Exploration of evolutionary pathways Aspergillus niger epoxide hydrolase [Elektronische Ressource] / vorgelegt von Yosephien Gumulya

De
Exploration of evolutionary pathways Aspergillus niger epoxide hydrolase Dissertation Zur Erlangung des Grades eines Doktors der Naturwissenschaften der Abteilung Chemie der Ruhr-Universität Bochum vorgelegt von M.Sc. Yosephine Gumulya Aus Indonesien Mülheim an der Ruhr 2010    Dedicated especially for my beloved parents and sister    Referent: Prof. Dr. Manfred T. Reetz Korreferent: Prof. Dr. Martin Feigel Tag der mündlichen Prüfung:   Diese vorliegende Arbeit wurde in der Zeit von April 2007 bis Marz 2010 am Max-Planck-Institut für Kohlenforschung im Mülheim an der Ruhr unter der Leitung von Herrn Prof. Dr. M. T. Reetz durchgeführt. Herrn Prof. Dr. M. T. Reetz danke ich herzlich für die interessante und herausfordernde Themenstellung, die gewähre Freiheit bei der Durchführung der Arbeit und jeder erdenklichen Unterstützung bei fachlichen und darüber hinausgehenden Fragen. Ich danke Herrn Prof. Dr. M. Feigel für die Übernahme des Korreferats sowie Herrn Prof. Dr. M. Müller für sein Auftreten als Nebenfachprüfer. Ich danke Frau R. Lohmer, Frau E. Enk, und Frau A. Rathofer für die außergewöhnliche Unterstützung und Hilfe in jeglicher Situation. Frau R. Barabasch danke ich für ihre Hilfe bei der Literaturarbeit. Dank an alle Mitgliedern des Arbeitskreises, besonders G. Mehler, P. Wedemann und M. Hermes für die Hilfsbereitschaft.
Publié le : vendredi 1 janvier 2010
Lecture(s) : 21
Source : WWW-BRS.UB.RUHR-UNI-BOCHUM.DE/NETAHTML/HSS/DISS/GUMULYAYOSEPHINE/DISS.PDF
Nombre de pages : 133
Voir plus Voir moins




Exploration of evolutionary pathways
Aspergillus niger epoxide hydrolase




Dissertation
Zur Erlangung des Grades eines Doktors der Naturwissenschaften der
Abteilung Chemie der Ruhr-Universität Bochum



vorgelegt von M.Sc.
Yosephine Gumulya
Aus Indonesien



Mülheim an der Ruhr
2010
 
 











Dedicated especially for my beloved parents and sister
 
 



















Referent: Prof. Dr. Manfred T. Reetz
Korreferent: Prof. Dr. Martin Feigel
Tag der mündlichen Prüfung:




 
 
Diese vorliegende Arbeit wurde in der Zeit von April 2007 bis Marz 2010 am Max-Planck-Institut für
Kohlenforschung im Mülheim an der Ruhr unter der Leitung von Herrn Prof. Dr. M. T. Reetz durchgeführt.
Herrn Prof. Dr. M. T. Reetz danke ich herzlich für die interessante und herausfordernde Themenstellung,
die gewähre Freiheit bei der Durchführung der Arbeit und jeder erdenklichen Unterstützung bei fachlichen
und darüber hinausgehenden Fragen.
Ich danke Herrn Prof. Dr. M. Feigel für die Übernahme des Korreferats sowie Herrn Prof. Dr. M. Müller für
sein Auftreten als Nebenfachprüfer.
Ich danke Frau R. Lohmer, Frau E. Enk, und Frau A. Rathofer für die außergewöhnliche Unterstützung
und Hilfe in jeglicher Situation.
Frau R. Barabasch danke ich für ihre Hilfe bei der Literaturarbeit.
Dank an alle Mitgliedern des Arbeitskreises, besonders G. Mehler, P. Wedemann und M. Hermes für die
Hilfsbereitschaft.
Ein besonderer Dank für den Mitarbeitern der HPLC Abteilung. A. Deege danke ich für die schnelle
Entwicklung der Trennungsmethode, Heike und Dina für die schnelle Messung meiner unzähligen Proben
(inklusiv am Wochenende).
Special thanks goes to Despina for all her helps in everything, in science (scientific discussions, giving
Maestro tutorials, updating the literature), in being a good company during the night shifts, and mainly for
her patience in reading and revising this thesis.
I thank Ximo, for his help in science (in helping me establishing the experimental platform, scientific
discussions) but mainly for giving the mental support during my tough initial PhD year.
I thank Dirk, for his companionship especially during my difficult times, for his patience in teaching me
AKTA and mainly for the critical input in thermo kinetics.
I would like to thank Layla and Soni who have helped me a lot during my tough first PhD year, Juan Pablo
for the help in MD simulations, Woyciech for the help in size exclusion chromatography, Zheng for being
the helpful epoxide hydrolase lab-mate, and Hermi, Layla, Carlos, Lore for being good office-mates.
I would like also to thank all the group members, the former and present coworkers in the group. It was
really a great experience to meet and to get to know all of you!!!
And last but not least, my parents and my sister. Their constant support and belief in me throughout all
these tough years are the ones that make me could finish my PhD. Without them, this thesis would not
even exist.
 
 
Table of Content
1. Introduction ............................................................................................................................................ 1
1.1. Epoxide hydrolases ........................................................................................................................... 2
1.2. Epoxide hydrolase-catalyzed reactions ............................................................................................. 2
1.3. Epoxide hydrolase from Aspergillus niger (ANEH) ............................................................................ 3
1.4. Directed evolution of Aspergillus niger epoxide hydrolase (ANEH) .................................................. 4
1.5. Hydrolytic kinetic resolution ............................................................................................................... 8
1.6. Protein thermostability ....................................................................................................................... 9
1.6.1. Thermodynamic stability and kinetic stability ............................................................................. 10
1.6.2. Factors responsible for enhanced thermal stability of proteins .................................................. 10
1.6.3. Methodologies that have been developed to evolve protein for better thermostability ............. 13
1.6.4. B-FIT (B-factor iterative test) ...................................................................................................... 17
1.6.5. Enzyme screening for stability .................................................................................................... 17
1.6.6. Enzyme thermal inactivation ...................................................................................................... 18
1.7. Protein fitness landscape ................................................................................................................ 20
1.8. Epistasis and accessibility of mutational paths ............................................................................... 22
1.9. Oversampling in directed evolution .................................................................................................. 23
1.10. Scope and outline of the thesis ..................................................................................................... 24
2. Saturation Mutagenesis for improving ANEH thermostability ........................................................ 26
2.1. Introduction ...................................................................................................................................... 26
2.2. Results and Discussion .................................................................................................................. 26
2.2.1. Development and validation of screening for improved ANEH thermostable mutants ............ 26
2.2.2. Initial round of iterative saturation mutagenesis ..................................................................... 27
2.3. Experimental .................................................................................................................................. 34
3. Exploration of evolutionary pathways in improving ANEH thermostability ................................... 36
3.1. Introduction ....................................................................................................................................... 36
3.2. Results and Discussion ................................................................................................................... 37
3.2.1. The second and third round of ISM to further increase ANEH thermal stability ................. 37
3.2.2. Influence of the sequential order of library generation ........................................................ 40
 
 
 
3.2.3. The fourth, fifth, and sixth round of ISM .............................................................................. 41
3.2.4. Combining mutations versus ISM approach ....................................................................... 45
3.2.5. Temperature dependence of the specific activity ................................................................ 47
3.2.6. Stability of purified ANEH wild type and thermo mutants ................................................... 49
3.2.7. Amino acid substitutions in thermostable ANEH mutants .................................................. 53
3.3.Experimental .................................................................................................................................... 55
4. Exploration of evolutionary pathways in improving ANEH enantioselectivity .............................. 57
4.1. Introduction ..................................................................................................................................... 57
4.2. Results and Discussion ................................................................................................................... 58
4.2.1. Pathways leading to the highly enantioselective mutants ................................................... 60
4.2.2. The fitness-pathway landscape .......................................................................................... 61
4.2.3. Sequence relationships among the best mutants obtained in each evolutionary pathway 63
4.2.4. Structural difference among the best mutants obtained in each evolutionary pathway ...... 65
4.3. Experimental ....................... 67
5. Combining B-FIT and CAST ................................................................................................................ 68
5.1. Introduction ..................................................................................................................................... 68
5.2. Results and Discussion ................................................................................................................... 68
5.3. Experimental ....................... 73
6. Saturation Mutagenesis for reversing ANEH enantioselectivity ..................................................... 74
6.1. Introduction ......................... 74
6.2. Results and Discussion ................. 75
6.2.1. Choice of parent for saturation mutagenesis ....................................................................... 75
6.2.2. Choice of sites for saturation mutagenesis ......................................................................... 76
6.3. Experimental ................................................................................................................................... 80
7. Summary and Outlook ......................................................................................................................... 81
8. Supplementary material ...................................................................................................................... 85
8.1. Experimental protocol ..................................................................................................................... 85
8.2. Experimental data ........................................................................................................................... 96
9. References .......................................................................................................................................... 116Chapter 1 1
 
 
1. Introduction
Enantiopure epoxides and vicinal diols are highly valuable intermediates in fine organic chemistry, which
have been used for the production of biologically active chemicals such as Nifenalol (a β-blocking agent)
[1], Bower’s compound (an insecticide) [2], and indene oxide (for Indinavir preparation, a HIV protease
inhibitor) [3]. The chemocatalytic routes for the production of these optically active epoxides are via
Sharpless/Katzuki or Jacobsen epoxidation. The titanium-based Sharpless catalysts succeeded to
epoxidize various allylic alcohols with high optical yield, albeit limited only for alkenes that have hydroxyl
group in the allylic position [4]. The optically active Jacobsen (salen) manganese (III) complexes eliminate
this requirement [5]. However, due to the steric nature of the catalysts, less satisfying results were
obtained with trans and terminal olefins. The biocatalytic approaches for the synthesis of enantiopure
epoxides are by direct epoxidation of alkenes by monooxygenases, dehalogenation by haloperoxidases
and halohydrin epoxidases, microbial reduction of α haloketones, or kinetic resolution of epoxides by
lipases or epoxide hydrolases [6; 7].
In general, there are three routes to obtain optically pure epoxides and diols via hydrolysis of epoxides
(Figure 1) : (a) kinetic resolution, in which only one of the two enantiomers of racemic epoxides is
hydrolyzed, yielding the remaining enantiomer in an optically pure form, (b) enanticonvergent, in which
one enantiomer is attacked at α-carbon while the other is attacked at β-carbon therefore giving a
complete conversion, 100% optically pure diol, and (c) asymmetric hydrolysis of a meso compound.

Figure 1 Routes to optically pure epoxides and diols by epoxide hydrolase catalyzed
[8]conversions .
 Chapter 1 2
 
 
1.1. Epoxide hydrolases
Epoxide hydrolases (EHs, EC 3.3.2.3) catalyze the hydrolysis of an epoxide to the corresponding vicinal
diol. The reaction proceeds via an SN-2 specific opening of the epoxide leading to in relevant cases the
formation of the respectively configurated 1,2-diols. These enzymes are ubiquitous in nature, have been
isolated from a wide range of sources, such as mammals [9], plants [10], insects [11], and various
microorganisms e.g. yeasts [12], fungi [13], and bacteria [14]. Five different mammalian EHs have been
identified: microsomal epoxide hydrolase (mEH) [15] , soluble epoxide hydrolase (sEH), also referred to
as cytosolic EH [16]), cholesterol EH [17], leukotriene A4 EH [18], and hepoxilin hydrolase [19]. mEH has
been known to participate in metabolism of numerous xenobiotics compounds (detoxification) [20; 21],
whereas sEH has important role in processing of signal molecules [22]. Direct sequence comparison
between mEH and sEH displays low similarity [23; 24], however they both belong to family of α/ β
hydrolase fold enzymes [25].
There are certain advantages of using EHs: (a) they are ubiquitous in nature, (b) they are cofactor
independent, (c) they can be produced easily and in large amounts from various microorganisms, (d) they
can be partially purified and used as an enzymatic powder without significant enzyme activity loss, (e)
they can act in the present of organic solvents, and (f) they very often lead to excellent enantiomeric
excess of the un-reacted epoxide substrate and the diol product [26].
1.2. Epoxide hydrolase-catalyzed reactions
The two main types of mammalian EHs, mEH and sEH, are complementary in their substrate scope. mEH
in general catalyzes the hydrolysis of cis-substituted epoxides as well as styrene oxide, while sEH shows
high activity towards trans-substituted epoxides and various fatty acid epoxides [21; 22; 27]. mEH readily
hydrolyze monosubstituted aryl- or alkyl- epoxides, giving preferentially the R-diol and leaving the S-
epoxide behind. Epoxides with straight chain substituent are, however, inefficiently resolved whereas the
branched ones are resolved with higher selectivity [28]. 1,2-disubstituted aliphatic epoxides are also good
substrates as long as they possess the cis-configuration [29] with the nucleophilic attack occurred on the
less hindered carbon atom. The more sterically encumbered substrates such as cis-2,3-disubstituted
epoxides are hydrolyzed slower and the trans-2,3-disubstituted epoxides are usually not at all accepted
[15; 30].
Unlike mammalian EH, fermentation processes enable production of microbial EH (bacterial or fungal EH)
in sufficient quantities for biotransformations. However the majority of biotransformation reactions are
performed with whole cells or crude cell free extracts instead of purified proteins. The enantioselectivities
of enzymes from microbial sources can be correlated to the substitution pattern of various types of
substrates [31], such as monosubstituted epoxides give best enantioselectivities with red yeast
(Rhodotorula or Rhodosporidium sp, R-preference [12; 32; 33; 34]), styrene type substrates with fungal Chapter 1 3
 
 
cells (Aspergillus or Beauveria sp, [35; 36; 37; 38]) and highly substituted 2,2- and 2,3-disubstituted
epoxides with bacterial enzymes (Rhodococcus or Nocardia sp, S-preference, [39; 40]).
The first enantioconvergent hydrolysis using EHs was reported for the preparation of (R)-phenyl-1,2-
ethanediol using two fungal EHs having the opposite regio- and enantioselectivity [13]. The Aspergillus
niger epoxide hydrolase (ANEH) hydrolyzed preferentially the R-enantiomer via attack at the less
hindered carbon atom, with retention of stereochemistry and thus formation of R-diol. On the contrary, the
Beauveria bassiana EH directs attack at the more hindered benzylic position (inversion of
stereochemistry), leading to formation of R-diol. Monterde et al. [41] has recently demonstrated
enantioconvergent hydrolysis using a single EH, Solanum tuberosum EH, which attacked predominantly
at the less hindered terminal carbon atom of the R-enantiomer and attacked preferentially at the more
hindered benzylic carbon of the S-enantiomer.
1.3. Epoxide hydrolase from Aspergillus niger (ANEH)
The work described in this thesis concerns the use of a fungal EH, which was originally isolated from A.
niger strain LCP521 and successfully cloned into E.coli by Arand and coworkers [42]. It shares significant
sequence similarity with mammalian mEH; however, it lacks the common N-terminal membrane anchor.
This enzyme is highly interesting as it shows exceptionally fast substrate turnover compared with other
EHs in addition to being highly enantioselective toward some industrially important epoxides [43]. Of
course, not all substrates are turned over with high enantioselectivity, and many react slowly or not at all.
Moreover, it is the first known soluble member of the family mEHs [44].
ANEH has a molecular mass of 44 kDa and is dimeric in solution. The X-ray structure has been solved
and shows that it belongs to the α/ β hydrolase fold family of enzymes [44]. The catalytic triad residues are
Asp 192, His 374, and Asp 348. It has a relatively narrow hydrophobic substrate binding tunnel, with the
catalytic nucleophile reside at the bottom, which explains its inability to hydrolyze bulky trans-substituted
epoxides. The catalytic mechanism comprises two steps (Figure 2). In the first step, the carboxylate
oxygen of a catalytic nucleophile (Asp 192) attacks the least hindered carbon atom of the oxirane ring,
forming a covalent enzyme-substrate ester intermediate, and opening the ring. This step is thought to be
the rate determining step [42]. In the second fast step, the intermediate is hydrolyzed through the attack
of water molecule, which is activated through proton abstraction using His 374 – Asp 348 charge relay.
The two conserved tyrosine residues (Tyr 251 and Tyr 314) help the initial binding and positioning of the
substrate (by making hydrogen bond between the epoxide ring oxygen and the hydroxyl groups), activate
the substrate, and serve as proton donors in the ring opening. Chapter 1 4
 
 

[44]Figure 2 Reaction mechanism of ANEH .
The wild type (WT) ANEH hydrolyzes styrene oxides and their derivatives rapidly with moderate to high
enantioselectivity, yielding the formed R-diol and the unreacted S-epoxide both in optically pure form [45].
The hydrolysis occurs via a trans opening of the oxirane ring, favoring the attack on the less sterically
hindered carbon atom. The hydrolytic kinetic resolutions of trifluoromethyl-substituted styrene oxide
derivatives and glycidaldehyde acetal derivatives have been performed using ANEH under very mild
experimental conditions (e.g. room temperature, in water / DMSO) [46; 47]. Moreover, several other
ANEH based kinetic resolutions have been reported, for instance 2-, 3-, or 4-pyridyloxirane (a key
building blocks for the synthesis of β-adrenergic receptor agonists or anti obesity drugs [48; 49]),
chloroepoxide (a key synthons for azole antifungal agents [50]), and trans-spiroepoxide, (a key building
block of 11 heterosteroids [51]).
1.4. Directed evolution of Aspergillus niger epoxide hydrolase (ANEH)
ANEH was first subjected to directed evolution experiment by applying epPCR (error prone polymerase
chain reaction) for the purpose of increasing its activity. The improved variant was shown to have 3.4
higher of expression level and 3.3 fold increased in catalytic efficiency on 4-(p-nitrophenoxy)-1,2-
epoxybutane [52]. In another experiment, hydrolytic kinetic resolution of racemic-GPE, the
enantioselectivity of ANEH was increased from E = 4.6 to E = 10.8, with the mutant containing three
amino acid exchanges (Ala217Val, Lys332Glu, and Ala390Glu) [53].
As an alternative to epPCR, our group has developed an efficient method for rapid directed evolution of
functional enzymes, called ISM (iterative saturation mutagenesis) [54]. It relies on a Cartesian view of the
3D protein structure and iterative cycles of saturation mutagenesis at rationally chosen sites in an
enzyme. The general scheme of ISM is shown in Figure 3 in which that the four randomization sites A, B,

Soyez le premier à déposer un commentaire !

17/1000 caractères maximum.