uu.seUppsala universitets publikasjoner
Endre søk
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Characterization and Directed Evolution of an Alcohol Dehydrogenase: A Study Towards Understanding of Three Central Aspects of Substrate Selectivity
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Many different chemicals are used in the everyday life, like detergents and pharmaceuticals. However, their production has a big impact on health and environment as much of the raw materials are not renewable and the standard ways of production in many cases includes toxic and environmentally hazardous components. As the population and as the life standard increases all over the planet, the demand for different important chemicals, like pharmaceuticals, will increase. A way to handle this is to apply the concept of Green chemistry, where biocatalysis, in the form of enzymes, is a very good alternative. Enzymes do not normally function in industrial processes and needs modifications through protein engineering to cope in such conditions. To be able to efficiently improve an enzyme, there is a need to understand the mechanism and characteristics of that enzyme.

Acyloins (α-hydroxy ketones) are important building blocks in the synthesis of pharmaceuticals. In this thesis, the enzyme alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber has been in focus, as it has been shown to display a wide substrate scope, also accepting aryl-substituted alcohols. The aim has been to study the usefulness of ADH-A as a biocatalyst towards production of acyloins and its activity with aryl-substituted vicinal diols and to study substrate-, regio-, and enantioselectivity of this enzyme.

This thesis is based on four different papers where the focus of the first has been to biochemically characterize ADH-A and determine its mechanism, kinetics and its substrate-, regio-, and enantioselectivity. The second and third paper aims towards deeper understanding of some aspects of selectivity of ADH-A. Non-productive binding and its importance for enantioselectivity is studied in the second paper by evolving ADH-A towards increased activity with the least favored enantiomer through protein engineering. In the third paper, regioselectivity is in focus, where an evolved variant displaying reversed regioselectivity is studied. In the fourth and last paper ADH-A is studied towards the possibility to increase its activity towards aryl-substituted vicinal diols, with R-1-phenyl ethane-1,2-diol as the model substrate, and the possibility to link ADH-A with an epoxide hydrolase to produce acyloins from racemic epoxides.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2017. , s. 95
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1497
Emneord [en]
Alcohol dehydrogenase, ADH-A, Biocatalysis, Directed evolution, Enantioselectivity, Enzyme kinetics, Enzyme mechanisms, Protein Engineering, Regioselectivity, Substrate selectivity
HSV kategori
Forskningsprogram
Biokemi
Identifikatorer
URN: urn:nbn:se:uu:diva-318984ISBN: 978-91-554-9875-7 (tryckt)OAI: oai:DiVA.org:uu-318984DiVA, id: diva2:1085659
Disputas
2017-05-19, BMC A1:111A, Husargatan 3, Uppsala, 09:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2017-04-28 Laget: 2017-03-30 Sist oppdatert: 2017-05-05
Delarbeid
1. Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A
Åpne denne publikasjonen i ny fane eller vindu >>Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A
2014 (engelsk)Inngår i: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 99, s. 68-78Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

An increasing interest in biocatalysis and the use of stereoselective alcohol dehydrogenases in synthetic asymmetric catalysis motivates detailed studies of potentially useful enzymes such as alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber. This enzyme is capable of catalyzing enantio-, and regioselective production of phenyl-substituted α-hydroxy ketones (acyloins) which are precursors for the synthesis of a range of biologically active compounds. In this study, we have determined the enzyme activity for a selection of phenyl-substituted vicinal diols and other aryl- or alkyl-substituted alcohols and ketones. In addition, the kinetic mechanism for the oxidation of (R)- and (S)-1-phenylethanol and the reduction of acetophenone has been identified as an Iso Theorell-Chance (hit and run) mechanism with conformational changes of the enzyme-coenzyme binary complexes as rate-determining for the oxidation of (S)-1-phenylethanol and the reduction of acetophenone. The underlying cause of the 270-fold enantiopreference for the (S)-enantiomer of 1-phenylethanol has been attributed to non-productive binding of the R-enantiomer. We have also shown that it is possible to tune the direction of the redox chemistry by adjusting pH with the oxidative reaction being favored at pH values above 7.

Emneord
alcohol dehydrogenase, kinetic mechanism, pre-steady state kinetics, product inhibition
HSV kategori
Forskningsprogram
Biokemi
Identifikatorer
urn:nbn:se:uu:diva-207474 (URN)10.1016/j.molcatb.2013.10.023 (DOI)000331340500010 ()
Tilgjengelig fra: 2013-09-15 Laget: 2013-09-15 Sist oppdatert: 2017-12-06bibliografisk kontrollert
2. Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer
Åpne denne publikasjonen i ny fane eller vindu >>Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer
Vise andre…
2017 (engelsk)Inngår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, nr 22, s. 3895-3914Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (1) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (2) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate binding modes.

Emneord
alcohol dehydrogenase, biocatalysis, stereoselectivity, directed evolution, crystal structures, enzyme kinetics
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-318981 (URN)10.1111/febs.14279 (DOI)000415877100011 ()
Forskningsfinansiär
Swedish Research Council, 621-2011-6055
Tilgjengelig fra: 2017-03-30 Laget: 2017-03-30 Sist oppdatert: 2019-10-21bibliografisk kontrollert
3. Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases
Åpne denne publikasjonen i ny fane eller vindu >>Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases
Vise andre…
(engelsk)Inngår i: Artikkel i tidsskrift (Annet vitenskapelig) Submitted
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-318982 (URN)
Tilgjengelig fra: 2017-03-30 Laget: 2017-03-30 Sist oppdatert: 2018-02-18
4. Laboratory Evolution of Alcohol Dehydrogenase ADH-A for Efficient Transformation of Vicinal Diols and Acyloins. Synthesis of 2-Hydroxy Acetophenone from Racemic Styrene Oxide
Åpne denne publikasjonen i ny fane eller vindu >>Laboratory Evolution of Alcohol Dehydrogenase ADH-A for Efficient Transformation of Vicinal Diols and Acyloins. Synthesis of 2-Hydroxy Acetophenone from Racemic Styrene Oxide
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-318983 (URN)
Tilgjengelig fra: 2017-03-30 Laget: 2017-03-30 Sist oppdatert: 2017-03-30

Open Access i DiVA

fulltext(2732 kB)314 nedlastinger
Filinformasjon
Fil FULLTEXT01.pdfFilstørrelse 2732 kBChecksum SHA-512
a5721ac03a187af89290421547522be983314d18f33f1adb892db775a22f338dc9e0299f1564ac8b53740878efccd51fa59ac2dacc2b7381569af5f5e0a1f91d
Type fulltextMimetype application/pdf
Kjøp publikasjonen >>

Personposter BETA

Hamnevik, Emil

Søk i DiVA

Av forfatter/redaktør
Hamnevik, Emil
Av organisasjonen

Søk utenfor DiVA

GoogleGoogle Scholar
Totalt: 314 nedlastinger
Antall nedlastinger er summen av alle nedlastinger av alle fulltekster. Det kan for eksempel være tidligere versjoner som er ikke lenger tilgjengelige

isbn
urn-nbn

Altmetric

isbn
urn-nbn
Totalt: 1512 treff
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf