The quinohaemoprotein alcohol dehydrogenase from Gluconacetobacter xylinus : molecular and catalytic properties
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ORIGINAL PAPER
The quinohaemoprotein alcohol dehydrogenase from Gluconacetobacter xylinus: molecular and catalytic properties J. L. Cha´vez-Pacheco • M. Contreras-Zentella • J. Membrillo-Herna´ndez R. Arreguı´n-Espinoza • G. Mendoza-Herna´ndez • S. Go´mez-Manzo • J. E. Escamilla
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Received: 30 November 2009 / Revised: 27 May 2010 / Accepted: 31 May 2010 / Published online: 18 June 2010 Ó Springer-Verlag 2010
Abstract Gluconacetobacter xylinus possesses a constitutive membrane-bound oxidase system for the use of ethanol. Its alcohol dehydrogenase complex (ADH) was purified to homogeneity and characterized. It is a 119-kDa heterodimer (68 and 41 kDa subunits). The peroxidase reaction confirmed the presence of haem C in both subunits. Four cytochromes c per enzyme were determined by pyridine hemochrome spectroscopy. Redox titrations of the purified ADH revealed the presence of four haem c redox centers, with apparent mid-point potential values (Em7) of -33, ?55, ?132 and ?310 mV, respectively. The ADH complex contains one mol of pyrroloquinoline quinone as determined by HPLC. The enzyme was purified in full reduced state; oxidation was induced by potassium ferricyanide and substrate restores full reduction. Activity responses to pH were sharp, showing two distinct optimal
Communicated by Timothy Donohue. J. L. Cha´vez-Pacheco M. Contreras-Zentella S. Go´mez-Manzo J. E. Escamilla (&) Departamento de Bioquı´mica, Instituto de Fisiologı´a Celular, Universidad Nacional Auto´noma de Me´xico, Apdo. Postal 70-242, 04510 Me´xico, Me´xico e-mail: [email protected] G. Mendoza-Herna´ndez Departamento de Bioquı´mica, Facultad de Medicina, Universidad Nacional Auto´noma de Me´xico, Apdo. Postal 70-242, 04510 Me´xico, Me´xico J. Membrillo-Herna´ndez Avenida Copı´lco 162, Coyoaca´n, 04314 Me´xico, Me´xico R. Arreguı´n-Espinoza Departamento de Bioquı´mica, Instituto de Quı´mica, Universidad Nacional Auto´noma de Me´xico, Apdo. Postal 70-242, 04510 Me´xico, Me´xico
pH values (i.e. pH 5.5 and 6.5) depending on the electron acceptor used. Purified ADH oxidizes primary alcohols (C2–C6) but not methanol. Noteworthy, aliphatic aldehydes (C1–C4) were also good substrates. Myxothiazol and antymicin A were powerful inhibitors of the purified ADH complex, most likely acting at the ubiquinone acceptor site in subunit II. Keywords Alcohol dehydrogenase Acetic acid bacteria Gluconacetobacter xylinus
Introduction Bacterial alcohol dehydrogenases can be classified into three types: Class I (ADHs-I) is similar to methanol dehydrogenase of methylotrophic bacteria, these enzymes usually have a soluble quinoprotein with a a2b2 structure in the periplasm as in the case of Pseudomonas aeruginosa (Schrover et al. 1993); however, the b-subunit was absent in the purified enzyme of Pseudomonas putida (Toyama et al. 1995). The enzyme has a high optimum pH, requires ammonia or alkylamines as activators, oxidizes a wide range of alcohol substrates, including secondary alcohols and uses specific c-type cytochrome as electron acceptor (Schrover e
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