Flavine-bevattende eiwitten zijn veelvoorkomende en veelzijdige natuurlijke katalysatoren. Deze eiwitten zijn in staat een grote verscheidenheid aan reductieve en oxidatieve reacties te versnellen. Om flavine-bevattende eiwitten beter te begrijpen, en hun potentiaal beter te kunnen benutten in een industriële setting, zijn gedetailleerde studies nodig. In dit proefschrift wordt de ontdekking, structuur opheldering en biochemische karakterisering van een aantal nieuwe (deaza)flavine-bevattende eiwitten beschreven. Deze enzymen zijn gevonden door middel van genome mining, en de behaalde resultaten laten zien hoe enzymologie en structurele biologie met elkaar verweven zijn. De ontdekte eiwitten zijn voorbeelden van robuuste biokatalysatoren die gemakkelijk op recombinante wijze verkregen en vervolgens gezuiverd kunnen worden. Met behulp van de opgehelderde structuur kunnen deze eiwitten verder ontwikkeld worden tot bruikbare oxidative enzymen om op grote schaal gebruikt te worden in biotechnologische toepassingen

Nguyen, Quoc-Thai

English

University of Groningen

   University of GroningenFlavoenzymes at the crossroads of biocatalysis and structural biologyNguyen, Quoc-ThaiIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2017Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Nguyen, Q-T. (2017). Flavoenzymes at the crossroads of biocatalysis and structural biology. [Groningen]:University of Groningen.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019SummaryF lavin-dependent enzymes are ubiquitous and versatile natural catalysts, ca-pable of catalyzing a plethora of reduction and oxidation reactions. Anoverview of recent ﬁndings on new ﬂavoenzymes with unprecedented chemistriesand mechanisms is presented in Chapter 1. A few newly discovered ﬂavoenzymeshave been study targets for this PhD project as can be read in the experimentalchapters (Chapters 2–5). All these chapters provide hints on new possibilitiesto exploit ﬂavoenzymes as eﬃcient (oxidative) biocatalysts of various classes andmicrobial origins.The discovery and evaluation of new ﬂavoenzymes requires a strong inter-play between various research ﬁelds, such as genome mining, protein chemistry,enzyme kinetics, and structural biology. For large-scale biotechnological applica-tions, robust oxidative enzymes that can be produced in a recombinant manner(e.g., by using Escherichia coli) and directly applied as whole-cell biocatalysts, orat least can be puriﬁed with ease, are preferred. The target enzymes of this thesismostly satisfy such prerequisites. The ﬁrst part (Chapters 2 and 3) describes theexplorative study on two FAD-containing ﬂavoprotein oxidases with interestingsubstrate scopes: eugenol oxidase from Rhodococcus jostii RHA1 (EUGO) andan alcohol oxidase from the white-rot fungus Phanerochaete chrysosporium. Thesecond part of the thesis involves the identiﬁcation of two newly discovered bac-terial F420H2 regenerating enzymes which are interesting candidates for use asbiocatalysts.EUGO represents an attractive target for the oxidative transformation of phe-nolic compounds as the enzyme can be produced in E. coli in large quantities (160mg of pure enzyme per liter culture). In addition, EUGO is highly thermostableover a wide range of pH values (at pH from 5.0 to 8.0, 𝑇m = 65 °C) and is tol-erant towards several cosolvents (Chapter 2). Substrate proﬁling revealed that,besides the oxidation of vanillyl alcohol and hydroxylation of eugenol, EUGOcan eﬃciently catalyze the dehydrogenation of various phenolic ketones such asthe natural products gingerone and raspberry ketone. Interestingly, EUGO wasalso found to perform the kinetic resolution of a racemic secondary alcohol 4-(1-hydroxyethyl)-2-methoxyphenol with its (𝑆)-enantiomer being selectively ox-idized. Crystal structures of the enzyme in complex with various products andinhibitors were determined. Intriguingly, structural comparison of EUGO withvanillyl alcohol oxidase (VAO) from Penicillium simplicissimum revealed a re-markable 180° ﬂipped orientation of the ligands, which suggests that EUGO mayaccept phenolic substrates carrying two o-methoxy substituents. Indeed, inspiredby these structural data, we tested 4-allyl-2,6-dimethoxyphenol, which is not asubstrate of VAO, and found that this dimethoxy compound is converted byEUGO (Chapter 2).The recent identiﬁcation of a novel AOX from the white-rot basidiomycete P.chrysosporium (PcAOX), that was suggested to eﬃciently oxidize glycerol, has8586 Summaryprompted us to further investigate this enzyme (Chapter 3). We demonstratedthat PcAOX can be produced with an impressive high yield: >600 mg of pureprotein from 1 L of E. coli culture can be obtained by merely one puriﬁcationstep. To our knowledge, PcAOX represents the ﬁrst fungal methanol oxidasewithin the glucose–methanol–choline oxidase superfamily that can be heterolo-gously expressed in E. coli. Whereas PcAOX displays high catalytic eﬃcienciestowards methanol and ethanol (𝑘cat around 18 s−1), activity towards glycerol, re-grettably, was found to be very poor (𝑘obs = 0.2 s−1 at 4 M glycerol). The crystalstructure of native PcAOX was solved at 2.40 Å and indicated that the substratebinding pocket is an exceptionally small and remarkably solvent-inaccessible cav-ity. These features highly correlate with a clear preference for methanol as thebest substrate and the observed low aﬃnity and activity towards glycerol. Basedon the available structural analysis, preliminary successful rational design hasbeen achieved in our laboratories (Chapter 3).F420H2-dependent reductases have potential as biocatalysts for performing se-lective hydrogenations of a wide range of organic compounds which are otherwiserecalcitrant to reductive activation such as enones and imines in various hetero-cycles. Two enzymes have been identiﬁed in previous studies that could serve arole in reducing F420—F420:NADPH oxidoreductase (FNO) and F420-dependentglucose-6-phosphate dehydrogenase (FGD). The developed E. coli-based expres-sion systems for these two enzymes would enable the routine production of FGDand FNO as tools for regeneration or synthesis of reduced F420 (Chapters 4 and5).In Chapter 4, we report on the identiﬁcation and expression of an FGD fromR. jostti RHA1 (Rh-FGD1), the ﬁrst F420-dependent enzyme described in thisbacterium. In contrast to the poorly expressed FGD from Mycobacterium tuber-culosis (Mtb-FGD1), we produced soluble Rh-FGD1 in a rather good yield: 80mg of pure protein per liter of culture. Rh-FGD1 is a relatively fast enzyme,with a 𝑘cat of 17 s−1 for glucose-6-phosphate (G6P). Inspection of the struc-ture of Rh-FGD1 in comparison with Mtb-FGD1 suggested that the phosphatemoiety of G6P occupies a positively charged pocket formed by the side chains ofLys157, Lys258, and Arg282. The crucial role of three positively charged residuesforming the phosphate-binding pocket was veriﬁed by site-directed mutagenesis.Interestingly, when glucose was used as a substrate instead of G6P, the mutantK197N showed a slightly improved 𝑘cat/𝐾m when compared with the wild-typeenzyme. By a more thorough engineering eﬀort, it may be possible to engineerRh-FGD1 into a more economical biocatalyst, e.g., an F420-dependent glucosedehydrogenase that can use a cheaper co-substrate (glucose). The elucidatedcrystal structure can guide such enzyme redesign project.We have identiﬁed a thermostable FNO from the mesophilic bacterium Ther-mobiﬁda fusca (Chapter 5). This work provides the ﬁrst experimental evidencefor F420-dependent enzymes in this bacterium. Similar to Rh-FGD1, Tfu-FNOcould be heterologously expressed in E. coli (yielding up to 200 mg recombinantenzyme per liter of culture). As NADPH is a more expensive and less stablethan NADH, we set out to engineer FNO towards NADH as substrate by modu-lating the residues involved in molecular recognition between the two cofactors.Based on the crystal structure of FNO in complex with NADP+ (obtained at 1.8Summary 87Å resolution), residues that directly interact with the 2′-phosphate of NADP+were identiﬁed: Thr28, Ser50, Arg51, and Arg55. Our mutagenesis data resultsconﬁrmed that these residues play an important role in discriminating betweenNADP+ and NAD+. Interestingly, the T28A mutant increases the kinetic eﬃ-ciency more than three-fold as compared to the wild type enzyme when NADHwas used as substrate, with a moderately high 𝑘cat (5.0 s−1). Thus Tfu-FNOor mutants thereof represent highly promising candidates for use in biocatalyticprocedures.

Flavoenzymes at the crossroads of biocatalysis and structural biology

Flavine-bevattende eiwitten zijn veelvoorkomende en veelzijdige natuurlijke katalysatoren. Deze eiwitten zijn in staat een grote verscheidenheid aan reductieve en oxidatieve reacties te versnellen. Om flavine-bevattende eiwitten beter te begrijpen, en hun potentiaal beter te kunnen benutten in een industriële setting, zijn gedetailleerde studies nodig. In dit proefschrift wordt de ontdekking, structuur opheldering en biochemische karakterisering van een aantal nieuwe (deaza)flavine-bevattende eiwitten beschreven. Deze enzymen zijn gevonden door middel van genome mining, en de behaalde resultaten laten zien hoe enzymologie en structurele biologie met elkaar verweven zijn. De ontdekte eiwitten zijn voorbeelden van robuuste biokatalysatoren die gemakkelijk op recombinante wijze verkregen en vervolgens gezuiverd kunnen worden. Met behulp van de opgehelderde structuur kunnen deze eiwitten verder ontwikkeld worden tot bruikbare oxidative enzymen om op grote schaal gebruikt te worden in biotechnologische toepassingen.Flavin-dependent enzymes are ubiquitous and versatile natural catalysts, capable of catalyzing a plethora of reduction and oxidation reactions. To obtain a better understanding on how flavin-dependent enzymes can boost chemical reactions and to fully exploit their potential as industrial biocatalysts, more detailed studies on such flavoenzymes are required. This thesis describes the discovery, structural analyses by X-ray crystallography, and biocatalytic evaluations of several newly found (deaza)flavoenzymes. The enzymes were identified by genome mining approaches and the obtained research results nicely illustrate the intimate blending between enzymology and structural biology. The discovered enzymes represent robust oxidative biocatalysts that can be produced in a recombinant manner and purified with ease. Together with the elucidated structures, they can be developed into useful oxidative enzymes for large-scale biotechnological applications

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 University of Groningen
Flavoenzymes at the crossroads of biocatalysis and structural biology
Nguyen, Quoc-Thai
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2017
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Nguyen, Q-T. (2017). Flavoenzymes at the crossroads of biocatalysis and structural biology [Groningen]:
University of Groningen
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
Flavoenzymes at the crossroads of
biocatalysis and structural biology
The research described in this thesis was carried out at the Groningen Biotech-
nology and Biomolecular Sciences Institute of the University of Groningen, the
Netherlands and at the Scuola Universitaria Superiore IUSS Pavia, Italy. The
research project was ﬁnancially supported by an Ubbo Emmius scholarship from
the University of Groningen, The Netherlands and by the European Union (EU)
project ROBOX (grant agreement n° 635734) under EU’s Horizon 2020 Pro-
gramme Research and Innovation actions H2020-LEIT BIO-2014-1.
Cover design: Quoc-Thai Nguyen. Front, back, and bookmark: Structure of
eugenol oxidase from Rhodococcus jostii RHA1 in complex with
the inhibitor isoeugenol (PDB ID code 5FXD).
Printed by: Ridderprint BV, the Netherlands
ISBN: 978-90-367-9893-8 (Printed version)
ISBN: 978-90-367-9892-1 (Electronic version)
Copyright © 2017 by Q.-T. Nguyen
Flavoenzymes at the crossroads of
biocatalysis and structural biology
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magniﬁcus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Friday 30 June 2017 at 16:15 hours
by
Nguyễn Quốc Thái
born on 29 November 1984
in Long An, Vietnam
Supervisors
Prof. M. W. Fraaije
Prof. A. Mattevi
Assessment Committee
Prof. W. J. H. van Berkel
Prof. N. S. Scrutton
Prof. P. Iadarola
Prof. G. J. Poelarends
Contents
1 Expanding the repertoire of ﬂavoenzyme-based biocatalysis 1
1.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Covalent ﬂavination . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.1 Importance of ﬂavin covalent formation . . . . . . . . . . . 4
1.2.2 Mechanisms of covalent ﬂavin attachment. . . . . . . . . . 6
1.3 The newly found FAD-N5-oxide cofactor in EncM and DszA . . . 7
1.4 Prenylated-FMN—a novel cofactor with a fourth ring . . . . . . . 8
1.5 Deazaﬂavins—resurrection of an old, long-neglected cofactor . . . 10
1.6 Concluding remarks and future perspectives . . . . . . . . . . . . 13
1.7 Aim and outline of the thesis . . . . . . . . . . . . . . . . . . . . 13
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2 Biocatalytic properties and structural analysis of eugenol oxi-
dase from Rhodococcus jostii RHA1 19
2.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2 Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.2.1 General materials and methods . . . . . . . . . . . . . . . 21
2.2.2 General procedure for the enzymatic oxidations catalyzed
by isolated EUGO . . . . . . . . . . . . . . . . . . . . . . 21
2.2.3 Kinetic resolution of racemic 4-(hydroxy-1-ethyl)-2-methoxy-
phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.2.4 Measurement of initial rates . . . . . . . . . . . . . . . . . 22
2.2.5 Determination of kinetic parameters for 4-allyl-2,6-dimethoxy-
phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.2.6 Analysis of EUGO thermostability . . . . . . . . . . . . . 22
2.2.7 Protein crystallization, X-ray data collection, and structure
determination . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . 23
2.3.1 Substrate speciﬁcity of eugenol oxidase . . . . . . . . . . . 23
2.3.2 Enzymatic dehydrogenation of raspberry ketone . . . . . . 25
2.3.3 EUGO stability . . . . . . . . . . . . . . . . . . . . . . . . 27
2.3.4 Kinetic resolution of 4-(1-hydroxyethyl)-2-methoxyphenol . 27
2.3.5 The overall three-dimensional structure of EUGO . . . . . 28
2.3.6 The active site of EUGO . . . . . . . . . . . . . . . . . . . 29
2.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5
6 Contents
3 Expression and structural determination of an alcohol oxidase
from Phanerochaete chrysosporium 35
3.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.2 Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.2.1 Protein expression and puriﬁcation . . . . . . . . . . . . . 37
3.2.2 Biochemical characterization . . . . . . . . . . . . . . . . . 37
3.2.3 Protein crystallization, X-ray data collection, and structure
determination . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . 39
3.3.1 Expression and puriﬁcation of PcAOX . . . . . . . . . . . 39
3.3.2 pH optimum and thermostability of PcAOX . . . . . . . . 40
3.3.3 Steady-state kinetics . . . . . . . . . . . . . . . . . . . . . 41
3.3.4 AOX overall structure . . . . . . . . . . . . . . . . . . . . 42
3.3.5 The active site of PcAOX . . . . . . . . . . . . . . . . . . 43
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4 Discovery and characterization of an F420-dependent glucose-
6-phosphate dehydrogenase from Rhodococcus jostii RHA1 49
4.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.2 Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.2.1 Expression and puriﬁcation of Rh-FGD1 in E. coli . . . . . 53
4.2.2 Thermostability . . . . . . . . . . . . . . . . . . . . . . . . 54
4.2.3 Spectrophotometric assay for FGD activity . . . . . . . . . 54
4.2.4 Substrate proﬁling . . . . . . . . . . . . . . . . . . . . . . 55
4.2.5 Crystallization, X-ray data collection, and structural de-
termination of Rh-FGD1 . . . . . . . . . . . . . . . . . . . 55
4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.3.1 Expression and puriﬁcation of Rh-FGDs in E. coli . . . . . 55
4.3.2 pH optimum and thermostability . . . . . . . . . . . . . . 56
4.3.3 Substrate proﬁling and steady-state kinetics . . . . . . . . 57
4.3.4 FGD1 overall structure . . . . . . . . . . . . . . . . . . . . 59
4.3.5 F420 binding site . . . . . . . . . . . . . . . . . . . . . . . 60
4.3.6 Glucose-6-phosphate binding site . . . . . . . . . . . . . . 62
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4.4.1 Physiological role of Rh-FGDs . . . . . . . . . . . . . . . . 63
4.4.2 FGDs as biocatalyst for cofactor regeneration . . . . . . . 64
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
5 Isolation and characterization of a thermostable F420:NADPH
oxidoreductase from Thermobiﬁda fusca 69
5.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
5.2 Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . 72
5.2.1 Cloning, expression, and puriﬁcation of Tfu-FNO . . . . . 72
5.2.2 Temperature, pH optima, and thermostability of Tfu-FNO 73
5.2.3 Steady-state kinetic analyses . . . . . . . . . . . . . . . . . 74
Contents 7
5.2.4 Crystallization, X-ray data collection, and structure deter-
mination of Tfu-FNO. . . . . . . . . . . . . . . . . . . . . 74
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
5.3.1 Puriﬁcation of Tfu-FNO . . . . . . . . . . . . . . . . . . . 75
5.3.2 Eﬀects of pH and temperature on activity . . . . . . . . . 76
5.3.3 Steady-state kinetics . . . . . . . . . . . . . . . . . . . . . 77
5.3.4 The overall structure of Tfu-FNO . . . . . . . . . . . . . . 78
5.3.5 NADP+ binding site . . . . . . . . . . . . . . . . . . . . . 80
5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.4.1 The role of FNO in generating reduced F420 . . . . . . . . 81
5.4.2 Structure and NADP(H) binding site of Tfu-FNO . . . . . 82
5.4.3 Potential applications in biocatalysis . . . . . . . . . . . . 82
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Summary 85
Samenvatting 89
Riassunto 93
List of publications 97
Acknowledgements 99



Flavoenzymes at the crossroads of biocatalysis and structural biology

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