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Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC.
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2017 (Engelska)Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, nr 22, s. 3895-3914Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2017. Vol. 284, nr 22, s. 3895-3914
Nyckelord [en]
alcohol dehydrogenase, biocatalysis, stereoselectivity, directed evolution, crystal structures, enzyme kinetics
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:uu:diva-318981DOI: 10.1111/febs.14279ISI: 000415877100011OAI: oai:DiVA.org:uu-318981DiVA, id: diva2:1085656
Forskningsfinansiär
Vetenskapsrådet, 621-2011-6055Tillgänglig från: 2017-03-30 Skapad: 2017-03-30 Senast uppdaterad: 2019-10-21Bibliografiskt granskad
Ingår i avhandling
1. Characterization and Directed Evolution of an Alcohol Dehydrogenase: A Study Towards Understanding of Three Central Aspects of Substrate Selectivity
Öppna denna publikation i ny flik eller fönster >>Characterization and Directed Evolution of an Alcohol Dehydrogenase: A Study Towards Understanding of Three Central Aspects of Substrate Selectivity
2017 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
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
Nyckelord
Alcohol dehydrogenase, ADH-A, Biocatalysis, Directed evolution, Enantioselectivity, Enzyme kinetics, Enzyme mechanisms, Protein Engineering, Regioselectivity, Substrate selectivity
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
Biokemi
Identifikatorer
urn:nbn:se:uu:diva-318984 (URN)978-91-554-9875-7 (ISBN)
Disputation
2017-05-19, BMC A1:111A, Husargatan 3, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2017-04-28 Skapad: 2017-03-30 Senast uppdaterad: 2017-05-05
2. Structure Function Relationships in Pyrimidine Degrading and Biocatalytic Enzymes, and Their Implications for Cancer Therapy and Green Chemistry
Öppna denna publikation i ny flik eller fönster >>Structure Function Relationships in Pyrimidine Degrading and Biocatalytic Enzymes, and Their Implications for Cancer Therapy and Green Chemistry
2018 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

This thesis includes the work of two separate projects, studies on pyrimidine degrading enzymes and studies on in vitro evolved enzymes. The common denominator of both projects was the use of structural information to explain functional effects, observed in the studied biocatalysts.

In humans, and other eukaryotic organisms, the nucleobases uracil and thymine are catabolized by the reductive pyrimidine degradation pathway. This pathway is one of the factors that control the pyrimidine nucleotide concentrations in a cell. Furthermore, it is the main clearance route for pyrimidine analogues, often used as cancer drugs, like 5-fluorouracil and other fluoropyrimidines. Deficiencies in any of the enzymes, involved in this pathway, can lead to a wide range of neurological disorders, and possibly fatal fluoropyrimidine toxicity in cancer patients. Two out of the three involved enzymes, dihydropyrimidine dehydrogenase (DPD) and β-ureidopropionase (βUP), were studied in the first project of this thesis. This resulted in the first crystal structure of a human β-ureidopropionase variant, which could be used to explain functional characteristics of the enzyme. Structural analyses on novel DPD variants, found in patients suffering from DPD deficiency, could explain the decrease in catalytic activity of these enzyme variants. This strategy, of using structural information to predict functional effects from sequential mutations, has the potential to be used as a cheap and fast first assessment of possible deficiencies in this pathway.

Enzymes are, however, not only involved in many diseases, but also used for industrial applications. The substitution of classical organic synthetic reactions with enzyme catalyzed reactions usually has a beneficial influence on environmental pollution, as illustrated in the principles of Green Chemistry. The major drawback of the use of enzymes for these purposes is their natural selectivity towards a small group of possible substrates and products, which often do not have the desired composition or conformation for an industrial application. In order to improve an enzyme for industrial purposes, the alcohol dehydrogenase ADH-A, from Rhodococcus ruber, was subjected to a semi-rational approach of directed evolution, using iterative saturation mutagenesis (ISM), in the second project of this thesis. This resulted in different enzyme variants that showed the desired improvements in activity. Most functional improvements could be rationalized with the help of structural information and molecular dynamics simulations. This showed that artificial protein design has the potential to produce enzyme variants capable of substituting many organic synthetic reactions, and that structural information can play a key role in the designing process.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2018. s. 129
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1633
Nyckelord
β-Ureidopropionase, Dihydropyrimidine Dehydrogenase, Alcohol Dehydrogenase, Pyrimidine catabolism, 5-Fluorouracil, CASTing, X-ray Crystallography
Nationell ämneskategori
Biokemi och molekylärbiologi Strukturbiologi
Identifikatorer
urn:nbn:se:uu:diva-341639 (URN)978-91-513-0240-9 (ISBN)
Disputation
2018-04-06, B41, Husargatan 3, Uppsala, Sweden, 09:30 (Engelska)
Opponent
Handledare
Tillgänglig från: 2018-03-15 Skapad: 2018-02-18 Senast uppdaterad: 2018-04-24
3. Engineered Alcohol Dehydrogenases for Stereoselective Chemical Transformations
Öppna denna publikation i ny flik eller fönster >>Engineered Alcohol Dehydrogenases for Stereoselective Chemical Transformations
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Enzymes are biomolecules built from amino acids and catalyze the chemical transformations in a cell. Enzymes are by nature stereoselective, biodegradable, environmentally friendly, and can perform catalysis in aqueous solutions and at ambient temperatures. Due to these advantages the use of enzymes as biocatalysts for chemical transformations has emerged as an attractive “greener” alternative to conventional chemical synthesis strategies. And, if naturally occurring enzymes cannot carry out the desired chemical transformations, the functional properties of enzymes can be modified by directed evolution or protein engineering techniques. Since enzymes are genetically encoded they can be optimized for desired traits such as substrate selectivity or improved catalytic efficiency. Considering these advantages and also keeping the synthetic and industrial application in mind, we have employed alcohol dehydrogenase-A (ADH-A) from Rhodococcus ruber DSM 44541 as a study object in engineering for new catalytic properties. ADH-A tolerates water miscible organic solvents, accepts a relatively wide range of aromatic sec-alcohols/ketones as substrates and is therefore a potentially useful biocatalyst for asymmetric synthesis of organic compounds.

 

Presented research work in this thesis has been primarily focused on engineering of ADH-A and characterization of resulting enzyme variants. The engineering efforts have aimed for altered substrate scope, as well as stereo- and regioselectivities. Furthermore, possible substrate promiscuity in engineered enzyme variants has also been addressed. In short, i). Paper I: three sub sites, each consisting of two-three amino acid residues within the active-site cavity were exposed to saturation mutagenesis in step-wise manner, coupled to an in vitro selection for improved catalytic activity with the unfavored (R)-1-phenylethanol. The observed stereoselectivity could be explained partly by a shift in nonproductive substrate binding. ii). Paper II is aimed specifically towards the improving the catalytic activity with aryl-substituted vicinal diols, such as (R)-1-phenylethane-1,2-diol, and the possibility to link the ADH-A reaction with a preceding epoxide hydrolysis to produce the acyloin 2-hydroxyacetophenone from rac-styrene oxide. iii). Paper III is mainly focused towards studies of regioselectivity. Here, ADH-A and engineered variants were challenged with a substrate containing two sec-alcohol functions and the cognate di-ketone. The regioselectivity in wild type as well as in engineered variants could in part be explained by a combination of experimental and computer simulations. iv). Paper IV is focused on elucidating possible effects on substrate promiscuities in engineered variants as compared to the wild type parent enzyme, when challenged with a spectrum of potential previously untested substrates.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2019. s. 79
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1872
Nyckelord
alcohol dehydrogenase-A, biocatalysts, protein engineering, enzyme kinetics, sec-alcohols, ketones, stereoselectivity, regioselectivity, substrate selectivity and promiscuity.
Nationell ämneskategori
Biokemi och molekylärbiologi Biokatalys och enzymteknik
Forskningsämne
Biokemi
Identifikatorer
urn:nbn:se:uu:diva-395527 (URN)978-91-513-0788-6 (ISBN)
Disputation
2019-12-06, A1:107a, BMC (Biomedicinskt centrum), Husargatan 3, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2019-11-15 Skapad: 2019-10-21 Senast uppdaterad: 2019-11-18

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