The Oxidoreductase DsbA1 negatively influences 2,4-diacetylphloroglucinol biosynthesis by interfering the function of Gcd in Pseudomonas fluorescens 2P24.

BACKGROUND: The polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG), produced by Pseudomonas fluorescens 2P24, is positively regulated by the GacS-GacA two-component system. RESULTS: Here we reported on the characterization of DsbA1 (disulfide oxidoreductase) as novel regulator of biocontrol activity in P. fluorescens. Our data showed that mutation of dsbA1 caused the accumulation of 2,4-DAPG in a GacA-independent manner. Further analysis indicated that DsbA1 interacts with membrane-bound glucose dehydrogenase Gcd, which positively regulates the production of 2,4-DAPG. Mutation of cysteine (C)-235, C275, and C578 of Gcd, significantly reduced the interaction with DsbA1, enhanced the activity of Gcd and increased 2,4-DAPG production. CONCLUSIONS: Our results suggest that DsbA1 regulates the 2,4-DAPG concentration via fine-tuning the function of Gcd in P. fluorescens 2P24.

Convenient and Universal Fabrication Method for Antibody-Enzyme Complexes as Sensing Elements Using the SpyCatcher/SpyTag System.

Antibody-enzyme complexes (AECs) are ideal sensing elements, especially when oxidoreductases are used as the enzymes in the complex, with the potential to carry out rapid electrochemical measurements. However, conventional methods for the fabrication of AECs, including direct fusion and chemical conjugation, are associated with issues regarding the generation of insoluble aggregates and production of homogeneous AECs. Here, we developed a convenient and universal method for the fabrication of homogeneous AECs using the SpyCatcher/SpyTag system. We used an anti-epidermal growth factor receptor (EGFR) variable domain of a heavy chain antibody (VHH) and a glucose dehydrogenase (GDH) derived from Aspergillus flavus ( AfGDH) as the model antibody and enzyme, respectively. Both SpyTag-fused VHH and SpyCatcher-fused AfGDH were successfully prepared using an Escherichia coli expression system, whereas anti-EGFR AECs were produced by simply mixing the two fusion proteins. A bivalent AEC, AfGDH with two VHH at both terminals, was also prepared and exhibited an increased affinity. A soluble EGFR was successfully detected in a dose-dependent manner using immobilized anti-EGFR immunoglobulin G (IgG) and bivalent AEC. We also confirmed the universality of this AEC fabricating method by applying it to another VHH. This method results in the convenient and universal preparation of sensing elements with the potential for electrochemical measurement.

Screening of peptide ligands for pyrroloquinoline quinone glucose dehydrogenase using antagonistic template-based biopanning.

We have developed a novel method, antagonistic template-based biopanning, for screening peptide ligands specifically recognizing local tertiary protein structures. We chose water-soluble pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH-B) as a model enzyme for this screening. Two GDH-B mutants were constructed as antagonistic templates; these have some point mutations to induce disruption of local tertiary structures within the loop regions that are located at near glucose-binding pocket. Using phage display, we selected 12-mer peptides that specifically bound to wild-type GDH-B but not to the antagonistic templates. Consequently, a peptide ligand showing inhibitory activity against GDH-B was obtained. These results demonstrate that the antagonistic template-based biopanning is useful for screening peptide ligands recognizing the specific local tertiary structure of proteins.

Effect of substrate inhibition and cooperativity on the electrochemical responses of glucose dehydrogenase. Kinetic characterization of wild and mutant types.

Thanks to its insensitivity to dioxygen and to its good catalytic reactivity, and in spite of its poor substrate selectivity, quinoprotein glucose dehydrogenase (PQQ-GDH) plays a prominent role among the redox enzymes that can be used for analytical purposes, such as glucose detection, enzyme-based bioaffinity assays, and the design of biofuel cells. A detailed kinetic analysis of the electrochemical catalytic responses, leading to an unambiguous characterization of each individual steps, seems a priori intractable in view of the interference, on top of the usual ping-pong mechanism, of substrate inhibition and of cooperativity effects between the two identical subunits of the enzyme. Based on simplifications suggested by extended knowledge previously acquired by standard homogeneous kinetics, it is shown that analysis of the catalytic responses obtained by means of electrochemical nondestructive techniques, such as cyclic voltammetry, with ferrocene methanol as a mediator, does allow a full characterization of all individual steps of the catalytic reaction, including substrate inhibition and cooperativity and, thus, allows to decipher the reason that makes the enzyme more efficient when the neighboring subunit is filled with a glucose molecule. As a first practical illustration of this electrochemical approach, comparison of the native enzyme responses with those of a mutant (in which the asparagine amino acid in position 428 has been replaced by a cysteine residue) allowed identification of the elementary steps that makes the mutant type more efficient than the wild type when cooperativity between the two subunits takes place, which is observed at large mediator and substrate concentrations. A route is thus opened to structure-reactivity relationships and therefore to mutagenesis strategies aiming at better performances in terms of catalytic responses and/or substrate selectivity.

Mutations that disrupt either the pqq or the gdh gene of Rahnella aquatilis abolish the production of an antibacterial substance and result in reduced biological control of grapevine crown gall.

Rahnella aquatilis HX2, a biocontrol agent for grapevine crown gall caused by Agrobacterium vitis, produces an antibacterial substance that inhibits the growth of A. vitis in vitro. In this study, we show that MH15 and MH16, two Tn5-induced mutants of HX2, have lost their abilities to inhibit A. vitis and have reduced biocontrol activities; they grow in logarithmic phase at a rate similar to that of the wild type and have single Tn5 insertions. They are also impaired in producing pyrroloquinoline quinone (PQQ) or glucose dehydrogenase (GDH). Complementation of MH15 and MH16 with cosmid clones of CP465 and CP104 from an HX2 DNA library restored the antibiosis, biocontrol, and PQQ or GDH production phenotypes. A 6.7-kb BamHI fragment from CP465 that fully restored the MH15-affected phenotypes was cloned and sequenced. Sequence analysis of the mutated DNA region resulted in the identification of seven open reading frames (ORFs), six of which share significant homology with PQQ-synthesizing genes in other bacteria, designated pqqA through pqqF. Meanwhile, A 5.5-kb PstI fragment from CP104 fully complemented the MH16 mutant and contained a single ORF highly similar to that of genes coding for GDHs. An in-frame gdh deletion mutant has the same phenotypes as the Tn5 mutant of MH16. Complementation of both deletion and Tn5 gdh mutants restored the affected phenotypes to wild-type levels. Our results suggest that an antibacterial substance plays a role in biocontrol of A. vitis by HX2.

Differential control by IHF and cAMP of two oppositely oriented genes, hpt and gcd, in Escherichia coli: significance of their partially overlapping regulatory elements.

The hpt gene, which encodes hypoxanthine phosphoribosyltransferase, is located next to, but transcribed in the opposite direction to, the gcd gene, which codes for a membrane-bound glucose dehydrogenase, at 3.1 min on the Escherichia coli genome. In their promoter-operator region, putative regulatory elements for integration host factor (IHF) and for the complex comprising 3', 5'-cyclic AMP (cAMP) and its receptor protein (CRP) are present, and they overlap the promoters for hpt and gcd, respectively. The involvement of IHF and cAMP-CRP, as well as the corresponding putative cis-acting elements, in the expression of the two genes was investigated by using lacZ operon fusions. In an adenylate cyclase-deficient strain, addition of cAMP increased the expression of hpt and reduced the expression of gcd. In agreement with this observation, the introduction of mutations into the putative binding element for the cAMP-CRP complex enhanced the expression of gcd. In contrast, mutations introduced into the putative IHF-binding elements increased the level of hpt expression. Similar results were obtained with IHF-defective strains. Thus, the expression of the two genes is regulated in a mutually exclusive manner. Additional experiments with mutations at the -10 sequence of the gcd promoter suggest that the binding of RNA polymerase to the hpt promoter interferes with the interaction of RNA polymerase with the gcd promoter, and vice versa.

Changes in gene transcription during a beta-mediated cell death.

The a beta peptide induces cell death in neurons grown in cell culture. Previous studies have shown that the mechanism of a beta-mediated cell death of central nervous system neurons appears to be via apoptosis. Apoptosis is an active process that involves both gene transcription and translation. Using semi-quantitative polymerase chain reaction, we have analyzed the levels of a variety of transcripts in primary neuronal cultures treated with a beta that are likely to play important roles in apoptosis. Following addition of 10 microM a beta 1-42 the immediate early response gene, c-fos, shows a rapid and sustained increase in transcript level while c-jun levels increase at a slower rate. Bcl-2 and its homologues, bcl-X and bax, also increase in amount with bcl-2 and bcl-X increasing more rapidly than bax. These data provide support indicating that a beta-mediated cell death in central nervous system neurons is an active process similar to that seen in apoptosis.

Continuous regeneration of NAD(H) covalently bound to a cysteine genetically engineered into glucose dehydrogenase.

We introduced a cysteine residue on the surface of glucose dehydrogenase from Bacillus subtilis using site-directed mutagenesis. To this mutant, an NAD-analogue was covalently attached by a disulphide bridge so that it was active intramolecularly. The glucose dehydrogenase-cys44-NAD complex, which contained one reactive NAD molecule per subunit of glucose dehydrogenase, was operated together with lactate dehydrogenase in a coupled enzymatic regeneration of NAD(H) in a hollow fiber reactor. L-lactate and gluconic acid were continuously produced from pyruvate and D-glucose, respectively, with a turnover number of 45 cycles per minute for each NAD molecule. The total turnover per coenzyme was 135,000 for the first 2.5 days.

Purification and site-specific immobilization of genetically engineered glucose dehydrogenase on thiopropyl-Sepharose.

The gene encoding glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was inserted in a plasmid 1.0 kb downstream from a lac promoter, resulting in a 70-fold higher production of the enzyme when expressed in Escherichia coli. A glucose dehydrogenase mutant containing a cysteine residue at position 44 could also be expressed at the same high level. This single cysteine residue was used as an 'affinity tag' to simplify the purification procedure as well as for site-specific immobilization of glucose dehydrogenase on Thiopropyl-Sepharose. This enzyme was purified to homogeneity with a final recovery of 65% and a specific activity of 240 U/mg. The oriented immobilization resulted in increased thermal stability.

The same domain motif for ubiquinone reduction in mitochondrial or chloroplast NADH dehydrogenase and bacterial glucose dehydrogenase.

The respiratory chain NADH:ubiquinone oxidoreductase (NADH dehydrogenase or Complex I) of mitochondria comprises some 30 different subunits, and one FMN and 4 or 5 iron-sulfur clusters as internal redox groups. The bacterial glucose dehydrogenase, which oxidizes glucose to gluconolactone in the periplasmatic space and transfers the electrons to ubiquinone, is a single polypeptide chain with pyrolloquinoline quinone as the only redox group. We report here that the two different enzymes have the same ubiquinone binding domain motif and we discuss the predicted membrane folding of this domain with regard to its role in the proton translocating function of the two enzymes.

Molecular structure and transformation of the glucose dehydrogenase gene in Drosophila melanogaster.

We have precisely mapped and sequenced the three 5' exons of the Drosophila melanogaster Gld gene and have identified the start sites for transcription and translation. The first exon is composed of 335 nucleotides and does not contain any putative translation start codons. The second exon is separated from the first exon by 8 kb and contains the Gld translation start codon. The inferred amino acid sequence of the amino terminus contains two unusual features: three tandem repeats of serine-alanine, and a relatively high density of cysteine residues. P element-mediated transformation experiments demonstrated that a 17.5-kb genomic fragment contains the functional and regulatory components of the Gld gene.