title: Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, SE-22100, Lund, Sweden. Marie-Francoise.Gorwa@tmb.lth.se.
authors.forename: Boaz
authors.initials: B
authors.lastname: Laadan
authors.affiliation: Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, SE-22100, Lund, Sweden. valeria.wallace@tmb.lth.se.
authors.forename: Valeria
authors.initials: V
authors.lastname: Wallace-Salinas
authors.affiliation: Department of Chemistry-BMC, Uppsala University, Box 576, SE-751 23, Uppsala, Sweden. asa.janfalkcarlsson@kemi.uu.se.
authors.forename: Åsa Janfalk
authors.initials: ÅJ
authors.lastname: Carlsson
authors.affiliation: Embrapa Agroenergy, Parque Estação Biológica, PqEB, W3 Norte (Final), 70770-901, Brasília, DF, Brazil. joao.almeida@embrapa.br.
authors.forename: João Rm
authors.initials: JR
authors.lastname: Almeida
authors.affiliation: Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, SE-22100, Lund, Sweden. peter.radstrom@tmb.lth.se.
authors.forename: Peter
authors.initials: P
authors.lastname: Rådström
authors.affiliation: Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, SE-22100, Lund, Sweden. Marie-Francoise.Gorwa@tmb.lth.se.
authors.forename: Marie F
authors.initials: MF
authors.lastname: Gorwa-Grauslund
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics*; D019943:Amino Acid Substitution; D005662:Furaldehyde / Q000737:chemistry*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D005662:Furaldehyde; C516801:ADH1 protein, S cerevisiae; D000426:Alcohol Dehydrogenase