Two binding sites of inhibitors in NADH: ubiquinone oxidoreductase (complex I). Relationship of one site with the ubiquinone-binding site of bacterial glucose:ubiquinone oxidoreductase. (65/68)

The effect of ten naturally occurring and two synthetic inhibitors of NADH:ubiquinone oxidoreductase (complex I) of bovine heart, Neurospora crassa and Escherichia coli and glucose:ubiquinone oxidoreductase (glucose dehydrogenase) of Gluconobacter oxidans was investigated. These inhibitors could be divided into two classes with regard to their specificity and mode of action. Class I inhibitors, including the naturally occurring piericidin A, annonin VI, phenalamid A2, aurachins A and B, thiangazole and the synthetic fenpyroximate, inhibit complex I from all three species in a partially competitive manner and glucose dehydrogenase in a competitive manner, both with regard to ubiquinone. Class II inhibitors including the naturally occurring rotenone, phenoxan, aureothin and the synthetic benzimidazole inhibit complex I from all species in an non-competitive manner, but have no effect on the glucose dehydrogenase. Myxalamid PI could not be classified as above because it inhibits only the mitochondrial complex I and in a competitive manner. All inhibitors affect the electron-transfer step from the high-potential iron-sulphur cluster to ubiquinone. Class I inhibitors appear to act directly at the ubiquinone-catalytic site which is related in complex I and glucose dehydrogenase.  (+info)

Function of multiple heme c moieties in intramolecular electron transport and ubiquinone reduction in the quinohemoprotein alcohol dehydrogenase-cytochrome c complex of Gluconobacter suboxydans. (66/68)

Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. ADH is a membrane-bound quinohemoprotein-cytochrome c complex which consists of subunits I (78 kDa), II (48 kDa), and III (14 kDa) and contains several hemes c as well as pyrroloquinoline quinone as prosthetic groups. To understand the role of the heme c moieties in the intramolecular electron transport and the ubiquinone reduction, the ADH complex of Gluconobacter suboxydans was separated into a subunit I/III complex and subunit II, then reconstituted into the complex. The subunit I/III complex, probably subunit I, contained 1 mol each of pyrroloquinoline quinone and heme c and exhibited significant ferricyanide reductase, but no Q1 reductase activities. Subunit II was a triheme cytochrome c and had no enzyme activity, but it enabled the subunit I/III complex to reproduce the Q1 and ferricyanide reductase activities. Hybrid ADH consisting of the subunit I/III complex of G. suboxydans ADH and subunit II of Acetobacter aceti ADH was constructed and it had showed a significant Q1 reductase activity, indicating that subunit II has a ubiquinone-binding site. Inactive ADH from G. suboxydans exhibiting only 10% of the Q1 and ferricyanide reductase activities of the active enzyme has been isolated separately from active ADH (Matsushita, K., Yakushi, T., Takaki, Y., Toyama, H., and Adachi, O (1995) J. Bacteriol. 177, 6552-6559). Using these active and inactive ADHs and also isolated subunit I/III complex, we performed kinetic studies which suggested that ADH contains four ferricyanide-reacting sites, one of which was detected in subunit I and the others in subunit II. One of the three ferricyanide-reacting sites in subunit II was defective in inactive ADH. The ferricyanide-reacting site remained inactive even after alkali treatment of inactive ADH and also after reconstituting the ADH complex from the subunits, in contrast to the restoration of Q1 reductase activity and the other ferricyanide reductase activities. Thus, the data suggested that the heme c in subunit I and two of the three heme c moieties in subunit II are involved in the intramolecular electron transport of ADH into ubiquinone, where one of the two heme c sites may work at, or close to, the ubiquinone-reacting site and another between that and the heme c site in subunit I. The remaining heme c moiety in subunit II may have a function other than the electron transfer from ethanol to ubiquinone in ADH.  (+info)

Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain. (67/68)

We have purified L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) from Gluconobacter oxydans T-100 that showed an ability to convert D-sorbitol to 2-keto-L-gulonate (2-KLGA). A genomic library of Gluconobacter oxydans T-100 was screened with a probe, a 180-bp PCR product which was obtained from degenerate oligodeoxyribonucleotides based on the elucidated sequence of the purified SDH (used as primers) and the genomic DNA of G. oxydans T-100 (used as a template). From sequencing of the DNA from a clone positive to the probe, the SNDH and the SDH were estimated to be coded in sequential open reading frames with 1,497 and 1,599 nucleotides, respectively, which was confirmed by expression of the DNA in Escherichia coli that showed both enzymatic activities. The DNA was introduced to a shuttle vector which was prepared from a plasmid of G. oxydans T-100 and pHSG298 to obtain an expression vector designated pSDH155. The production of 2-KLGA by pSDH155 in G. oxydans G624, an L-sorbose-accumulating strain, was improved to 230% compared to that of G. oxydans T-100. Chemical mutation of the host strain to suppress the L-idonate pathway and replacement of the original promoter with that of E. coli tufB resulted in improving the production of 2-KLGA. Consequently, high-level production from D-sorbitol to 2-KLGA (130 mg/ml) was achieved by simple fermentation of the recombinant Gluconobacter.  (+info)

Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans. (68/68)

Decaprenyl diphosphate (decaprenyl-PP) synthase catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce decaprenyl-PP, which is used for the side chain of ubiquinone (Q)-10. We have cloned the synthase gene, designated ddsA, from Gluconobacter suboxydans and expressed it in Escherichia coli. Sequence analysis revealed the presence of an ORF of 948 bp capable of encoding a 33,898-Da polypeptide that displays high similarity (30-50%) to other prenyl diphosphate synthases. Expression of the ddsA gene complemented the lethality resulting from a defect in the octaprenyl diphosphate synthase gene of E. coli and produced Q-10, indicating that Q-10 can substitute for the function of Q-8. The His-tagged DdsA protein was purified to characterize its enzymatic properties. This enzyme required detergent (0.05% Triton X-100) and 10 mM Mg2+, for full activity. The Michaelis constants for geranyl diphosphate, all-E-farnesyl diphosphate and all-E-geranylgeranyl diphosphate were 7.00, 0.50 and 0.32 microM, respectively. Nine single-amino-acid substitutions were introduced upstream of conserved region II or VI. Most of the mutants showed a considerable decrease in catalytic activity or shortening of the ultimate chain length. However, the A70G mutant produced a longer-chain-length product than wild-type decaprenyl-PP synthase, and the A70Y mutant completely abolished the decaprenyl-PP synthase function, indicating that Ala70 is important for enzyme activity and the determination of the chain-length properties of DdsA.  (+info)