Quaternary structure of quinoprotein ethanol dehydrogenase from <i>Pseudomonas aeruginosa</i> and its reoxidation with a novel cytochrome <i>c</i> from this organism

Schrover, J M J; Frank, Johannes; Wielink, John E. van; Duine, Johannis A.

doi:10.1042/bj2900123

Cited by 41 publications

(27 citation statements)

References 29 publications

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“…ble glucose dehydrogenase through cytochrome b 562 (5,13,19). In other type I quinoproteins, cytochrome c EDH (31) and cytochrome c 550 (29) have been reported to serve as electron acceptors. BOH appears to function like the ADH in P. putida HK5.…”

Section: Discussionmentioning

confidence: 99%

“…The molecular structure of type I ADH found in Pseudomonas aeruginosa and Pseudomonas putida (15,16,37) resembles that of MDH but has very low affinity for methanol. Type I ADH uses a c-type cytochrome (cytochrome c QEDH or cytochrome c 550 ) as the electron acceptor (29,31), which subsequently reacts with another c-type cytochrome or coppercontaining protein (azurin) (4,26). Type II ADHs are soluble periplasmic quinohemoproteins having PQQ and heme c as prosthetic groups and have been found in Comamonas testosteroni (12,17), P. putida (37), and Ralstonia eutropha (40).…”

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confidence: 99%

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Roles for the Two 1-Butanol Dehydrogenases ofPseudomonas butanovorain Butane and 1-Butanol Metabolism

Vangnai¹,

Sayavedra-Soto

Arp

2002

J Bacteriol

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Pseudomonas butanovora grown on butane or 1-butanol expresses two 1-butanol dehydrogenases, a quinoprotein (BOH) and a quinohemoprotein (BDH). BOH exhibited high affinity towards 1-butanol (K m ‫؍‬ 1.7 ؎ 0.2 M). BOH also oxidized butyraldehyde and 2-butanol (K m ‫؍‬ 369 ؎ 85 M and K m ‫؍‬ 662 ؎ 98 M, respectively). The mRNA induction profiles of BOH and BDH at three different levels of 1-butanol, a nontoxic level (0.1 mM), a growth-supporting level (2 mM), and a toxic level (40 mM), were similar. When cells were grown in citrate-containing medium in the presence of different levels of 1-butanol, wild-type P. butanovora could tolerate higher levels of 1-butanol than the P. butanovora boh::tet strain and the P. butanovora bdh::kan strain. A model is proposed in which the electrons from 1-butanol oxidation follow a branched electron transport chain. BOH may be coupled to ubiquinone, with the electrons being transported to a cyanidesensitive terminal oxidase. In contrast, electrons from BDH may be transferred to a terminal oxidase that is less sensitive to cyanide. The former pathway may function primarily in energy generation, while the latter may be more important in the detoxification of 1-butanol.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Roles for the Two 1-Butanol Dehydrogenases ofPseudomonas butanovorain Butane and 1-Butanol Metabolism

Vangnai¹,

Sayavedra-Soto

Arp

2002

J Bacteriol

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“…Three different antibodies for ADHs IIB and IIG purified from P. putida HK5 and for ADH from P. aeruginosa (19) were prepared in this study. The enzymes (1 mg each) were injected separately into different rabbits with Freund's complete adjuvant.…”

Section: Methodsmentioning

confidence: 99%

“…ADHs other than methanol dehydrogenase, found in nonmethylotrophic bacteria, have been classified into three groups (types I through III) according to their molecular properties (17). The type I ADH has been purified from Pseudomonas aeruginosa (11,18,19) and Pseudomonas putida (10). Its molecular structure resembles that of methanol dehydrogenase in many respects, but it has a low affinity for methanol.…”

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confidence: 99%

Three distinct quinoprotein alcohol dehydrogenases are expressed when Pseudomonas putida is grown on different alcohols

et al. 1995

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A bacterial strain that can utilize several kinds of alcohols as its sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida HK5. Three distinct dye-linked alcohol dehydrogenases (ADHs), each of which contained the prosthetic group pyrroloquinoline quinone (PQQ), were formed in the soluble fractions of this strain grown on different alcohols. ADH I was formed most abundantly in the cells grown on ethanol and was similar to the quinoprotein ADH reported for P. putida (H. Görisch and M. Rupp, Antonie Leeuwenhoek 56:35-45, 1989) except for its isoelectric point. The other two ADHs, ADH IIB and ADH IIG, were formed separately in the cells grown on 1-butanol and 1,2-propanediol, respectively. Both of these enzymes contained heme c in addition to PQQ and functioned as quinohemoprotein dehydrogenases. Potassium ferricyanide was an available electron acceptor for ADHs IIB and IIG but not for ADH I. The molecular weights were estimated to be 69,000 for ADH IIB and 72,000 for ADH IIG, and both enzymes were shown to be monomers. Antibodies raised against each of the purified ADHs could distinguish the ADHs from one another. Immunoblot analysis showed that ADH I was detected in cells grown on each alcohol tested, but ethanol was the most effective inducer. ADH IIB was formed in the cells grown on alcohols of medium chain length and also on 1,3-butanediol. Induction of ADH IIG was restricted to 1,2-propanediol or glycerol, of which the former alcohol was more effective. These results from immunoblot analysis correlated well with the substrate specificities of the respective enzymes. Thus, three distinct quinoprotein ADHs were shown to be synthesized by a single bacterium under different growth conditions.

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“…Apparently, the binding is not so strong since substoichiometric amounts (0.82 and 0.51 Ca2+/holoenzyme and apoenzyme molecule, respectively) were detected. This is probably due to removal in Caz+-lacking buffers (required for the determination), as has also been observed for PQQ-containing ethanol dehydrogenases from Pseudomonas aeruginosa [27]. The small difference in Ca content between the apoenzyme and holoenzyme indicates that the presence of PQQ hardly contributes to Caz+ binding to the enzyme.…”

Section: Discussionmentioning

confidence: 86%

Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni

Jong

Geerlof

Stoorvogel

et al. 1995

European Journal of Biochemistry

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Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDSPAGE. Analysis of the Nterminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occumng at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could he separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o-quinonelo-quinol moiety is not essential for binding but it is for activity.Keywords. Quinoprotein ; pyrroloquinoline quinone ; heme c ; alcohol dehydrogenase ; Comarnonas testosteroni. Quinohaemoprotein ethanol dehydrogenase (QH-EDH) fromCornamonas testosteroni catalyses the NAD-independent oxidation of a broad range of primary alcohols to the corresponding aldehydes and the (subsequent) oxidation of aldehydes to carboxylic acids. The soluble enzyme can be routinely isolated as the inactive, pyrroloquinoline-quinone (PQQ)-free, haem-c-containing apoenzyme from ethanol-grown cells cultivated at ambient temperatures. In the absence of PQQ in the medium, growth on alcohol still occurs and is most probably catalyzed via a NAD-dependent alcohol dehydrogenase [ 2 ] . Nevertheless it should be stressed that under this condition the QH-EDH apoenzyme is still produced [I]. Active holoenzyme is obtained by addition of equimolar amounts of PQQ (the structure of this cofactor is depicted in Fig. 7) in the presence of Ca2+. Alternatively, holoenzyme can be isolated directly from cells grown in the presence of PQQ. Small-scale isolation and preliminary characterization of apo-QH-EDH and holo-QH-EDH have been Enzyme. Quinohaemoprotein ethanol dehydrogenase (EC 1. I .99.-).described [I]. In the meantime, it was found that medium-scale and large-scale cultivation of C. testosteroni at different conditions of pH, tem...

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Quaternary structure of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa and its reoxidation with a novel cytochrome c from this organism

Cited by 41 publications

References 29 publications

Roles for the Two 1-Butanol Dehydrogenases ofPseudomonas butanovorain Butane and 1-Butanol Metabolism

Roles for the Two 1-Butanol Dehydrogenases ofPseudomonas butanovorain Butane and 1-Butanol Metabolism

Three distinct quinoprotein alcohol dehydrogenases are expressed when Pseudomonas putida is grown on different alcohols

Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni

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