Glucose dehydrogenase of a rhizobacterial strain of <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae

Glucose dehydrogenase (GDH) of Gram-negative bacteria is a membrane bound enzyme catalyzing the oxidation of glucose to gluconic acid and is involved in the solubilization of insoluble mineral phosphate complexes. A 2.4 kb glucose dehydrogenase gene (gcd) of Enterobacter asburiae sharing extensive homology to the gcd of other enterobacteriaceae members was cloned in a PCR-based directional genome walking approach and the expression confirmed in Escherichia coli YU423 on both MacConkey glucose agar and hydroxyapatite (HAP) containing media. Mineral phosphate solubilization by the cloned E. asburiae gcd was confirmed by the release of significant amount of phosphate in HAP containing liquid medium. gcd was over expressed in E. coli AT15 (gcd::cm) and the purified recombinant protein had a high affinity to glucose, and oxidized galactose and maltose with lower affinities. The enzyme was highly sensitive to heat and EDTA, and belonged to Type I, similar to GDH of E. coli.     

Variation in the Nature of Organic Acid Secretion and Mineral Phosphate Solubilization by Citrobacter sp. DHRSS in the Presence of Different Sugars

A novel phosphate solubilizing bacterium (PSB) was isolated from the rhizosphere of sugarcane and is capable of utilizing sucrose and rock phosphate as the sole carbon and phosphate source, respectively. This PSB exhibited mineral phosphate solubilizing (MPS) phenotype on sugars such as sucrose and fructose, which are not substrates for enzyme glucose dehydrogenase (GDH), along with GDH substrates, viz., glucose, xylose, and maltose, as carbon sources. PCR amplification of the rRNA gene and sequence analysis identified this bacterium as Citrobacter sp. DHRSS. On sucrose and fructose Citrobacter sp. DHRSS liberated 170 and 100 μM free phosphate from rock phosphate and secreted 49 mM (2.94 g/L) and 35 mM (2.1 g/L) acetic acid, respectively. Growth of Citrobacter sp. DHRSS on sucrose is mediated by an intracellular inducible neutral invertase. Interestingly, in the presence of GDH substrates like glucose and maltose, Citrobacter sp. DHRSS produced approximately 20 mM (4.36 g/L) gluconic acid and phosphate released was 520 and 570 μM, respectively. Citrobacter sp. DHRSS GDH activity was found when grown on GDH and non-GDH substrates, indicating that it is constitutive and could act on a wide range of aldose sugars. This study demonstrates the role of different organic acids in mineral phosphate solubilization by rhizobacteria depending on the nature of the available carbon source.     

Repression of oxalic acid-mediated mineral phosphate solubilization in rhizospheric isolates of Klebsiella pneumoniae by succinate

Two strains of Klebsiella (SM6 and SM11) were isolated from rhizospheric soil that solubilized mineral phosphate by secretion of oxalic acid from glucose. Activities of enzymes for periplasmic glucose oxidation (glucose dehydrogenase) and glyoxylate shunt (isocitrate lyase and glyoxylate oxidase) responsible for oxalic acid production were estimated. In presence of succinate, phosphate solubilization was completely inhibited, and the enzymes glucose dehydrogenase and glyoxylate oxidase were repressed. Significant activity of isocitrate lyase, the key enzyme for carbon flux through glyoxylate shunt and oxalic acid production during growth on glucose suggested that it could be inducible in nature, and its inhibition by succinate appeared to be similar to catabolite repression.     

Gamma-radiation induced modifications in substrate specificity of glucose dehydrogenase and carbon source utilization pattern of phosphate-solubilizing <Emphasis Type="Italic">Pantoea</Emphasis> strains

Glucose, maltose, and mannose as sole carbon sources, induced synthesis of glucose dehydrogenase (GDH) in three strains of Pantoea with specific activities from 0.14 to 0.6 U/mg proteins. Utilization of lactose indicated that the enzyme belongs to GDH type B isozyme. Of mutant clones, developed through radiation mutagenesis, P2-M2 utilized ribose with GDH specific activity of 0.57 U/mg protein, P4-M3 grown on glucose gave 1.5 U/mg protein and P4-M5 had high activities, when grown on galactose, maltose, and lactose. Clones P3-M2 and P2-M5 had versatile utilization of sugars and released higher amounts of P from tri-calcium phosphate and can be efficiently used for biofertilization.     

Repression of mineral phosphate solubilizing phenotype in the presence of weak organic acids in plant growth promoting fluorescent pseudomonads

Two phosphate solubilizing bacteria (PSB), M3 and SP1, were obtained from the rhizosphere of mungbean and sweet potato, respectively and identified as strains of Pseudomonas aeruginosa. Their rock phosphate (RP) solubilizing abilities were found to be due to secretion high amount of gluconic acid. In the presence of malate and succinate, individually and as mixture, the P solubilizing ability of both the strains was considerably reduced. This was correlated with a nearly 80% decrease in the activity of the glucose dehydrogenase (GDH) but not gluconate dehydrogenase (GAD) in both the isolates. Thus, GDH enzyme, catalyzing the periplasmic production of gluconic acid, is under reverse catabolite repression control by organic acids in P. aeruginosa M3 and SP1. This is of relevance in rhizospheric conditions and is a new explanation for the lack of field efficacy of such PSB.     

The in vivo and in vitro substrate specificity of quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus LMD79.41

Quinoprotein glucose dehydrogenase (GDH; EC 1.1.99.17) was partially purified from cell-free extracts of Acinetobacter calcoaceticus LMD79.41. The enzyme oxidized monosaccharides (d-glucose, d-allose, 2-deoxy-d-glucose, d-galactose, d-mannose, d-xylose, d-ribose and l-arabinose) as well as disaccharides (d-lactose, d-maltose and d-cellobiose).Intact cells of A. calcoaceticus LMD79.41 also oxidized these monosaccharides, but not the disaccharides.The difference in substrate specificity can not be explained by impermeability of the outer membrane for disaccharides, since right-side-out membrane vesicles did not oxidize disaccharides either. Destruction of the cytoplasmic membrane strongly affected the catalytic properties of GDH. Not only did the affinity towards some monosaccharides change substantially, but disaccharides also became good substrates upon solubilization of the enzyme. Thus, at least in A. calcoaceticus LMD79.41, the oxidation of disaccharides by GDH can be considered as an in vitro ‘artefact’ caused by the removal of the enzyme from its natural environment.     

Stabilization of pyrroloquinoline quinone glucose dehydrogenase by cross-linking chemical modification

Summary Cross-linking chemical modification of pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH) by glutaraldehyde was carried out and its stability was analyzed. Although native PQQGDH was inactivated within 30 min at a higher temperature than 50 °C, cross-linked PQQGDH retained more than 40% of initial activity even after 30 min of incubation at 54 °C. In addition to the increase in thermal stability, cross-linked PQQGDH gained high EDTA tolerance. The stabilization may be achieved by increased the rigidity of PQQGDH holo enzyme conformation.     

Effects of a GTP-insensitive mutation of glutamate dehydrogenase on insulin secretion in transgenic mice

Glutamate dehydrogenase (GDH) plays an important role in insulin secretion as evidenced in children by gain of function mutations of this enzyme that cause a hyperinsulinism-hyperammonemia syndrome (GDH-HI) and sensitize beta-cells to leucine stimulation. GDH transgenic mice were generated to express the human GDH-HI H454Y mutation and human wild-type GDH in islets driven by the rat insulin promoter. H454Y transgene expression was confirmed by increased GDH enzyme activity in islets and decreased sensitivity to GTP inhibition. The H454Y GDH transgenic mice had hypoglycemia with normal growth rates. H454Y GDH transgenic islets were more sensitive to leucine- and glutamine-stimulated insulin secretion but had decreased response to glucose stimulation. The fluxes via GDH and glutaminase were measured by tracing 15N flux from [2-15N]glutamine. The H454Y transgene in islets had higher insulin secretion in response to glutamine alone and had 2-fold greater GDH flux. High glucose inhibited both glutaminase and GDH flux, and leucine could not override this inhibition. 15NH4Cl tracing studies showed 15N was not incorporated into glutamate in either H454Y transgenic or normal islets. In conclusion, we generated a GDH-HI disease mouse model that has a hypoglycemia phenotype and confirmed that the mutation of H454Y is disease causing. Stimulation of insulin release by the H454Y GDH mutation or by leucine activation is associated with increased oxidative deamination of glutamate via GDH. This study suggests that GDH functions predominantly in the direction of glutamate oxidation rather than glutamate synthesis in mouse islets and that this flux is tightly controlled by glucose.     

Inactivation of pqq genes of Enterobacter intermedium 60-2G reduces antifungal activity and induction of systemic resistance

Enterobacter intermedium 60-2G, a phosphate solubilizing bacterium, has the ability to induce systemic resistance in plants against soft rot pathogen Erwinia carotovora. Glucose dehydrogenase, an enzyme that utilizes pyrroloquinoline quinone (PQQ) as a cofactor, is required for the synthesis of gluconic acid by E. intermedium 60-2G. Here, we report that the pqqA and pqqB genes are required for phosphate solubilization and induced systemic resistance against a soft rot pathogen in tobacco. Mutations in either the pqqA or pqqB gene abolished the production of 2-ketogluconic acid and eliminated the ability of E. intermedium to solubilize hydroxyapatite. Addition of gluconic acid to the growth media restored the ability of the pqqA mutant to produce 2-ketogluconic acid. Interestingly, both pqqA and pqqB mutants of E. intermedium lost their ability to inhibit the growth of the rice pathogen Magnaporthe grisea KI-409. Additionally, induced systemic resistance against the soft rot pathogen was attenuated in the pqq mutants. These functions were restored by complementation with the wild-type pqq gene cluster. Our findings suggest that PQQ plays an important function in beneficial traits including phosphate solubilization, antifungal activity, and induced systemic resistance of E. intermedium, possibly by acting as a cofactor for several enzymes including glucose dehydrogenase.     

Characterization and Engineering of a Novel Pyrroloquinoline Quinone Dependent Glucose Dehydrogenase from <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> So ce56

A novel pyrroloquinoline quinone dependent glucose dehydrogenase like enzyme (PQQ GDH) was isolated from Sorangium cellulosum So ce56. The putative coding region was cloned, over expressed in E. coli and the resulting enzyme was characterized. The recombinant protein has a relative molecular mass of 63 kDa and shows 43% homology to PQQ GDH-B from Acinetobacter calcoaceticus. In the presence of PQQ and CaCl₂ the enzyme has dehydrogenase activity with the substrate glucose as well as with other mono- and disaccharides. The thermal stability and its pH activity profile mark the enzyme as a potential glucose biosensor enzyme. In order to decrease the activity on maltose, which is unwanted for a potential application in biosensors, the protein was rationally modified at three specified positions. The best variant showed a 59% reduction in activity on maltose compared to the wild type enzyme. The catalytic efficiency (k _cat/K _M) was reduced fivefold but the specific activity still amounted to 63% of the wild type activity.     

Construction of Engineered Water-soluble PQQ Glucose Dehydrogenase with Improved Substrate Specificity

This was the first study that achieved a narrowing of the substrate specificity of water soluble glucose dehydrogenase harboring pyrroloquinoline quinone as their prosthetic group, PQQGDH-B. We conducted the introduction of amino acid substitutions into the loop 6BC region of the enzyme, which made up the active site cleft without directly interacting with the substrate, and constructed a series of site directed mutants. Among these mutants, Asn452Thr showed the least narrowed substrate specificity while retaining a similar catalytic efficiency, thermal stability and EDTA tolerance as the wild-type enzyme. The relative activities of mutant enzyme with lactose were lower than that of the wild-type enzyme. The altered substrate specificity profile of the mutant enzyme was found to be mainly due to increase in Km value for substrate than glucose. The predicted 3D structures of Asn452Thr and the wild-type enzyme indicated that the most significant impact of the amino acid substitution was observed in the interaction between the 6BC loop region with lactose.     

Construction of Engineered Water-soluble PQQ Glucose Dehydrogenase with Improved Substrate Specificity

This was the first study that achieved a narrowing of the substrate specificity of water soluble glucose dehydrogenase harboring pyrroloquinoline quinone as their prosthetic group, PQQGDH-B. We conducted the introduction of amino acid substitutions into the loop 6BC region of the enzyme, which made up the active site cleft without directly interacting with the substrate, and constructed a series of site directed mutants. Among these mutants, Asn452Thr showed the least narrowed substrate specificity while retaining a similar catalytic efficiency, thermal stability and EDTA tolerance as the wild-type enzyme. The relative activities of mutant enzyme with lactose were lower than that of the wild-type enzyme. The altered substrate specificity profile of the mutant enzyme was found to be mainly due to increase in Km value for substrate than glucose. The predicted 3D structures of Asn452Thr and the wild-type enzyme indicated that the most significant impact of the amino acid substitution was observed in the interaction between the 6BC loop region with lactose.     

Residues in the alpha helix 7 of the bacterial maltose binding protein which are important in interactions with the Mal FGK2 complex.

The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex.     

Transconjugation studies in <Emphasis Type="Italic">Azospirillum</Emphasis> sp. negative to mineral phosphate solubilization

It has been previously shown that certain gram-negative bacteria do not have the ability to solubilize insoluble phosphates due to the lack of pyrroloquinoline-quinone synthesis genes (pqq). PQQ is required as cofactor for the assembly of the glucose dehydrogenase (GDH) holoenzyme, which acts in the oxidation of glucose to gluconic acid. In this context the transconjugation and expression of pqq genes in Azospirillum sp. was studied using the construct pMCG 898. pMCG 898 containing pqq gene/s was mobilized into an Azospirillum strain negative to mineral phosphate solubilization by biparental mating. The presence of the construct was also confirmed by minipreps of the transconjugants. The transconjugants were able to solubilize dicalcium phosphate while the wild type was not able to do so. The nitrogen-fixing ability of the transconjugants was also examined and they retained the ability to fix nitrogen. Further detailed studies are required to confirm the utility of such strains in releasing inorganic P from fixed phosphates in soil.     

Substrate specificity of glucose dehydrogenase (GDH) of Enterobacter asburiae PSI3 and rock phosphate solubilization with GDH substrates as C sources

Enterobacter asburiae PSI3 is a rhizospheric isolate that solubilizes mineral phosphates by the action of a phosphate starvation-inducible GDH (EC 1.1.5.2). We report here that GDH activity of this isolate shows broad substrate range, being able to act on mono and disaccharides. Enterobacter asburiae PSI3 was proficient at bringing about a drop in pH and solubilization of RP with the use of 75 mmol/L of each of the GDH substrate sugars tested as the sole C source. It liberated amounts of P ranging from 450 micromol/L (on arabinose) to 890 micromol/L (on glucose). When grown on a mixture of 7 GDH substrates at concentrations of 15 mmol/L each, the bacterium solubilized RP equivalent to 46% of the value when 75 mmol glucose/L was the C source. HPLC analysis of the culture supernatant under these conditions showed that the acidification of the media is primarily due to the production of organic acids. The significance of these results on the efficacy of E. asburiae PSI3 at solubilizing phosphates under rhizospheric conditions is discussed.     

Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).     

Biochemical analyses of natural and induced null variants of Drosophila enzymes.

For the characterization of null mutants identified in Drosophila populations, several Drosophila enzymes including alcohol dehydrogenase, cytoplasmic malate dehydrogenase, alpha-glycerol phosphate dehydrogenase, and phosphoglucose isomerase were co-purified to homogeneity using an 8-(6-aminohexyl)-amino-ATP-Sepharose affinity column followed by DEAE-Sepharose column chromatography. Mitochondrial malate dehydrogenase was purified by the use of CM-Sepharose and the same ATP affinity column. Alcohol dehydrogenase and alpha-glycerol phosphate dehydrogenase were mapped on a two-dimensional gel. Antiserum was raised in rabbits against these Drosophila enzymes. The presence of cross-reacting material in null mutants was characterized by double immunodiffusion, immunoelectrophoresis, and two-dimensional gel electrophoresis. By immunological techniques, two natural null variants of malic enzyme and one of phosphoglucose isomerase were shown to be negative to cross-reacting material. Two low-dose-rate gamma-radiation-induced null mutants of cytoplasmic malate dehydrogenase were shown to be positive to cross-reacting material. Two-dimensional gel analyses enabled the characterization of three natural null variants of alpha-glycerol phosphate dehydrogenase. The viability of some null mutants with homozygous null or null/deficiency genotypes is discussed in terms of the in vivo metabolic roles of the related enzymes.     

Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity

An engineered Escherichia coli PQQ glucose dehydrogenase (PQQGDH) with improved enzymatic characteristics was constructed by substituting and combining the gene-encoding protein regions responsible for EDTA tolerance, thermal stability and substrate specificity. The protein region responsible for complete EDTA tolerance in Acinetobacter calcoaceticus, which is recognized as the indicator of high stability in co-factor binding, was elucidated. The region is located between 32 and 59% from the N-terminus of A. calcoaceticus PQQGDH(A27 region) and also corresponds to the same position from 32 to 59% from the N-terminus in E. coli PQQGDH, though E. coli PQQGDH is EDTA sensitive. We previously reported that the C-terminal 3% region of A. calcoaceticus (A3 region) played an important role in the increase of thermal stability, and that His775Asn substitution in E. coli PQQGDH resulted in an increase in the substrate specificity of E. coli PQQGDH towards glucose. Based on these findings, chimeric and/or mutated PQQGDHs, E97A3 H775N, E32A27E41 H782N, E32A27E38A3 and E32A27E38A3 H782N were constructed to investigate the compatibility of two protein regions and one amino acid substitution. His775 substitution to Asn corresponded to His782 substitution to Asn (H782N) in chimeric enzymes harbouring the A27 region. Since all the chimeric PQQGDHs harbouring the A27 region were EDTA tolerant, the A27 region was found to be compatible with the other region and substituted amino acid responsible for the improvement of enzymatic properties. The contribution of the A3 region to thermal stability complemented the decrease in the thermal stability due to the His775 or His782 substitution to Asn. E32A27E38A3 H782N, which harbours all the above mentioned three regions, showed improved EDTA tolerance, thermal stability and substrate specificity. These results suggested a strategy for the construction of a semi-artificial enzyme by substituting and combining the gene-encoding protein regions responsible for the improvement of enzyme characteristics. The characteristics of constructed chimeric PQQGDH are discussed based on the predicted model, beta-propeller structure.     

C-terminal periplasmic domain of Escherichia coli quinoprotein glucose dehydrogenase transfers electrons to ubiquinone

Membrane-bound quinoprotein glucose dehydrogenase (GDH) in Escherichia coli donates electrons directly to ubiquinone during the oxidation of d-glucose as a substrate, and these electrons are subsequently transferred to ubiquinol oxidase in the respiratory chain. To determine whether the specific ubiquinone-reacting site of GDH resides in the N-terminal transmembrane domain or in the large C-terminal periplasmic catalytic domain (cGDH), we constructed a fusion protein between the signal sequence of beta-lactamase and cGDH. This truncated GDH was found to complement a GDH gene-disrupted strain in vivo. The signal sequence of the fused protein was shown to be cleaved off, and the remaining cGDH was shown to be recovered in the membrane fraction, suggesting that cGDH has a membrane-interacting site that is responsible for binding to membrane, like peripheral proteins. Kinetic analysis and reconstitution experiments revealed that cGDH has ubiquinone reductase activity nearly equivalent to that of the wild-type GDH. Thus, it is likely that the C-terminal periplasmic domain of GDH possesses a ubiquinone-reacting site and transfers electrons directly to ubiquinone.     

Mutants of Escherichia coli producing pyrroloquinoline quinone.

In glucose minimal medium a PTS- strain of Escherichia coli [delta (ptsH ptsI crr)] could grow slowly (doubling time, d = 10 h). When the population reached 5 x 10(6) to 2 x 10(7) cells ml-1, mutants growing rapidly (d = 1.5 h) appeared and rapidly outgrew the initial population. These mutants (EF mutants) do not use a constitutive galactose permease for glucose translocation. They synthesize sufficient pyrroloquinoline quinone (PQQ) to yield a specific activity of glucose dehydrogenase (GDH) equivalent to that found in the parent strain grown in glucose minimal medium supplemented with 1 nM-PQQ. Membrane preparations containing an active GDH oxidized glucose to gluconic acid, which was also present in the culture supernatant of EF strains in glucose minimal medium. Glucose utilization is the only phenotypic trait distinguishing EF mutants from the parent strain. Glucose utilization by EF mutants was strictly aerobic as expected from a PQQ-dependent catabolism. The regulation of PQQ production by E. coli is discussed.