Characterization of the Gene Encoding Quinohaemoprotein Ethanol Dehydrogenase of <i>Comamonas testosteroni</i>

Stoorvogel, J.; Kraayveld, Diana E.; Sluis, C.A. van; Jongejan, Jaap A.; Vries, Simon de; Duine, Johannis A.

doi:10.1111/j.1432-1033.1996.00690.x

Cited by 28 publications

(30 citation statements)

References 53 publications

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“…A Shine-Dalgarno-like ribosome binding site sequence (GGAG) is localized six bases upstream of the start codon. The amino acid sequence deduced from bdh is similar to other quinohemoproteins (Table 2) (37).…”

Section: Resultsmentioning

confidence: 87%

Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism byPseudomonas butanovora

Vangnai¹,

Arp

Sayavedra-Soto

2002

J Bacteriol

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The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence). The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors. BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced. When induced with butane, the gene for BOH was expressed earlier than the gene for BDH. Insertional disruption of bdh or boh affected adversely, but did not eliminate, butane utilization by P. butanovora. The P. butanovora mutant with both genes boh and bdh inactivated was unable to grow on butane or 1-butanol. These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol. The enzyme activity of BOH was characterized in cell extracts of the P. butanovora strain with bdh disrupted. Unlike BDH, BOH oxidized 2-butanol. The results support the involvement of two distinct NAD ؉ -independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway of P. butanovora.Pseudomonas butanovora (ATCC 43655) is an aerobic gramnegative proteobacterium closely related to the genera Thauera and Azoarcus as shown by analysis of its 16S rRNA (1). This organism has been classified in the genus Pseudomonas based on its morphology, physiology, and biochemistry (39, 40). P. butanovora was isolated from activated sludge from an oil-refining company for the purpose of generating biomass from n-alkanes (39, 40). P. butanovora can derive energy for growth from C 2 to C 9 n-alkanes and any of their oxidation products as well as from a variety of other carbon sources (39, 40). Butane-grown P. butanovora can oxidize some chlorinated hydrocarbons by cometabolism through the action of a monooxygenase (18) and thus may have applications in bioremediation schemes.The pathway for the oxidation of butane in P. butanovora proceeds primarily from butane to 1-butanol, to butyraldehyde, to butyrate (2), and then probably to the ␤-oxidation pathway of fatty acid oxidation. As in other alkane utilizers (3, 27, 36), in P. butanovora the oxidation of the alkane (butane) is initiated by the action of a monooxygenase (19). Each intermediate in the pathway accumulated in the presence of appropriate inhibitors, supported cell growth, and stimulated O 2 consumption (2). The presence of a terminal butane oxidation pathway (i.e., production of 1-butanol) in P. butanovora was indicated (2). Although butane-grown cells consumed 2-butanol, 2-butanol production (indicative of a subterminal oxidation pathway) was not demonstrated, even in the presence of appropriate inhibitors of 2-b...

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Section: Resultsmentioning

confidence: 87%

Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism byPseudomonas butanovora

Vangnai¹,

Arp

Sayavedra-Soto

2002

J Bacteriol

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“…The N-terminal domain binds PQQ and calcium in the active site and is homologous to the PQQ-binding domains of all quinoprotein ADHs. The C-terminal domain has a covalently attached heme c and is similar to some cytochrome c proteins (33). QH-ADH oxidizes primary alcohols and aldehydes in the PQQ-binding domain (31).…”

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confidence: 99%

Crystal Structure of Quinohemoprotein Alcohol Dehydrogenase from Comamonas testosteroni

Oubrie¹,

Rozeboom²,

Kalk

et al. 2002

Journal of Biological Chemistry

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Quinoprotein alcohol dehydrogenases are redox enzymes that participate in distinctive catabolic pathways that enable bacteria to grow on various alcohols as the sole source of carbon and energy. The x-ray structure of the quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni has been determined at 1.44 Å resolution. It comprises two domains. The N-terminal domain has a ␤-propeller fold and binds one pyrroloquinoline quinone cofactor and one calcium ion in the active site. A tetrahydrofuran-2-carboxylic acid molecule is present in the substrate-binding cleft. The position of this oxidation product provides valuable information on the amino acid residues involved in the reaction mechanism and their function. The C-terminal domain is an ␣-helical type I cytochrome c with His 608 and Met 647 as heme-iron ligands. This is the first reported structure of an electron transfer system between a quinoprotein alcohol dehydrogenase and cytochrome c. The shortest distance between pyrroloquinoline quinone and heme c is 12.9 Å, one of the longest physiological edge-to-edge distances yet determined between two redox centers. A highly unusual disulfide bond between two adjacent cysteines bridges the redox centers. It appears essential for electron transfer. A water channel delineates a possible pathway for proton transfer from the active site to the solvent.Bacteria have versatile metabolic pathways that enable them to adapt to different environmental conditions. Many Gram-negative bacteria, for example, can grow on compounds as different as methylamine, ethanol, and glucose as their sole source of carbon and energy (1-3). The crucial, first step in the catabolism of such compounds is often an oxidation reaction catalyzed by a class of periplasmic enzymes called quinoproteins. Quinoproteins are oxidoreductases that possess one of four different quinone compounds instead of nicotinamide or flavin cofactors (4 -7). They oxidize a wide variety of alcoholand amine-containing substrates to the corresponding aldehydes or ketones. Proteins containing the pyrroloquinoline quinone (PQQ) 1 cofactor form the best characterized and largest quinoprotein subclass (8). Two different types of PQQ-containing alcohol dehydrogenases (ADHs) have been characterized. The first type includes quinoprotein ethanol dehydrogenases (EDHs) from several Pseudomonas species (9 -11) and quinoprotein methanol dehydrogenases (MDHs) from methylotrophic bacteria (12). The second type of PQQ-dependent ADHs is the quinohemoprotein alcohol dehydrogenases (QH-ADHs). In addition to PQQ, these latter enzymes contain a covalently bound heme c. Both soluble monomeric QH-ADHs (2, 11, 13-16) and membrane-associated enzymes that consist of several subunits (17-19) have been described.No three-dimensional structures of QH-ADHs are known. In contrast, several x-ray structures have been reported of type I quinoprotein ADHs (20 -24) and a soluble quinoprotein glucose dehydrogenase (sGDH) (25). sGDH-inhibitor (26) and sGDHsubstrate (27) complexes have provided detail...

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“…eutropha strain Bo is able to synthesize PQQ; however, the sequence located up-and downstream from the tfaA gene (ϳ800 bp) did not reveal any similarity to genes encoding enzymes involved in PQQ biosynthesis as was found for Pseudomonas aeruginosa (40). Furthermore, no similarity was obtained to genes which might be involved in the further oxidation of formed aldehydes, as was shown for C. testosteroni and P. aeruginosa (40,45).…”

Section: Discussionmentioning

confidence: 69%

“…It became obvious from the structural data that the type I dehydrogenases resemble the ␣-subunit of type II enzymes (22,45).…”

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confidence: 99%

Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001

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The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparent k cat /K m and K i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat /K m value of 788 ؋ 10 4 M ؊1 s ؊1 . The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

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Characterization of the Gene Encoding Quinohaemoprotein Ethanol Dehydrogenase of Comamonas testosteroni

Cited by 28 publications

References 53 publications

Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism byPseudomonas butanovora

Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism byPseudomonas butanovora

Crystal Structure of Quinohemoprotein Alcohol Dehydrogenase from Comamonas testosteroni

Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

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