Studies on the production of (S)-(+)-solketal (2,2-dimethyl-1,3-dioxolane-4-methanol) by enantioselective oxidation of racemic solketal with Comamonas testosteroni

Geerlof, A.; Stoorvogel, J.; Jongejan, Jaap A.; Leenen, E.J.T.M.; Dooren, T. J. G. M. van; Tweel, W.J.J. van den; Duine, Johannis A.

doi:10.1007/s002530050208

Applied Microbiology and Biotechnology

1994

DOI: 10.1007/s002530050208

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Studies on the production of (S)-(+)-solketal (2,2-dimethyl-1,3-dioxolane-4-methanol) by enantioselective oxidation of racemic solketal with Comamonas testosteroni

A. Geerlof

J. Stoorvogel

Jaap A. Jongejan

et al.

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“…All strains of C. testosteroni investigated so far contain QH-EDH, those of C. acidovorans or C. terrigena do not (Geerlof et al, 1994b). QH-EDH is produced in the apo form, that is one haem c molecule/enzyme molecule is present but PQQ is not (Groen et al, 1986).…”

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Description of the Kinetic Mechanism and the Enantioselectivity of Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni in the Oxidation of Alcohols and Aldehydes

Geerlof

Rakels

Straathof

et al. 1994

European Journal of Biochemistry

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Initial rate studies were performed on the oxidation of (racemic) alcohols as well as aldehydes by quinohaemoprotein ethanol dehydrogenase, type 1, from Comamonas testosteroni with potassium ferricyanide as electron acceptor. The data could be fitted with an equation derived for a mechanism (hexa-uni ping-pong) in which alcohols are oxidized to the corresponding carboxylic acids and the intermediate aldehyde becomes released from the enzyme. However, for some substrates it was necessary to assume that they exert uncompetitive inhibition. The same model was used to fit the data of conversion processes. Reversible inactivation of the enzyme takes place during the conversion, the extent being inversely proportional to the concentration of ferricyanide present at the start. From the values of the kinetic parameters obtained for (R)-and (S)-solketal [2,2-dimethyl-4-(hydroxymethyl)-l,3-dioxolane] and their corresponding aldehydes, it appeared that the second step in (S)-solketal conversion is much faster than the first one and that opposite enantiomeric preferences exist for the alcohol and the aldehyde substrates. Since the initial rate measurements as well as the progress curve analysis gave similar kinetic parameter values and product analysis revealed intermediates in the amounts predicted, it is concluded that the kinetic and enantioselective behaviour of the enzyme is adequately described by the model presented here. Finally, the results indicate that both kinetic approaches should be used in conversions with consecutive reactions since they provide complementary information.Quinohaemoprotein alcohol dehydrogenases catalyze the oxidation of a broad range of (primary) alcohols as well as aldehydes. As reflected by their name, they contain two different types of cofactor, namely pyrroloquinoline quinone (PQQ) and haem c. This type of alcohol dehydrogenase occurs in Acetobacter (Adachi et al., 1978a), Gluconobacter (Adachi et al., 1978b), Pseudomonas (Matsushita et al., 1993), and Comamonas (Groen et al., 1986) bacteria grown on alcohols with oxygen as electron acceptor. Significant differences exist between the enzymes of this group with respect to structure and specificity. For that reason, the enzyme from Comamonas testosteroni is indicated as quinohaemo- Abbreviations. E, enantiomeric ratio; EL, midpoint potential under physiological conditions (at 1 mM, 25°C and pH 7); QH-EDH, quinohaemoprotein ethanol dehydrogenase; (R)-BrMePrOH, (R)-3-bromo-2-methyl-1 -propano1 ; PQQ, pyrroloquinoline quinone (2,7,9-tricarboxy-lH-pyrrolo[2,3-flquinoline-4,5-dione).

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confidence: 96%

Description of the Kinetic Mechanism and the Enantioselectivity of Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni in the Oxidation of Alcohols and Aldehydes

Geerlof

Rakels

Straathof

et al. 1994

European Journal of Biochemistry

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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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Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni. Purification, Characterization, and Reconstitution of the Apoenzyme with Pyrroloquinoline Quinone Analogues

Jong¹,

Geerlof²,

Stoorvogel³

et al. 1995

Eur J Biochem

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Studies on the production of (S)-(+)-solketal (2,2-dimethyl-1,3-dioxolane-4-methanol) by enantioselective oxidation of racemic solketal with Comamonas testosteroni

Cited by 4 publications

References 0 publications

Description of the Kinetic Mechanism and the Enantioselectivity of Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni in the Oxidation of Alcohols and Aldehydes

Description of the Kinetic Mechanism and the Enantioselectivity of Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni in the Oxidation of Alcohols and Aldehydes

Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni

Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni. Purification, Characterization, and Reconstitution of the Apoenzyme with Pyrroloquinoline Quinone Analogues

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