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Enugala, Thilak Reddy
Publications (9 of 9) Show all publications
Al-Smadi, D., Enugala, T. R., Kessler, V., Mhasal, A. R., Kamerlin, S. C., Kihlberg, J., . . . Widersten, M. (2019). Chemical and Biochemical Approaches for the Synthesis of Substituted Dihydroxybutanones and Di-, and Tri-Hydroxypentanones. Journal of Organic Chemistry, 84(11), 6982-6991
Open this publication in new window or tab >>Chemical and Biochemical Approaches for the Synthesis of Substituted Dihydroxybutanones and Di-, and Tri-Hydroxypentanones
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2019 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 84, no 11, p. 6982-6991Article in journal (Refereed) Published
Abstract [en]

Polyhydroxylated compounds are building blocks for the synthesis of carbohydrates and other natural products. Their synthesis is mainly achieved by different synthetic versions of aldol-coupling reactions, catalyzed either by organocatalysts, enzymes or metal-organic catalysts. We have investigated the formation of 1,4-substituted 2,3-dihydroxybutan-1-one derivatives from para- and meta-substituted phenylacetaldehydes by three distinctly different strategies. The first involved a direct aldol reaction with hydroxyacetone, dihydroxyacetone or 2-hydroxyacetophenone, catalyzed by the cinchona derivative cinchonine. The second was reductive cross-coupling with methyl or phenyl glyoxal promoted by SmI2 resulting in either 5-substituted 3,4-dihydroxypentan-2-ones or 1,4 bis-phenyl substituted butanones, respectively. Finally, in the third case, aldolase catalysis was employed for synthesis of the corresponding 1,3,4-trihydroxylated pentan-2-one derivatives. The organocatalytic route with cinchonine generated distereomerically enriched syn products (de = 60−99 %), with moderate enantiomeric excesses (ee = 43−56%), but did not produce aldols with either hydroxyacetone or dihydroxyacetone as donor ketones. The SmI2-promoted reductive cross-coupling generated product mixtures with diastereomeric and enantiomeric ratios close to unity. This route allowed for the production of both 1-methyl- and 1-phenylsubstituted 2,3-dihydroxybutanones, at yields between 40−60%. Finally, the biocatalytic approach resulted in enantiopure syn (3R,4S) 1,3,4-trihydroxypentan-2-ones.

National Category
Organic Chemistry
Research subject
Chemistry with specialization in Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-383068 (URN)10.1021/acs.joc.9b00742 (DOI)000471212000043 ()
Available from: 2019-05-08 Created: 2019-05-08 Last updated: 2019-07-05Bibliographically approved
Enugala, T. R. (2019). Engineered Alcohol Dehydrogenases for Stereoselective Chemical Transformations. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Engineered Alcohol Dehydrogenases for Stereoselective Chemical Transformations
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 79
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1872
Keywords
alcohol dehydrogenase-A, biocatalysts, protein engineering, enzyme kinetics, sec-alcohols, ketones, stereoselectivity, regioselectivity, substrate selectivity and promiscuity.
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-395527 (URN)978-91-513-0788-6 (ISBN)
Public defence
2019-12-06, A1:107a, BMC (Biomedicinskt centrum), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-11-15 Created: 2019-10-21 Last updated: 2019-11-18
Hamnevik, E., Maurer, D., Enugala, T. R., Chu, T., Löfgren, R., Dobritzsch, D. & Widersten, M. (2018). Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-Diol and Its Corresponding Acyloin. Biochemistry, 57, 1059-1062
Open this publication in new window or tab >>Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-Diol and Its Corresponding Acyloin
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2018 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, p. 1059-1062Article in journal (Refereed) Published
Abstract [en]

Laboratory evolution of alcohol dehydrogenase produced enzyme variants with improved turnover numbers with a vicinal 1,2-diol and its corresponding hydroxyketone. Crystal structure and transient kinetics analysis aids in rationalizing the new functions of these variants.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-340574 (URN)10.1021/acs.biochem.8b00055 (DOI)000426013300003 ()29384657 (PubMedID)
Funder
Stiftelsen Olle Engkvist Byggmästare, 183-358
Available from: 2018-01-31 Created: 2018-01-31 Last updated: 2019-10-21Bibliographically approved
Ma, H., Engel, S., Enugala, T. R., Al-Smadi, D. & Widersten, M. (2018). New Stereoselective Biocatalysts for Carboligation and Retro-Aldol Cleavage Reactions Derived from D-Fructose 6-Phosphate Aldolase. Biochemistry, 57(40), 5877-5885
Open this publication in new window or tab >>New Stereoselective Biocatalysts for Carboligation and Retro-Aldol Cleavage Reactions Derived from D-Fructose 6-Phosphate Aldolase
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2018 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, no 40, p. 5877-5885Article in journal (Refereed) Published
Abstract [en]

D-Fructose 6-phosphate aldolase (FSA) catalyzes the asymmetric cross-aldol addition of phenylacetaldehyde and hydroxyacetone. We conducted structure guided saturation mutagenesis of noncatalytic active-site residues to produce new FSA variants, with the goal of widening the substrate scope of the wild-type enzyme toward a range of para- and meta-substituted arylated aldehydes. After a single generation of mutagenesis and selection, enzymes with diverse substrate selectivity scopes were identified. The kinetic parameters and stereoselectivities for a subset of enzyme/substrate combinations were determined for the reactions in both the aldol addition and cleavage reaction directions. The achieved collection of new aldolase enzymes provides new tools for controlled asymmetric synthesis of substituted aldols.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-360283 (URN)10.1021/acs.biochem.8b00814 (DOI)000447238100012 ()30204427 (PubMedID)
Funder
Stiftelsen Olle Engkvist ByggmästareCarl Tryggers foundation
Available from: 2018-09-11 Created: 2018-09-11 Last updated: 2018-12-10Bibliographically approved
Maurer, D., Enugala, T. R., Hamnevik, E., Bauer, P., Lüking, M., Petrovic, D., . . . Widersten, M. (2018). Stereo- and Regioselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases. ACS Catalysis, 8(8), 7526-7538
Open this publication in new window or tab >>Stereo- and Regioselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases
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2018 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 8, no 8, p. 7526-7538Article in journal (Refereed) Published
Abstract [en]

ADH-A from Rhodococcus ruber DSM 44541 catalyzes the oxidation of (S)-1-phenylethanol 3000-fold more efficiently as compared with the 2-hydroxylated derivative (R)-phenylethane-1,2-diol. The enzyme is also highly selective for sec-alcohols with comparably low activities with the corresponding primary alcohols. When challenged with a substrate containing two secondary alcohols, such as 1-phenylpropane-(1R,2S)-diol, ADH-A favors the oxidation of the benzylic carbon of this alcohol. The catalytic efficiency, however, is modest in comparison to the activity with (S)-1-phenylethanol. To investigate the structural requirements for improved oxidation of vicinal diols, we conducted iterative saturation mutagenesis combined with activity screening. A first-generation variant, B1 (Y54G, L119Y) displays a 2-fold higher kcat value with 1-phenylpropane-(1R,2S)-diol and a shift in the cooperative behavior in alcohol binding, from negative in the wild type, to positive in B1, suggesting a shift from a less active enzyme form (T) in the wild type to a more active form (R) in the B1 variant. Also, the regiopreference changed to favor oxidation of C-2. A second-generation variant, B1F4 (F43T, Y54G, L119Y, F282W), shows further improvement in the turnover and regioselectivity in oxidation of 1-phenylpropane-(1R,2S)-diol. The crystal structures of the B1 and B1F4 variants describe the structural alterations to the active site, the most significant of which is a repositioning of a Tyr side-chain located distal to the coenzyme and the catalytic zinc ion. The links between the changes in structures and stereoselectivities are rationalized by molecular dynamics simulations of substrate binding at the respective active sites.

Keywords: alcohol dehydrogenase; alcohol oxidation; biocatalysis; crystal structure; directed evolution; enzyme engineering; molecular dynamics simulations; stereoselectivity

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-355854 (URN)10.1021/acscatal.8b01762 (DOI)000441112400074 ()
Funder
Stiftelsen Olle Engkvist ByggmästareSwedish Research Council, 2015-04928Knut and Alice Wallenberg Foundation, KAW 2013.0124EU, FP7, Seventh Framework Programme, 283570Swedish National Infrastructure for Computing (SNIC), 2015/16-12Swedish National Infrastructure for Computing (SNIC), 2016/34-27
Available from: 2018-07-05 Created: 2018-07-05 Last updated: 2019-10-21Bibliographically approved
Al-Smadi, D., Enugala, T. R., Norberg, T., Kihlberg, J. & Widersten, M. (2018). Synthesis of substrates for aldolase-catalyzed reactions: A comparison of methods for the synthesis of substituted phenylacetaldehydes. Synlett: Accounts and Rapid Communications in Synthetic Organic Chemistry, 29(9), 1187-1190
Open this publication in new window or tab >>Synthesis of substrates for aldolase-catalyzed reactions: A comparison of methods for the synthesis of substituted phenylacetaldehydes
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2018 (English)In: Synlett: Accounts and Rapid Communications in Synthetic Organic Chemistry, ISSN 0936-5214, E-ISSN 1437-2096, Vol. 29, no 9, p. 1187-1190Article in journal (Refereed) Published
Abstract [en]

Methods for the synthesis of phenylacetaldehydes (oxidation, one-carbon chain extension) were compared by using the synthesis of 4-methoxyphenylacetaldehyde as a model example. Oxidations of 4-methoxyphenylethanol with activated DMSO (Swern oxidation) or manganese dioxide gave unsatisfactory results; whereas oxidation with 2-iodoxybenzoic add (IBX) produced 4-methoxyphenylacetaldehyde in reasonable (75%) yield. However, Wittig-type one-carbon chain extension with methoxymethylene-triphenylphosphine followed by hydrolysis gave an excellent (81% overall) yield of 4-methoxyphenylacetaldehyde from 4-methoxybenzaldehyde (a cheap starting material). This approach was subsequently used to synthesise a set of 10 substituted phenylacetaldehydes in good to excellent yields.

National Category
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-342939 (URN)10.1055/s-0036-1591963 (DOI)000432738600011 ()
Funder
Stiftelsen Olle Engkvist Byggmästare
Available from: 2018-02-23 Created: 2018-02-23 Last updated: 2018-10-11Bibliographically approved
Hamnevik, E., Enugala, T. R., Maurer, D., Ntuku, S., Oliveira, A., Dobritzsch, D. & Widersten, M. (2017). Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer. The FEBS Journal, 284(22), 3895-3914
Open this publication in new window or tab >>Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer
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2017 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 22, p. 3895-3914Article in journal (Refereed) 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.

Keywords
alcohol dehydrogenase, biocatalysis, stereoselectivity, directed evolution, crystal structures, enzyme kinetics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-318981 (URN)10.1111/febs.14279 (DOI)000415877100011 ()
Funder
Swedish Research Council, 621-2011-6055
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2019-10-21Bibliographically approved
Ma, H., Enugala, T. R. & Widersten, M. (2015). A micro-plate format assay for real-time screening for new aldolases accepting aryl-substituted acceptor substrates. ChemBioChem (Print), 16(18), 2595-2598
Open this publication in new window or tab >>A micro-plate format assay for real-time screening for new aldolases accepting aryl-substituted acceptor substrates
2015 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 18, p. 2595-2598Article in journal (Refereed) Published
Abstract [en]

Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose-6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been presented as a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with these type of bulky acceptor substrates is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here, we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an in vitro evolved aldehyde reductase that reduces the aldehyde product formed after FSA-afforded cleavage of the aldol. The assay is suitable both for steady state enzyme kinetics and real-time activity screening in a 96-well format.

Keywords
aldolase, dehydrogenase, directed evolution, retro-aldol, screening
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-264195 (URN)10.1002/cbic.201500466 (DOI)000367720300008 ()26449620 (PubMedID)
Funder
Swedish Research Council, 621-2011-6055
Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2017-12-01Bibliographically approved
Maurer, D., Enugala, T. R., Hamnevik, E., Bauer, P., Lüking, M., Hillier, H., . . . Widersten, M. Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases.
Open this publication in new window or tab >>Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases
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(English)In: Article in journal (Other academic) Submitted
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-318982 (URN)
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2018-02-18
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