thus, our study provides valuable new insights on chickpea-H.armigera interactions at the protein level.

5-Keto-D-fructose production from sugar alcohol by isolated wild strain Gluconobacter frateurii CHM 43.

GLUCONOBACTER FRATEURII: CHM 43 have D-mannitol dehydrogenase (quinoprotein glycerol dehydrogenase) and flavoprotein D-fructose dehydrogenase in the membranes. When the two enzymes are functional, D-mannitol is converted to 5-keto-D-fructose with 65% yield when cultivated on D-mannitol. 5-Keto-D-fructose production with almost 100% yield was realized with the resting cells. The method proposed here should give a smart strategy for 5-keto-D-fructose production.

MiRNAs differentially expressed in skeletal muscle of animals with divergent estimated breeding values for beef tenderness.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs of approximately 22 nucleotides, highly conserved among species, which modulate gene expression by cleaving messenger RNA target or inhibiting translation. MiRNAs are involved in the regulation of many processes including cell proliferation, differentiation, neurogenesis, angiogenesis, and apoptosis. Beef tenderness is an organoleptic characteristic of great influence in the acceptance of meat by consumers. Previous studies have shown that collagen level, marbling, apoptosis and proteolysis are among the many factors that affect beef tenderness. Considering that miRNAs can modulate gene expression, this study was designed to identify differentially expressed miRNAs that could be modulating biological processes involved with beef tenderness. RESULTS: Deep sequence analysis of miRNA libraries from longissimus thoracis muscle allowed the identification of 42 novel and 308 known miRNAs. Among the known miRNAs, seven were specifically expressed in skeletal muscle. Differential expression analysis between animals with high (H) and low (L) estimated breeding values for shear force (EBVSF) revealed bta-mir-182 and bta-mir-183 are up-regulated (q value < 0.05) in animals with L EBVSF, and bta-mir-338 is up-regulated in animals with H EBVSF. The number of bovine predicted targets for bta-mir-182, bta-mir-183 and bta-mir-338 were 811, 281 and 222, respectively, which correspond to 1204 unique target genes. Among these, four of them, MEF2C, MAP3K2, MTDH and TNRC6B were common targets of the three differentially expressed miRNAs. The functional analysis identified important pathways related to tenderness such as apoptosis and the calpain-calpastatin system. CONCLUSION: The results obtained indicate the importance of miRNAs in the regulatory mechanisms that influence muscle proteolysis and meat tenderness and contribute to our better understanding of the role of miRNAs in biological processes associated with beef tenderness.

Effect of the pat, fk, stpk Gene Knock-out and mdh Gene Knock-in on Mannitol Production in Leuconostoc mesenteroides.

Leuconostoc mesenteroides can be used to produce mannitol by fermentation, but the mannitol productivity is not high. Therefore, in this study modify the chromosome of Leuconostoc mesenteroides by genetic methods to obtain high-yield strains of mannitol production. In this study, gene knock-out strains and gene knock-in strains were constructed by a two-step homologous recombination method. The mannitol productivity of the pat gene (which encodes phosphate acetyltransferase) deleteon strain (Δpat::amy), fk gene (which encodes fructokinase) deleteon strain (Δfk::amy) and stpk gene (which encodes serine-threonine protein kinase) deleteon strain (Δstpk::amy) were all increased compared to the wild type, and the productivity of mannitol for each strain was 84.8%, 83.5% and 84.1% respectively. The mannitol productivity of the mdh gene (which encodes mannitol dehydrogenase) knock-in strains (Δpat::mdh, Δfk::mdh and Δstpk::mdh) was increased to a higher level than that of the single-gene deletion strains, and the productivity of mannitol for each was 96.5%, 88% and 93.2%, respectively. The multi-mutant strain ΔdtsΔldhΔpat::mdhΔstpk::mdhΔfk::mdh had mannitol productivity of 97.3%. This work shows that multi-gene knock-out and gene knock-in strains have the greatest impact on mannitol production, with mannitol productivity of 97.3% and an increase of 24.7% over wild type. This study used the methods of gene knock-out and gene knock-in to genetically modify the chromosome of Leuconostoc mesenteroides. It is of great significance that we increased the ability of Leuconostoc mesenteroides to produce mannitol and revealed its broad development prospects.

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans.

Gluconobacter (G.) oxydans is able to incompletely oxidize various sugars and polyols for the production of biotechnologically important compound. Recently, we have shown that the organism produces and accumulates mannitol as compatible solute under osmotic stress conditions. The present study describes the role of two cytoplasmic mannitol dehydrogenases for osmotolerance of G. oxydans. It was shown that Gox1432 is a NADP+-dependent mannitol dehydrogenase (EC 1.1.1.138), while Gox0849 uses NAD+ as cofactor (EC 1.1.1.67). The corresponding genes were deleted and the mutants were analyzed for growth under osmotic stress and non-stress conditions. A severe growth defect was detected for Δgox1432 when grown in high osmotic media, while the deletion of gox0849 had no effect when cells were exposed to 450 mM sucrose in the medium. Furthermore, the intracellular mannitol content was reduced in the mutant lacking the NADP+-dependent enzyme Gox1432 in comparison to the parental strain and the Δgox0849 mutant under stress conditions. In addition, transcriptional analysis revealed that Gox1432 is more important for mannitol production in G. oxydans than Gox0849 as the transcript abundance of gene gox1432 was 30-fold higher than of gox0849. In accordance, the activity of the NADH-dependent enzyme Gox0849 in the cell cytoplasm was 10-fold lower in comparison to the NADPH-dependent mannitol dehydrogenase Gox1432. Overexpression of gox1432 in the corresponding deletion mutant restored growth of the cells under osmotic stress, further strengthening the importance of the NADP+-dependent mannitol dehydrogenase for osmotolerance in G. oxydans. These findings provide detailed insights into the molecular mechanism of mannitol-mediated osmoprotection in G. oxydans and are helpful engineering strains with improved osmotolerance for biotechnological applications.

Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica.

Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase.

Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses.

The mannitol utilization system of the marine bacterium Zobellia galactanivorans.

Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. In particular, mannitol can account for as much as 20 to 30% of the dry weight of brown algae and is likely to be an important source of carbon for marine heterotrophic bacteria. Zobellia galactanivorans (Flavobacteriia) is a model for the study of pathways involved in the degradation of seaweed carbohydrates. Annotation of its genome revealed the presence of genes potentially involved in mannitol catabolism, and we describe here the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) and a fructokinase (FK). Among the observations, the M2DH of Z. galactanivorans was active as a monomer, did not require metal ions for catalysis, and featured a narrow substrate specificity. The FK characterized was active on fructose and mannose in the presence of a monocation, preferentially K(+). Furthermore, the genes coding for these two proteins were adjacent in the genome and were located directly downstream of three loci likely to encode an ATP binding cassette (ABC) transporter complex, suggesting organization into an operon. Gene expression analysis supported this hypothesis and showed the induction of these five genes after culture of Z. galactanivorans in the presence of mannitol as the sole source of carbon. This operon for mannitol catabolism was identified in only 6 genomes of Flavobacteriaceae among the 76 publicly available at the time of the analysis. It is not conserved in all Bacteroidetes; some species contain a predicted mannitol permease instead of a putative ABC transporter complex upstream of M2DH and FK ortholog genes.

MTDH mediates trastuzumab resistance in HER2 positive breast cancer by decreasing PTEN expression through an NFκB-dependent pathway.

BACKGROUND: Trastuzumab resistance is almost inevitable in the management of human epidermal growth factor receptor (HER) 2 positive breast cancer, in which phosphatase and tensin homolog deleted from chromosome 10 (PTEN) loss is implicated. Since metadherin (MTDH) promotes malignant phenotype of breast cancer, we sought to define whether MTDH promotes trastuzumab resistance by decreasing PTEN expression through an NFκB-dependent pathway. METHODS: The correlations between MTDH and PTEN expressions were analyzed both in HER2 positive breast cancer tissues and trastuzumab resistant SK-BR-3 (SK-BR-3/R) cells. Gene manipulations of MTDH and PTEN levels by knockdown or overexpression were utilized to elucidate molecular mechanisms of MTDH and PTEN implication in trastuzumab resistance. For in vivo studies, SK-BR-3 and SK-BR-3/R cells and modified derivatives were inoculated into nude mice alone or under trastuzumab exposure. Tumor volumes, histological examinations as well as Ki67 and PTEN expressions were revealed. RESULTS: Elevated MTDH expression indicated poor clinical benefit, shortened progression free survival time, and was negatively correlated with PTEN level both in HER2 positive breast cancer patients and SK-BR-3/R cells. MTDH knockdown restored PTEN expression and trastuzumab sensitivity in SK-BR-3/R cells, while MTDH overexpression prevented SK-BR-3 cell death under trastuzumab exposure, probably through IκBα inhibition and nuclear translocation of p65 which subsequently decreased PTEN expression. Synergized effect of PTEN regulation were observed upon MTDH and p65 co-transfection. Forced PTEN expression in SK-BR-3/R cells restored trastuzumab sensitivity. Furthermore, decreased tumor volume and Ki67 level as well as increased PTEN expression were observed after MTDH knockdown in subcutaneous breast cancer xenografts from SK-BR-3/R cells, while the opposite effect were found in grafts from MTDH overexpressing SK-BR-3 cells. CONCLUSIONS: MTDH overexpression confers trastuzumab resistance in HER2 positive breast cancer. MTDH mediates trastuzumab resistance, at least in part, by PTEN inhibition through an NFκB-dependent pathway, which may be utilized as a promising therapeutic target for HER2 positive breast cancer.

Time-averaged distributions of solute and solvent motions: exploring proton wires of GFP and PfM2DH.

Proton translocation pathways of selected variants of the green fluorescent protein (GFP) and Pseudomonas fluorescens mannitol 2-dehydrogenase (PfM2DH) were investigated via an explicit solvent molecular dynamics-based analysis protocol that allows for direct quantitative relationship between a crystal structure and its time-averaged solute-solvent structure obtained from simulation. Our study of GFP is in good agreement with previous research suggesting that the proton released from the chromophore upon photoexcitation can diffuse through an extended internal hydrogen bonding network that allows for the proton to exit to bulk or be recaptured by the anionic chromophore. Conversely for PfM2DH, we identified the most probable ionization states of key residues along the proton escape channel from the catalytic site to bulk solvent, wherein the solute and high-density solvent crystal structures of binary and ternary complexes were properly reproduced. Furthermore, we proposed a plausible mechanism for this proton translocation process that is consistent with the state-dependent structural shifts observed in our analysis. The time-averaged structures generated from our analyses facilitate validation of MD simulation results and provide a comprehensive profile of the dynamic all-occupancy solvation network within and around a flexible solute, from which detailed hydrogen-bonding networks can be inferred. In this way, potential drawbacks arising from the elucidation of these networks by examination of static crystal structures or via alternate rigid-protein solvation analysis procedures can be overcome. Complementary studies aimed at the effective use of our methodology for alternate implementations (e.g., ligand design) are currently underway.

Characterization of mannitol-2-dehydrogenase in Saccharina japonica: evidence for a new polyol-specific long-chain dehydrogenases/reductase.

Mannitol plays a crucial role in brown algae, acting as carbon storage, organic osmolytes and antioxidant. Transcriptomic analysis of Saccharina japonica revealed that the relative genes involved in the mannitol cycle are existent. Full-length sequence of mannitol-2-dehydrogenase (M2DH) gene was obtained, with one open reading frame of 2,007 bp which encodes 668 amino acids. Cis-regulatory elements for response to methyl jasmonic acid, light and drought existed in the 5'-upstream region. Phylogenetic analysis indicated that SjM2DH has an ancient prokaryotic origin, and is probably acquired by horizontal gene transfer event. Multiple alignment and spatial structure prediction displayed a series of conserved functional residues, motifs and domains, which favored that SjM2DH belongs to the polyol-specific long-chain dehydrogenases/reductase (PSLDR) family. Expressional profiles of SjM2DH in the juvenile sporophytes showed that it was influenced by saline, oxidative and desiccative factors. SjM2DH was over-expressed in Escherichia coli, and the cell-free extracts with recombinant SjM2DH displayed high activity on D-fructose reduction reaction. The analysis on SjM2DH gene structure and biochemical parameters reached a consensus that activity of SjM2DH is NADH-dependent and metal ion-independent. The characterization of SjM2DH showed that M2DH is a new member of PSLDR family and play an important role in mannitol metabolism in S. japonica.

Medium-chain dehydrogenases with new specificity: amino mannitol dehydrogenases on the azasugar biosynthetic pathway.

Azasugar biosynthesis involves a key dehydrogenase that oxidizes 2-amino-2-deoxy-D-mannitol to the 6-oxo compound. The genes encoding homologous NAD-dependent dehydrogenases from Bacillus amyloliquefaciens FZB42, B. atrophaeus 1942, and Paenibacillus polymyxa SC2 were codon-optimized and expressed in BL21(DE3) Escherichia coli. Relative to the two Bacillus enzymes, the enzyme from P. polymyxa proved to have superior catalytic properties with a Vmax of 0.095 ± 0.002 µmol/min/mg, 59-fold higher than the B. amyloliquefaciens enzyme. The preferred substrate is 2- amino-2-deoxy-D-mannitol, though mannitol is accepted as a poor substrate at 3% of the relative rate. Simple amino alcohols were also accepted as substrates at lower rates. Sequence alignment suggested D283 was involved in the enzyme's specificity for aminopolyols. Point mutant D283N lost its amino specificity, accepting mannitol at 45% the rate observed for 2-amino-2-deoxy-D-mannitol. These results provide the first characterization of this class of zinc-dependent medium chain dehydrogenases that utilize aminopolyol substrates.

Yarrowia lipolytica dehydrogenase/reductase: an enzyme tolerant for lipophilic compounds and carbohydrate substrates.

Yarrowia lipolytica short chain dehydrogenase/reductase (YlSDR) was expressed in Escherichia coli, purified and characterized in vitro. The substrate scope for YlSDR mediated oxidation was investigated with alcohols and unprotected carbohydrates spectrophotometrically, revealing a preference for secondary compared to primary alcohols. In reduction direction, YlSDR was highly active on ribulose and fructose, suggesting that the enzyme is a mannitol-2-dehydrogenase. In order to explore substrate tolerance especially for space-demanding, lipophilic protecting groups, 5-O-trityl-D-ribitol and 5-O-trityl-α,ß-D-ribose were investigated as substrates: YlSDR oxidized 5-O-trityl-D-ribitol and 5-O-trityl-α,ß-D-ribose and reduced the latter at the expense of NADP(H).

[Production of D-mannitol by metabolically engineered Escherichia coli].

D-Mannitol has wide applications in food, pharmaceutical, and chemical industries. In this study, we constructed a genetically stable Escherichia coli strain for D-mannitol production by integrating mannitol dehydrogenase (mdh) and fructose permease (fupL) genes of Leuconostoc pseudomesenteroides ATCC 12291 into chromosome of E. coli ATCC 8739 and inactivating other fermentation pathways (including pyruvate formate-lyase, lactate dehydrogenase, fumarate reductase, alcohol dehydrogenase, methylglyoxal synthase and pyruvate oxidase). Using mineral salts medium with glucose and fructose as carbon sources, the engineered strain could produce 1.2 mmol/L D-mannitol after anaerobic fermentation for 6 days. Based on the coupling of cell growth and D-mannitol production, metabolic evolution was used to improve D-mannitol production. After evolution for 80 generations, D-mannitol titer increased 2.6-fold and mannitol dehydrogenase activity increased 2.8-fold. Genetically stable strains constructed in this work could ferment sugars to produce D-mannitol without the addition of antibiotics, inducers and formate, which was favorable for industrial production.

Mannitol is not involved in protective reactions of Acanthamoeba.

Genes for mannitol-metabolizing enzymes, mannitol phosphate dehydrogenase (MPDH) and mannitol dehydrogenase (MDH), have been recently identified in the genome of Acanthamoeba castellanii and their potential role in stress tolerance was proposed. Using qRT-PCR, comparison has been made of mRNA levels of the enzymes for mannitol metabolism at various time intervals during the stress defence reactions of encystation and pseudocyst formation. Gradual decrease of both enzymes during encystation and slight increases at the beginning of pseudocyst formation were observed. Detailed analysis of mRNA sequences of the two genes revealed similarities with various alcohol dehydrogenases rather than mannitol dehydrogenases. Our results indicate there is probably no protective role for mannitol in Acanthamoeba as no mannitol was detected using HILIC ESI MS, in any Acanthamoeba life cycle stage. Possible misinterpretation of previously published sequences as encoding enzymes of the mannitol metabolic pathway is discussed.

Primary roles of two dehydrogenases in the mannitol metabolism and multi-stress tolerance of entomopathogenic fungus Beauveria bassiana.

Knockout and complement mutants of mannitol-1-phosphate dehydrogenase (MPD) and mannitol dehydrogenase (MTD) were constructed to probe the roles of both enzymes in the mannitol metabolism and multi-stress tolerances of entomopathogenic fungus Beauveria bassiana. Compared with wild-type and complement mutants, ΔBbMPD lost 99.5% MPD activity for reducing fructose-6-phosphate to mannitol-1-phosphate while ΔBbMTD lost 78.9% MTD activity for oxidizing mannitol to fructose. Consequently, mannitol contents in mycelia and conidia decreased 68% and 83% for ΔBbMPD, and 16% and 38% for ΔBbMTD, accompanied by greatly enhanced trehalose accumulations due to 81-87% decrease in their neutral trehalase expression. Mannitol as mere carbon source in a nitrate-based minimal medium suppressed the colony growth of ΔBbMTD instead of ΔBbMPD, and delayed more conidial germination of ΔBbMTD than ΔBbMPD. Based on median lethal responses, conidial tolerances to H(2) O(2) oxidation, UV-B irradiation and heat stress at 45°C decreased 38%, 39% and 22% in ΔBbMPD, and 18%, 16% and 11% in ΔBbMTD respectively. Moreover, ΔBbMPD and ΔBbMTD lost 14% and 7% of their virulence against Spodoptera litura larvae respectively. Our findings highlight the primary roles of MPD and MTD in mannitol metabolism and their significant contributions to multi-stress tolerances and virulence influential on the biocontrol potential of B.bassiana.

Dynamic mechanism of proton transfer in mannitol 2-dehydrogenase from Pseudomonas fluorescens: mobile GLU292 controls proton relay through a water channel that connects the active site with bulk solvent.

The active site of mannitol 2-dehydrogenase from Pseudomonas fluorescens (PfM2DH) is connected with bulk solvent through a narrow protein channel that shows structural resemblance to proton channels utilized by redox-driven proton pumps. A key element of the PfM2DH channel is the "mobile" Glu(292), which was seen crystallographically to adopt distinct positions up and down the channel. It was suggested that the "down → up" conformational change of Glu(292) could play a proton relay function in enzymatic catalysis, through direct proton shuttling by the Glu or because the channel is opened for water molecules forming a chain along which the protons flow. We report evidence from site-directed mutagenesis (Glu(292) → Ala) substantiated by data from molecular dynamics simulations that support a role for Glu(292) as a gate in a water chain (von Grotthuss-type) mechanism of proton translocation. Occupancy of the up and down position of Glu(292) is influenced by the bonding and charge state of the catalytic acid base Lys(295), suggesting that channel opening/closing motions of the Glu are synchronized to the reaction progress. Removal of gatekeeper control in the E292A mutant resulted in a selective, up to 120-fold slowing down of microscopic steps immediately preceding catalytic oxidation of mannitol, consistent with the notion that formation of the productive enzyme-NAD(+)-mannitol complex is promoted by a corresponding position change of Glu(292), which at physiological pH is associated with obligatory deprotonation of Lys(295) to solvent. These results underscore the important role of conformational dynamics in the proton transfer steps of alcohol dehydrogenase catalysis.