[31] d-Gluconate dehydrogenase from bacteria, 2-keto-d-gluconate-yielding, membrane-bound

Matsushita, Kazunobu; Shinagawa, Emiko; Ameyama, Minoru

doi:10.1016/s0076-6879(82)89033-2

Cited by 73 publications

(43 citation statements)

References 6 publications

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“…GADH activity at the various stages of purification was measured spectrophotometrically at 25°C by determining the initial rate of 2,6-dichlorophenolindophenol (DCPIP) reduction at 540 nm as described previously (17). One unit of enzyme activity was defined as the amount of enzyme which catalyzed D-gluconate-dependent reduction of 1 mol of DCPIP per min.…”

Section: Methodsmentioning

confidence: 99%

“…Membrane-bound gluconate dehydrogenases (GADHs), which catalyze the production of 2KDG from D-gluconate, have been purified and characterized from Pseudomonas aeruginosa (16,19), P. fluorescens (17), Klebsiella pneumoniae (17), Serratia marcescens (17), and Gluconobacter dioxyacetonicus (28). The subunit structures and enzymatic properties of these GADHs were found to be very similar (17). These GADHs consisted of three subunits; namely, flavoprotein, cytochrome c, and a third subunit whose function is unknown.…”

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confidence: 99%

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Cloning and expression of a gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase from Erwinia cypripedii ATCC 29267 in Escherichia coli

1997

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We have cloned the gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase (GADH) from Erwinia cypripedii ATCC 29267 in Escherichia coli by performing a direct-expression assay. The positive clone converted D-gluconate to 2-keto-D-gluconate (2KDG) in the culture medium. Nucleotide sequence analysis of the GADH clone revealed that the cloned fragment contained the complete structural genes for a 68-kDa dehydrogenase subunit, a 47-kDa cytochrome c subunit, and a 24-kDa subunit of unknown function and that the genes were clustered with the same transcriptional polarity. Comparison of the deduced amino acid sequences and the NH 2 -terminal sequences determined for the purified protein indicated that the dehydrogenase, cytochrome c, and 24-kDa subunits contained typical signal peptides of 22, 19, and 42 amino acids, respectively. The molecular masses of the processed subunits deduced from the nucleotide sequences (65, 45, and 20 kDa) coincided well with the molecular masses of subunits estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. In E. cypripedii and recombinant E. coli, the GADH was constitutively formed and the activity of GADH was enhanced more than twofold by addition of D-gluconate to the medium. The conversion of D-glucose to D-gluconate, 2-keto-D-gluconate (2KDG), and 2,5-diketo-D-gluconate (25DKG) in several species of the genera Acetobacter, Gluconobacter, and Erwinia is mediated by membrane-bound dehydrogenases linked to the cytochrome chains located in the cytoplasmic membrane of the bacterium (1, 31). Membrane-bound gluconate dehydrogenases (GADHs), which catalyze the production of 2KDG from D-gluconate, have been purified and characterized from Pseudomonas aeruginosa (16,19), P. fluorescens (17), Klebsiella pneumoniae (17), Serratia marcescens (17), and Gluconobacter dioxyacetonicus (28). The subunit structures and enzymatic properties of these GADHs were found to be very similar (17). These GADHs consisted of three subunits; namely, flavoprotein, cytochrome c, and a third subunit whose function is unknown. Despite these findings, no further work on gene structures has been reported.In the present work, we describe the direct-expression cloning of the gene cluster encoding three subunits of membranebound GADH from Erwinia cypripedii ATCC 29267. E. coli K-12 derivatives are capable of synthesizing the apo-glucose dehydrogenase (apo-GDH) but not the cofactor, pyrroloquinoline quinone (PQQ), which is essential for the formation of the holoenzyme (3, 5). When PQQ is present in the medium, the holoenzyme is reconstituted and then E. coli is capable of oxidizing glucose to gluconate (7). It has also been reported that the expression of PQQ synthase genes in E. coli resulted in GDH activity in the absence of exogenous PQQ (13). Therefore, we tried to convert D-glucose to 2KDG via D-gluconate by using recombinant E. coli harboring the cloned GADH gene in the presence of PQQ. MATERIALS AND METHODSBacterial strains, media, and culture conditions. E. cypripedii ATCC ...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Cloning and expression of a gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase from Erwinia cypripedii ATCC 29267 in Escherichia coli

1997

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“…The enzyme kinetic assay of native and mutant Ga5DH was performed by a previously described procedure. 27 …”

Section: Cloning Expression and Purification Of Ga5dhmentioning

confidence: 99%

Structural insight into the catalytic mechanism of gluconate 5‐dehydrogenase from Streptococcus suis: Crystal structures of the substrate‐free and quaternary complex enzymes

Zhang¹,

Peng²,

Gao³

et al. 2009

Protein Science

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Gluconate 5-dehydrogenase (Ga5DH) is an NADP(H)-dependent enzyme that catalyzes a reversible oxidoreduction reaction between D-gluconate and 5-keto-D-gluconate, thereby regulating the flux of this important carbon and energy source in bacteria. Despite the considerable amount of physiological and biochemical knowledge of Ga5DH, there is little physical or structural information available for this enzyme. To this end, we herein report the crystal structures of Ga5DH from pathogenic Streptococcus suis serotype 2 in both substrate-free and liganded (NADP 1 /Dgluconate/metal ion) quaternary complex forms at 2.0 Å resolution. Structural analysis reveals that Ga5DH adopts a protein fold similar to that found in members of the short chain dehydrogenase/ reductase (SDR) family, while the enzyme itself represents a previously uncharacterized member of this family. In solution, Ga5DH exists as a tetramer that comprised four identical $29 kDa subunits. The catalytic site of Ga5DH shows considerable architectural similarity to that found in other enzymes of the SDR family, but the S. suis protein contains an additional residue (Arg104) that plays an important role in the binding and orientation of substrate. The quaternary complex structure provides the first clear crystallographic evidence for the role of a catalytically important serine residue and also reveals an amino acid tetrad RSYK that differs from the SYK triad found in the majority of SDR enzymes. Detailed analysis of the crystal structures reveals important contributions of Ca 21 ions to active site formation and of specific residues at the C-termini of subunits to tetramer assembly. Because Ga5DH is a potential target for therapy, our findings Additional Supporting Information may be found in the online version of this article. provide insight not only of catalytic mechanism, but also suggest a target of structure-based drug design.

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“…Determination of gluconate content. Gluconate content in the culture medium was determined using gluconate dehydrogenase, purified from Pseudomonas aeruginosa (Matsushita et al, 1979), which has a high specificity for gluconate (Matsushita et al, 1982). Gluconate dehydrogenase activity was measured spectrophotometrically at 25 OC in a reaction mixture which contained 50 mM PIPES/NaOH buffer (pH 6.5), gluconate dehydrogenase (approximately 0 1 unit), 0-4mM PMS, 0.2 mM DCIP and the sample, obtained from the medium after centrifugation, or a standard gluconate solution (&lo mM).…”

Section: / Omentioning

confidence: 99%

Escherichia coli is unable to produce pyrroloquinoline quinone (PQQ)

et al. 1997

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Many bacteria can synthesize the cofactor pyrroloquinoline quinone (PQQ), a cofactor of several dehydrogenases, including glucose dehydrogenase (GCD). Among the enteric bacteria, Klebsiella pneumoniae has been shown to contain the genes required for PQQ biosynthesis. Escherichia coli and Salmonella typhimurium were thought to be unable to synthesize PQQ but it has been reported that strain EF260, a derivative of E. coli FB8, can synthesize PQQ after mutation and can oxidize glucose to gluconate via the GCD/PQQ pathway (F. Biville, E. Turlin & F. Gasser, 1991, J Gen Micmbioll37,1775-1782). We have reinvestigated this claim and conclude that it is most likely erroneous. (i) Strain EF260, isolated originally by Biville and coworkers, was unable to synthesize a holo-enzyme GCD unless PQQ was supplied to the growth medium. No GCD activity could be detected in membrane fractions. (ii) The amount of PQQ detected in the growth medium of EF260 was very low and not very different from that found in a medium with its parent strain or in a medium containing no cells. (iii) EF260 cells were unable to produce gluconate from glucose via the PQQ/GCD pathway. (iv) Introduction of a gcd::Cm deletion in EF260, eliminating GCD, did not affect glucose metabolism. This suggested a pathway for glucose metabolism other than the PQQ/GCD pathway. (v) Glucose uptake and metabolism in EF260 involved a low-affinity transport system of unknown identity, followed most likely by phosphorylation via glucokinase. It is concluded that E. coli cannot synthesize PQQ and that it lacks genes required for PQQ biosynthesis.

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[31] d-Gluconate dehydrogenase from bacteria, 2-keto-d-gluconate-yielding, membrane-bound

Cited by 73 publications

References 6 publications

Cloning and expression of a gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase from Erwinia cypripedii ATCC 29267 in Escherichia coli

Cloning and expression of a gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase from Erwinia cypripedii ATCC 29267 in Escherichia coli

Structural insight into the catalytic mechanism of gluconate 5‐dehydrogenase from Streptococcus suis: Crystal structures of the substrate‐free and quaternary complex enzymes

Escherichia coli is unable to produce pyrroloquinoline quinone (PQQ)

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