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Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
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2014 (English)In: Microbial Cell Factories, ISSN 1475-2859, Vol. 13, 112- p.Article in journal (Other academic) Published
Abstract [en]


A previously discovered mutant of Saccharomyces cerevisiae alcohol dehydrogenase 1 (Adh1p) was shown to enable a unique NADH-dependent reduction of 5-hydroxymethylfurfural (HMF), a well-known inhibitor of yeast fermentation. In the present study, site-directed mutagenesis of both native and mutated ADH1 genes was performed in order to identify the key amino acids involved in this substrate shift, resulting in Adh1p-variants with different substrate specificities.


In vitro activities of the Adh1p-variants using two furaldehydes, HMF and furfural, revealed that HMF reduction ability could be acquired after a single amino acid substitution (Y295C). The highest activity, however, was reached with the double mutation S110P Y295C. Kinetic characterization with both aldehydes and the in vivo primary substrate acetaldehyde also enabled to correlate the alterations in substrate affinity with the different amino acid substitutions.


We demonstrated the key role of Y295C mutation in HMF reduction by Adh1p. We generated and kinetically characterized a group of protein variants using two furaldehyde compounds of industrial relevance. Also, we showed that there is a threshold after which higher in vitro HMF reduction activities do not correlate any more with faster in vivo rates of HMF conversion, indicating other cell limitations in the conversion of HMF.

Place, publisher, year, edition, pages
2014. Vol. 13, 112- p.
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-229869DOI: 10.1186/s12934-014-0112-5ISI: 000340803100001OAI: oai:DiVA.org:uu-229869DiVA: diva2:738158
Available from: 2014-08-15 Created: 2014-08-15 Last updated: 2014-09-22Bibliographically approved

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Publisher's full texthttp://www.microbialcellfactories.com/content/13/1/112

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