Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants

Microbial Cell Factories

Research

Valeria Wallace-Salinas¹  Peter Rådström¹  Marie F Gorwa-Grauslund¹  Boaz Laadan¹  Åsa Janfalk Carlsson²  João RM Almeida³ 

[1] Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, SE-22100, Lund, Sweden;Department of Chemistry-BMC, Uppsala University, Box 576, SE-751 23, Uppsala, Sweden;Embrapa Agroenergy, Parque Estação Biológica, PqEB, W3 Norte (Final), 70770-901, Brasília, DF, Brazil;

关键词: Alcohol dehydrogenase;    NADH;    Site-directed mutagenesis;    Furfural;    HMF;   

DOI  :  10.1186/s12934-014-0112-5

 received in 2013-11-19, accepted in 2014-07-23,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundA 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.ResultsIn 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.ConclusionsWe 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.

【 授权许可】

Unknown   
© Laadan et al. 2014. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.