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

Laadan, Boaz LU ; Wallace, Valeria LU ; Carlsson, Asa Janfalk; Almeida, Joao R. M.; Rådström, Peter LU and Gorwa-Grauslund, Marie-Francoise LU (2014) In Microbial Cell Factories 13.
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Alcohol dehydrogenase, NADH, Site-directed mutagenesis, Furfural, HMF
in
Microbial Cell Factories
volume
13
publisher
BioMed Central
external identifiers
  • wos:000340803100001
  • scopus:84905989815
ISSN
1475-2859
DOI
10.1186/s12934-014-0112-5
language
English
LU publication?
yes
id
34543a45-f217-462c-a7f8-f08e586cefa1 (old id 4652860)
date added to LUP
2014-09-24 09:48:40
date last changed
2017-01-22 03:43:03
@article{34543a45-f217-462c-a7f8-f08e586cefa1,
  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.},
  articleno    = {112},
  author       = {Laadan, Boaz and Wallace, Valeria and Carlsson, Asa Janfalk and Almeida, Joao R. M. and Rådström, Peter and Gorwa-Grauslund, Marie-Francoise},
  issn         = {1475-2859},
  keyword      = {Alcohol dehydrogenase,NADH,Site-directed mutagenesis,Furfural,HMF},
  language     = {eng},
  publisher    = {BioMed Central},
  series       = {Microbial Cell Factories},
  title        = {Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants},
  url          = {http://dx.doi.org/10.1186/s12934-014-0112-5},
  volume       = {13},
  year         = {2014},
}