Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/4652860
- author
- Laadan, Boaz LU ; Wallace, Valeria LU ; Carlsson, Asa Janfalk ; Almeida, Joao R. M. ; Rådström, Peter LU and Gorwa-Grauslund, Marie-Francoise LU
- organization
- publishing date
- 2014
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Alcohol dehydrogenase, NADH, Site-directed mutagenesis, Furfural, HMF
- in
- Microbial Cell Factories
- volume
- 13
- article number
- 112
- publisher
- BioMed Central (BMC)
- external identifiers
-
- wos:000340803100001
- scopus:84905989815
- pmid:25287956
- 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
- 2016-04-01 13:16:23
- date last changed
- 2022-02-04 03:20:29
@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.}}, 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}}, keywords = {{Alcohol dehydrogenase; NADH; Site-directed mutagenesis; Furfural; HMF}}, language = {{eng}}, publisher = {{BioMed Central (BMC)}}, 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}}, doi = {{10.1186/s12934-014-0112-5}}, volume = {{13}}, year = {{2014}}, }