Exploring Conjugate Addition Activity in Pseudozyma

antarctica Lipase B

Maria Svedendahl

Licentiate Thesis

Division of Biochemistry School of Biotechnology

Royal Institute of Technology Stockholm, Sweden 2009

© Maria Svedendahl 2009

Royal Institute of Technology

School of Biotechnology

AlbaNova University Center

SE-106 91 Stockholm

Sweden

ISBN 978-91-7415-435-1 TRITA-BIO-Report 2009:20 ISSN 1654-2312

Printed in Stockholm, September 2009

Universitetsservice US-AB

Drottning Kristinas väg 53 B

100 44 Stockholm

ABSTRACT

Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis-/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).

SAMMANFATTNING

Multifunktionella enzymer har alternativa funktioner eller aktiviteter, kända som “extraknäckande” eller “promiskuösa”, vilka ofta är dolda bakom ett enzyms naturliga aktivitet och är därför endast synliga under särskilda betingelser. I denna avhandling undersöks Pseudozyma (tidigare Candida) antarctica lipas B för en promiskuös konjugatadditionsaktivitet. Pseudozyma antarctica lipas B är ett lipas som används industriellt för hydrolys- och transacyleringsreaktioner. Detta enzym innehåller en katalytisk triad, Ser105-His224-Asp187, där en nukleofil attack från Ser105 på ett karboxylsyra/estersubstrat bildar acylenzym. För att erhålla konjugatadditionsaktivitet i Pseudozyma antarctica lipas B antogs att den nukleofila Ser105 behövde bytas ut för att undvika konkurrerande hemiacetalbildning. Experiment visade på konjugatadditionsaktivitet i både vildtypsenzymet och Ser105Ala varianten. Enzymkatalyserade konjugatadditioner utfördes genom att addera en sec-amin, tioler eller 1,3-dikarbonylföreningar till olika α,β-omättade karbonylföreningar i vatten eller organiskt lösningsmedel. De enzymkatalyserade reaktionerna följde Michaelis-Menten kinetik och den ursprungliga ping pong bi bi reaktionmekanismen för hydrolys-/transacyleringsaktivitet i Pseudozyma antarctica lipas B omdirigerades till en ordnad bi uni mekanism för dess nya konjugatadditionsaktivitet (Artikel I, II, III). Lipasets hydrolytiska aktivitet undertrycktes mer än 1000 gånger genom att ersätta den nukleofila Ser105 med Ala (Artikel III).

LIST OF PAPERS

This thesis is based on the following papers which are referred to by their roman numerals.

I Peter Carlqvist*, Maria Svedendahl*, Cecilia Branneby, Karl Hult, Tore Brinck and Per Berglund.

Exploring the active-site of a rationally redesigned lipase for catalysis of Michael-type additions.

ChemBioChem 2005 6: 331-336.

II Maria Svedendahl, Karl Hult and Per Berglund.

Fast carbon-carbon bond formation by a promiscuous lipase.

J. Am. Chem. Soc. 2005 127: 17988-17989.

III Maria Svedendahl, Biljana Jovanović, Linda Fransson and Per Berglund.

Suppressed native hydrolytic activity of a lipase to reveal promiscuous Michael addition activity in water.

ChemCatChem 2009 1: DOI: 10.1002/cctc.200900041.

* Equal contribution

TABLE OF CONTENTS

1. INTRODUCTION 1

1.1 Multifunctional enzymes 2 1.1.1 Moonlighting enzymes 2 1.1.2 Promiscuous enzymes 3

1.2 Pseudozyma antarctica lipase B 5 1.2.1 General 5 1.2.2 Structure 6 1.2.3 Reaction mechanism 6 1.2.4 Pseudozyma antarctica lipase B promiscuity 8

1.3 Conjugate Additions 9 1.3.1 General 9 1.3.2 Chemical catalysts 10 1.3.3 Enzyme catalysts 10

2. PRESENT INVESTIGATION 12

2.1 Paper I: Exploring the active-site of a rationally redesigned lipase for catalysis of Michael-type additions 13

2.2 Paper II: Fast carbon-carbon bond formation by a promiscuous lipase 20

2.3 Paper III: Suppressed native hydrolytic activity of a lipase to reveal promiscuous Michael addition activity in water 25

3. OUTLOOK AND FUTURE ASPECTS 35

4. ACKNOWLEDGEMENTS 36

5. REFERENCES 37

1. INTRODUCTION

Proteins are complex molecules constructed by amino acids connected by peptide bonds to form chains of repeating amide planes. These planes can twist to form different secondary structures, commonly α-helixes or β-sheets, before an overall three-dimensional protein structure is brought together.

The specific order of amino acids in a protein gives each protein a specific fold with unique properties. All proteins are dependent on a correct three-dimensional structure for proper function. Amino acids can be replaced by other amino acids by mutations. This could change the protein structure, affect the protein activity or remain unnoticed, which depends on the nature of the protein and the position of the altered amino acid.

Some proteins are specialized to catalyze (i.e. increase the rate of) chemical reactions. These proteins are called enzymes. Enzyme catalysis takes place within an active-site, where substrates bind and are turned over to products by catalytically active amino acid residues. The shape and type of amino acids within the active-site aid the enzyme to discriminate between different substrates or regions of a substrate, which is a requirement for cell metabolism and life.

Living organisms contain networks of biochemical cascade reactions mediated and regulated by enzymes. To control these systems, enzymes must work independently and simultaneously, while being both selective and specific for its reaction and substrate. This is achieved by the three-dimensional enzyme structure that gives each type of enzyme its specificity to select the right substrate among many. The shape of the active-site holds the substrate in a precise orientation to perform the correct chemical transformation. Enzyme selectivity is generally known as the possibility of an enzyme to distinguish between different substrates as well as different chemical functionalities on a substrate. Enzyme specificity is defined by the kinetic constant ( )Mcat Kk .

1

1.1 Multifunctional enzymes

Multifunctional enzymes are an ancient phenomenon, but have during the last decade been highlighted within the biocatalysis society. Today, many enzymes are recognized as “moonlighting” or “promiscuous”. Moonlighting enzymes can occasionally have structural or regulatory functions due to environmental changes, while promiscuous enzymes show alternative catalytic abilities. These multifunctional behaviours are often hidden behind a native transformation and only visible under special conditions and are difficult to discover if not searched for (Jeffery 1999, 2003, 2009; Copley et al. 2003; Nobeli et al. 2009).

1.1.1 Moonlighting enzymes

Moonlighting enzymes can show different function depending on changes in the molecular environment, such as cellular location, cell type, oligomeric state or cellular concentration of ligand, cofactor or product. These enzymes either use parts of the enzyme native catalytic machinery or other structural features for its moonlighting functions. Phosphoglucose isomerase and cytochrome c are examples of moonlighting enzymes (Jeffery 1999, 2003, 2009; Copley et al. 2003).

Phosphoglucose isomerase is a moonlighting enzyme having one enzymatic activity inside the cell and other functions outside the cell. In the cell cytoplasm, the enzyme catalyzes reversible interconversion of glucose 6-phosphate and fructose 6-phosphate in the glycolysis and the gluconeogenesis pathways as well as membrane protein glycosylation. Outside the cell, phosphoglucose isomerase moonlights as cytokine, growth factor and inhibitor (Jeffery et al. 2000).

The native function of cytochrome c is to shuttle electrons between complex III and complex IV in the mitochondrial electron transport chain. The moonlighting function of cytochrome c is to stimulate apoptosis (a controlled form of cell death that kills cells in response to infection or DNA damage). Cells exposed to damage or to stimulation of the death domain receptor, release cytochrome c from the mitochondrial intermembrane space to the cytosol, where apoptosis

2

is stimulated by an interaction or binding of an apoptosis protease activation factor-1 (Copley et al. 2003).

1.1.2 Promiscuous enzymes

Promiscuous enzymes catalyze reactions that differ from their biological activities. There are different types of enzyme promiscuity; (I) enzyme condition promiscuity, (II) enzyme substrate promiscuity, (III) enzyme catalytic promiscuity and (IV) enzyme alternate-site promiscuity (Hult and Berglund, 2007; Taglieber et al. 2007). Enzyme catalytic promiscuity is of special interest, since it can be a part of in vivo and in vitro evolution of new enzymes capable of catalyzing reactions of special interests, for example, detoxifying antibiotics, pesticides and other pollutants cased by human activities found in the environment (Copley et al. 2003). It is not clear why modern enzymes show catalytic promiscuity. One reason could be that it is not possible for Nature to create an enzyme that can only catalyze one specific transformation. The promiscuous activity is normally not affecting an organism if its promiscuous reaction does not affect the rate of the native activity or if the substrate for the promiscuous reaction is non-natural. Then, there is no selective pressure to remove the promiscuous reaction (O´Brian and Herschlag et al. 1999; Copley et al. 2003; James and Tawfik, 2003; Bornscheuer and Kazlauskas, 2004; Kazlauskas et al. 2005; Khersonsky et al. 2005; DePristo 2007; Hult and Berglund, 2007; Toscano et al. 2007; Nobeli et al. 2009; Toscano et al. 2007).

Enzyme condition promiscuity is shown by enzymes with activity in unnatural reaction conditions, such as organic solvent, extreme temperature or altered pH. Enzyme condition promiscuity is shown by lipases, since they are active and stable in organic solvent and at different temperatures (Uppenberg et al. 1994) to be used in a number of industrial lipase applications (Schmid et al. 2001; Liese et al. 2006). Pseudozyma antarctica lipase B can even perform transacylation in solid-gas bioreactors, without any liquid phase (Lamare et al. 2004).

Enzyme substrate promiscuity is shown by enzymes with broad substrate acceptance. This is a common behaviour of extracellular hydrolases. Lipases accept a wide range of acids and esters. Another enzyme, glucose dehydrogenase (an oxidoreductase) is promiscuous with regard to substrates. This enzyme normally catalyzes oxidation of

3

glucose to gluconate, but also accepts substrates like galactose, xylose and L-arabinose (Milburn et al. 2006).

Enzyme catalytic promiscuity is shown by enzymes catalyzing a reaction that differ from the chemical transformation the enzyme was evolved for. Transformations are altered if the bonds that are broken or cleaved differ from the native transformation, as well as the transition state structure. This enzyme property can be further divided into accidental (reaction catalyzed by a wild-type enzyme) and induced (new activity established by mutations) enzyme catalytic promiscuity (Hult and Berglund, 2007). Examples of accidental catalytic promiscuity are; aldol additions catalyzed by Pseudozyma antarctica lipase B (Branneby et al. 2003 and 2004), conjugate additions catalyzed by different serine hydrolases (Kitazume et al. 1986, 1987, 1988; Torre et al. 2004, 2005; Cai et al. 2004, 2006; Paper I, II, III; Xu et al. 2005, 2007a, 2007b; Qian et al. 2007; Strohmeier et al. 2009), direct epoxidations catalyzed by Pseudozyma antarctica lipase B (Svedendahl et al. 2008) and Markovnikov additions performed by different hydrolases (Lou et al. 2008, 2009). Induced enzyme catalytic promiscuity has for instance been shown by Seebeck and Hilvert, who reduced the native activity of an alanine racemase to favour a retro-aldol activity not seen in the wild-type enzyme by a single-point mutation (Seebeck and Hilvert, 2003).

A promiscuous enzyme-catalyzed reaction that does not involve any of the native catalytic amino acid residues nor take place in the normal active-site was shown by Taglieber et al. (2007). In this study, one subunit of the enzyme tHisF/tHisH from Thermotoga maritima displayed promiscuous hydrolytic activity.

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1.2 Pseudozyma antarctica lipase B 1.2.1 General

Lipases belong to the enzyme class of hydrolases and normally hydrolyze triglycerides into free fatty acids and glycerol at an oil-water interface. The hydrolysis can be replaced by transacylation, if the oil-water medium is replaced by a dry organic solvent and if another nucleophile than water (alcohol, sugar, thiol or amine) is available (Martinelle and Hult, 1995). Most lipases show interfacial activation (Sarda et al. 1957); the opening of a lid covering the active-site at an oil-water interface (Tilbeurgh et al. 1993).

Pseudozyma, formerly Candida (Boekhout, 1995), antarctica lipase B (PalB) is suggested to be a crossbreed between an esterase and a lipase because of several reasons; the enzyme displays no significant homology to other known lipase sequences (Uppenberg et al. 1994), low activity against large triglyceride substrates and no interfacial activation (Martinelle et al. 1995)

Pseudozyma antarctica lipase B shows broad substrate acceptance, high stability and activity in different types of media and at different temperatures. These properties, in combination with its high enantio-, chemo- and regioselectivity are important qualities for an industrial biocatalyst (Uppenberg et al. 1994). Today, Pseudozyma antarctica lipase B are used in a number of industrial hydrolysis and transacylation applications (Liese et al. 2006).

5

Figure 1. The overall structure of Pseudozyma antarctica lipase B derived from the 1TCA crystal structure (Uppenberg et al. 1994) from the Protein Data Bank (www.rcsb.org) displayed in blue. The amino acids constructing the catalytic triad (Ser105-His224-Asp187) and oxyanion hole (Thr40 and Gln106) are depicted in light yellow sticks. Molecular graphics created with PyMOL (www.pymol.org).

1.2.2 Structure

The structure of Pseudozyma antarctica lipase B was solved in 1994 by Uppenberg. Pseudozyma antarctica lipase B has α/β-hydrolase fold (Ollis et al. 1992) with an approximate dimension of 30×40×50 Å. A solvent exposed hydrophobic channel leads down to the enzyme active-site, where a catalytic triad consisting of Ser105-His224-Asp187 and an oxyanion hole built up by Thr40 and Gln106, are found. An aromatic amino acid residue, Trp104, forms the bottom of the catalytic cavity (Uppenberg et al. 1994). The overall structure of Pseudozyma antarctica lipase B with the catalytically active amino acids depicted are shown in Figure 1.

6

NNH

OH

O

O

NH

O

O

HNO H

His224

Gln106

Thr40

R2OHOHR1

O

Asp187

H2O

Ser105

NNH

OH

O

O

NH

O

O

HNO H

His224

Gln106

Thr40

O

O

Ser105 HN

NH

O

O

NH

O

O

HNO H

His224

Gln106

Thr40

Ser105

R2O

O

Asp187

O

O

O

OR2

R1

NNH

HO

NH

O

O

HNO H

His224

Gln106

Thr40

Asp187

Ser105

O

O

R1

R1R1

Asp187

Scheme 1. The ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis of an ester to carboxylic acid. The nucleophilic Ser105 forms an acyl enzyme and an acid is formed after an attack of water (Hult, 1992). A transacylation reaction can take place if the water molecule is replaced by an alcohol (Martinelle and Hult, 1995).

1.2.3 Reaction mechanism

The reaction mechanism for hydrolysis of an ester by Pseudozyma antarctica lipase B is shown in Scheme 1. Pseudozyma antarctica lipase B displays a ping pong bi bi type of kinetics that includes the formation of two tetrahedral intermediates and an acyl enzyme (Hult, 1992). The ester substrate enters the active-site and becomes coordinated to an oxyanion hole. The oxyanion hole is formed by two amino acids, Thr40 and Gln106, which provides three possible hydrogen bonds to coordinate the carbonyl oxygen of the substrate. The nucleophilic Ser105 attacks the carbonyl carbon of the substrate to form a tetrahedral intermediate that is stabilized by the oxyanion hole. As an

7

alcohol is released from the intermediate an acyl enzyme is formed. Water attacks the acyl enzyme to form a second tetrahedral intermediate. The product is released and the enzyme is ready to act on a new substrate. A transacylation reaction can occur if water is excluded from the system and replaced by another nucleophile, such as an alcohol (Martinelle and Hult, 1995).

1.2.4 Pseudozyma antarctica lipase B promiscuity Pseudozyma antarctica lipase B is nowadays known as a highly

promiscuous enzyme. As described above in section 1.1, Pseudozyma antarctica lipase B displays condition-, substrate- and catalytic promiscuity, since it is active in different solvents, has a broad substrate acceptance and can catalyze various alternative reactions that proceed in the enzyme active-site via transition states that differ from the native transformation.

8

1.3 Conjugate Additions 1.3.1 General

Conjugate additions are 1,4-additions of a nucleophile to an unsaturated compound in conjunction with an activating group (Perlmutter, 1992). An example of a conjugate addition using a carbonyl function as activating group is shown in Scheme 2.

R

O

R+

X R R R

X RO

R R

X ROH

Scheme 2. A conjugate addition of any nucleophile (donor) to an activated α,β-unsaturated carbonyl compound (acceptor).

1,4-Addition originally refers to a metal ion added to position number 1 and a nucleophilic addition on position number 4 (Figure 2a). Alternatively, to indicate the positions of the unsaturated system the Greek letters, α and β, are used (Figure 2b) (Perlmutter, 1992).

R

O

R

1

2 3 4R

O

Rα β

a b

Figure 2. a) Originally, the conjugate addition or 1,4-addition was defined for a metal ion added to position number 1 and a nucleophilic addition on position number 4. b) The carbon atoms of the unsaturated system of the acceptor molecule are often indicated by the Greek letters, α and β.

Michael published in 1887 and 1894 the first two papers dealing with conjugate additions using carbon nucleophiles, i.e. malonate salts to α,β-unsaturated esters (Michael, 1887 and 1894). Since then, a lot of different nucleophiles and acceptor molecules have been discovered for the conjugate addition. Today, conjugate additions are standard reactions in organic chemistry textbooks.

In the literature, different terms are used for conjugate additions, such as Michael-type addition, Michael addition, Michael reaction and more. These names refers to conjugate additions or 1,4-additions of any nucleophile to an unsaturated system conjugated with an activating group. The term Michael-type addition refers to a conjugate addition

9

where a nucleophile other then a carbon anion is used. Michael addition, after Arthur Michael, is the name for a conjugate addition of a carbanion to an unsaturated system conjugated with an activating group. The term, Michael reaction, should be used for a conjugate addition using a stabilized carbon anion to an unsaturated system conjugated with a carbonyl group (Perlmutter, 1992).

1.3.2 Chemical catalysts

Conjugate additions are traditionally catalyzed by bases, such as alkali metal oxides or hydroxides, which can cause unwanted side reactions. Therefore, milder reaction conditions are applied. During the last decades, researchers have developed efficient organometallic catalysts. High enantioselectivities can be achieved by using organometallic reagents in combination with a chiral ligand. This approach gives high enantioselectivity, but the catalysis requires stoichiometric amounts of transition metal salt and the chiral ligand is usually not recovered. The catalyst is highly substrate specific and can only be used for one or a few types of acceptor molecules (Christoffers 1998; Krause and Hoffmann-Röder, 2001; Christoffers et al. 2007; Harutyunyan et al. 2008). Enantioselective conjugate additions using thiol nucleophiles were achieved with chiral lanthanide catalysts (Emori et al. 1998).

Additionally, catalysts free of metals are often desired for synthesis of agrochemical and pharmaceutical compounds. Organocatalysts like, amino acid derivates, chiral amines, oligopeptides and phosphines, catalyze conjugate additions efficiently and with enantioselectivity (Krause and Hoffmann-Röder, 2001; Mather et al. 2006; Pellissier, 2007).

1.3.3 Enzyme catalysts

Enzyme catalysts are environmentally compatible alternatives to traditional chemical catalysts. Enzyme-catalyzed conjugate additions are rarely reported within synthesis. In the literature, two enzyme-catalyzed enantioselective approaches are found for conjugate additions. In the approach of Kitazume et al. (1986, 1987, 1988) conjugate additions using various nucleophiles to trifluorinated α,β-unsaturated carbonyl compounds were performed with different

10

11

hydrolases (Scheme 3). Today, a number of enzymes (acylases, lipases, proteases) have been found to display promiscuous conjugate addition activity in vitro, but in contradiction to the results of Kitazume et al., without showing stereoselectivity. In the second approach, O-acetylserine sulfhydrylase catalyzes conjugate addition using various nucleophiles to L-O-acetylserine via pyridoxal-5´-phosphate intermediate (Ikegami and Murakoshi, 1994; Flint et al. 1996).

HO

O

CF3

NuH EnzO

O

CF3

Nu H2O HO

O

CF3

Nu

Enzyme

Scheme 3. Enantioselective conjugate addition of various nucleophiles (sec-amines and thiols) to trifluorinated α,β-unsaturated carbonyl compounds catalyzed by different hydrolases. Highest stereoselectivity (71% ee) was achieved using Candida rugosa lipase in buffer solution at 40˚C (Kitazume et al. 1986, 1987, 1988). Scheme adapted from Faber 2000.

In vivo, certain enzymes within the pyrimidine metabolism, such as thymidylate synthase, form transient conjugate addition intermediates to increase the reactivity of the pyrimidine for further transformation (Ivanetich and Santi, 1992).

2. PRESENT INVESTIGATION The objective of this project was to explore the active-site of Pseudozyma antarctica lipase B for promiscuous reactions. Focus was on understanding enzyme and substrate interactions. For this, a collaboration project was initiated with division of Physical Chemistry at KTH to combine practical and theoretical experiments.

Initially, a theoretical study investigated the possibility of using the active-site of Pseudozyma antarctica lipase B as a scaffold for catalysis of various promiscuous reactions like aldol additions, Bayer-Villiger oxidations, conjugate additions and direct epoxidation reactions. The substrate interactions in Pseudozyma antarctica lipase B were explored by molecular modeling and reaction mechanisms were suggested for each reaction by quantum chemical calculations. To prevent hemiacetal formation by Ser105, two variants of Pseudozyma antarctica lipase B were suggested (Ser105Ala and Ser105Gly). Early laboratory work revealed aldol addition activity in Pseudozyma antarctica lipase B wild-type and variants (Branneby et al. 2003). The two variant enzymes displayed increased specific activities compared to wild-type enzyme. However, Pseudozyma antarctica lipase B wild-type and Ser105Ala was chosen for further explorations of promiscuous reactions.

This thesis focuses on the promiscuous conjugate addition activity of Pseudozyma antarctica lipase B wild-type and Ser105Ala. Three papers on this topic will be further discussed in the following sections.

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2.1 Paper I: Exploring the active-site of a rationally redesigned lipase for catalysis of Michael-type additions

In Paper I, the active-site of Pseudozyma antarctica lipase B Ser105Ala was explored for promiscuous conjugate addition activity by both practical and theoretical experiments. A reaction mechanism for a model conjugate addition reaction using methanethiol and 2-propenal in Pseudozyma antarctica lipase B Ser105Ala was determined by quantum chemical calculations at the division of Physical Chemistry at KTH (Scheme 4). According to the mechanism, the native ping pong bi bi reaction mechanism for hydrolysis/transacylation activity is altered to an ordered bi uni reaction mechanism for conjugate addition activity. 2-Propenal coordinated to the oxyanion hole formed by Thr40 and Gln106, while methanethiol coordinated to His224. His224 activates methanethiol by proton abstraction for nucleophilic attack on the β-carbon of 2-propenal. A conjugate addition intermediate is formed which is stabilized by the oxyanion hole. The catalytic reaction ends by a final proton transfer from His224 to the α-carbon of the conjugate addition intermediate. The product leaves the active-site and the enzyme is ready to turnover another pair of substrates. The rate determinating step of this reaction mechanism is the proton transfer from His224 to the α-carbon of the conjugate addition intermediate (Paper I).

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O

NH

O

O

HN

OH

Gln106

Thr40NH

O

O

HN

O

HGln106

Thr40

NNH

O

NH

O

O

HN

OH

His224

Gln106

Thr40

O

O

Asp187

NNH

O

NH

O

O

HN

OH

His224

Gln106

Thr40

Asp187

O

O

H

H

HH S

O

H S

NNH

His224

O

O

Asp187

O

H

NNH

His224

O

O

Asp187

H

Ala105 Ala105

Ala105Ala105

HS

S

Scheme 4. Proposed ordered bi uni reaction mechanism for conjugate addition of methanethiol to 2-propenal catalyzed by Pseudozyma antarctica lipase B Ser105Ala (Paper I).

The conjugate addition activity of Pseudozyma antarctica lipase B wild-type and Ser105Ala was explored using various nucleophiles and α,β-unsaturated carbonyl compounds (Scheme 5). Nucleophiles like, alcohols, amines, thiols and water were selected for evaluation, but only one sec-amine and various thiols produced conjugate additions. This was explained by the concept of soft and hard electrophiles and nucleophiles (Jacobs, 1997). α,β-unsaturated carbonyl compounds are considered as ambident electrophiles that may react on different sites depending on the nucleophile. Generally for α,β-unsaturated carbonyl compounds, hard nucleophiles react on the carbonyl carbon in a 1,2-addition and soft nucleophiles react with the β-carbon in a 1,4-addition

14

(conjugate addition). Alcohols and water are hard nucleophiles and prefer 1,2-addition to α,β-unsaturated carbonyl compounds. Thiols are soft nucleophiles that react in 1,4-fashion to α,β-unsaturated carbonyl compounds. Aldehydes, ketons and esters were evaluated as α,β-unsaturated carbonyl compounds.

O

R2R3R1

HS R4Enzyme

Organic solventR1

R2R3

O SR4

or

NH

R1

R2R3

O N

or

Scheme 5. General reaction scheme for conjugate additions catalyzed by Pseudozyma antarctica lipase B wild-type and Ser105Ala. Aldehydes, ketons and esters were able to undergo enzyme-catalyzed conjugate additions using various thiols or one sec-amine in organic solvent. The following substrates were used for conjugate additions: (1) 2-Propenal, R1=H, R2=H, R3=H; (2) 2-Butenal, R1=H, R2=H, R3=Me; (3) 2-Pentenal, R1=H, R2=H, R3=Et; (4) 2-Methyl-2-pentenal, R1=H, R2=Me, R3=Et; (5) 3-Phenyl-2-propenal, R1=H, R2=H, R3=Ph; (6) 2-Cyclohexenone, R1=Me, R2=H, R3=Et; (7) Methyl acrylate, R1=OMe, R2=H, R3=H; (8) Ethanethiol, R4=Et; (9) tert-Butylthiol, R4=tBu; (10) 2-Butylthiol, R4=2-Bu; (11) 2-Pentanethiol, R4=2-Pent; (12) Benzylthiol, R4=Bz; (13) Thiophenol, R4=Ph and (14) Diethyl amine.

Both Pseudozyma antarctica lipase B wild-type and Pseudozyma antarctica lipase B Ser105Ala produced conjugate adducts using the substrates described in Scheme 5 and Table 1, at similar rates. But, conjugate additions of thiols to methyl acrylate in organic solvent were only catalyzed by Pseudozyma antarctica lipase B Ser105Ala (Table 1, No 21 and 22). No product was detected using the wild-type enzyme, since the nucleophilic Ser105 forms an acyl enzyme that blocks the enzyme-catalysis. Unexplained and in contradiction to these results, the addition of diethyl amine to methyl acrylate in organic solvent proceeded faster using wild-type enzyme compared to the Ser105Ala variant (Table 1, No 23).

The catalytic proficiency ( ) nonMcat kKk of Pseudozyma antarctica lipase B Ser105Ala was determined for a conjugate addition of thiophenol to 2-cyclohexenone in toluene (Table 2). Using an apparent specificity constant ( )appMcat Kk and the background reaction rate ( ), the catalytic proficiency was determined to 24 millions, which is comparable to those values of some native enzyme-catalyzed reactions (Radzicka and Wolfenden, 1995).

nonk

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Table 1. Catalytic events per enzyme and time, indicated by apparent kinetic constants ( ) for Pseudozyma antarctica lipase B Ser105Ala (mt) and appcatkPseudozyma antarctica lipase B wild-type (wt) catalyzed conjugate addition using different combinations of substrates. The background reaction rates ( non ) are kshown for comparison. Reactions were performed in organic solvents at 20˚C.

No Acceptor[a] Donor[b] appmt cat,k

[min-1]

app wtcat,k

[min-1] appwt,cat

appmt cat,

kk

nonk

[min-1 M-1]

1 1 9 3.5 1.5 × 10-5

2 2 8 4.3 4.6 0.94 3.0 × 10-5

3 2 9 0.6 3.3 × 10-6

4 2 10 0.18 1.1 × 10-6

5 2 11 1.03 0.67 1.5 9.9 × 10-7

6 2 12 1.3 1.3 × 10-4

7 3 8 1.03 0.93 1.1 3.8 × 10-6

8 3 9 0.0077 3.3 × 10-6

9 3 10 0.044 6.5 × 10-8

10 3 11 0.056 0.018 3.1 5.0 × 10-6

11 3 12 1.3 3.8 × 10-5

12 4 8 0.41 2.0 × 10-7

13 4 12 0.21 0.22 0.96 3.3 × 10-5

14 5 8 0.13 6.5 × 10-5

15 5 9 0.024 2.3 × 10-8

16 5 10 0.058 2.0 × 10-7

17 5 11 0.27 0.097 2.8 9.9 × 10-8

18 5 12 1.2 1.0 × 10-6

19 6 11 0.27 0.48 0.56 1.9 × 10-5

20 6 13 1.4 1.1 × 10-7

21 7 8 0.064 6.4 2.0 × 10-7

22 7 11 0.028 1600.0 2.3 × 10-8

23 7 14

Irreversible inhibition of Pseudozyma antarctica lipase B wild-type was applied to experimentally demonstrate the requirement of the enzyme active-site for conjugate addition. The inhibitor, n-hexylphosphonate ethyl ester, reacts with the catalytic Ser105 to form an irreversible complex. A control reaction containing inhibited wild-type enzyme was just as slow as the spontaneous background reaction.

Table 2. Apparent kinetic constants ( ) for Pseudozyma antarctica lipase B appcatkSer105Ala catalyzed conjugate addition of thiophenol (13) to 2-cyclohexenone (6) in toluene. The background reaction rate ( ) is shown for comparison. nonk

appcatk

[s-1]

( )appMcat Kk [s-1 M-1]

nonk [s-1 M-1]

appcatk / nonk

[M]

( ) nonappMcat kKk

2.2 × 10-2 3.4 1.4 × 10-7 1.6 × 105 2.4 × 107

Despite the chiral environment provided by the active-site of Pseudozyma antarctica lipase B, no enantioselectivity was displayed for the reactions in Table 1. In a recent paper, the shortage of enantiomeric preference in Pseudozyma antarctica lipase B for conjugate additions was explored by molecular modeling. No specific binding modes were revealed using an enantiomer pair of a chiral conjugate adduct and the lack enantioselectivity was explained by a too voluminous enzyme active-site (Strohmeier et al. 2009). However, various enzymes acylases (Xu et al. 2005, 2007a, 2007b; Qian et al. 2007), lipases (Torres et al. 2004, 2005; Paper I, II, III; Xu et al. 2005, 2007a, 2007b; Cai et al. 2006), lyase (unpublished data by Svedendahl) and proteases (Cai et al. 2004, 2006) containing different catalytic amino acids catalyze conjugate additions to produce racemic products. It is possible that all these enzymes have too large active-sites for enantioselective conjugate addition, but Kitazume et al. (1986, 1987, 1988) performed enantioselective conjugate additions catalyzed by various nucleophiles to trifluorinated α,β-unsaturated carboxyl acids and esters using different hydrolytic enzymes in buffer solution (Scheme 3). These results have not been repeated within the literature. The reason for enantioselectivity in those cases can be the formation of an acyl enzyme complex that keeps the substrate fixed during enzyme-catalysis. The enzymatic reaction ends by a hydrolysis step to release the conjugate adduct from the enzyme. The use of trifluorinated compounds seems to increase the rate of the

17

conjugate addition to be competitive with the hydrolysis activity that would otherwise be too fast to compete with, for the promiscuous reaction. Conjugate additions of aldehydes and ketons are performed by Pseudozyma antarctica lipase B wild-type without the formation of an acyl enzyme, since no possible leaving group is present. Pseudozyma antarctica lipase B wild-type will most probably form an acyl enzyme with methyl acrylate. Since there is no substrate to react with the acyl enzyme, the enzyme is blocked for further transformations. A noncovalent coordinated substrate is free to move in a totally different way in the active-site compared to a covalently coordinated substrate such as in ordinary lipase catalyzed hydrolysis and transacylation reactions and the conjugate addition shown by Kitazume et al. The difference between a covalently coordinated intermediate in the active-site of Pseudozyma antarctica lipase B wild-type and a Michael addition intermediate with only hydrogen bond coordination in the active-site of Pseudozyma antarctica lipase B Ser105Ala is shown in Figure 3a and 3b.

Finally, in Paper I, Pseudozyma antarctica lipase B wild-type and the Ser105Ala variant displayed conjugate addition activity. A reaction mechanism for the conjugate addition was established, where the native ping pong bi bi type of kinetics of Pseudozyma antarctica lipase B was rerouted to an ordered bi uni kinetics for conjugate addition activity. Both Pseudozyma antarctica lipase B variants catalyzed conjugate additions of one sec-amine or various thiols to α,β-unsaturated carbonyl compounds like aldehydes, ketons or esters in organic solvent at 20˚C. The enzyme-catalyzed conjugate addition displayed Michaelis-Menten kinetics. The catalytic transformation was shown to take place in the enzyme active-site, but despite this, no enantioselectivity was displayed. The shortage of enantiopreference of the Pseudozyma antarctica lipase B catalyzed conjugate additions can be due to the lack of covalent binding of the substrates during enzyme-catalysis.

18

Tetrahedral intermediate

Ser105 Asp187

His224

Gln106

Thr40

Figure 3a. A covalently bound tetrahedral intermediate in the active-site of Pseudozyma antarctica lipase B wild-type. The catalytically active amino acid residues (Thr40, Asp187, Ser105, Gln106 and His224) of Pseudozyma antarctica lipase B wild-type with a tetrahedral intermediate are shown in sticks. The structure of Pseudozyma antarctica lipase B was derived from the 1LBS crystal structure (Uppenberg et al. 1995) from the Protein Data Bank (www.rcsb.org). The tetrahedral intermediate was constructed by modifications of the inhibitor structure adopted from the crystal structure. The hydrogen bonds are shown by dashes. Molecular graphics created with YASARA (www.yasara.org) and PovRay (www.povray.org).

Asp187

His224

Thr40

Ala105

Gln106

Michael addition intermediate

Figure 3b. A noncovalently coordinated Michael addition intermediate in the active-site of Pseudozyma antarctica lipase B Ser105Ala. The catalytically active amino acid residues (Thr40, Asp187, Gln106 and His224) and Ala105 of Pseudozyma antarctica lipase B Ser105Ala with a Michael addition intermediate are shown in sticks. The structure of Pseudozyma antarctica lipase B was derived from the 1TCA crystal structure (Uppenberg et al. 1994) from the Protein Data Bank (www.rcsb.org). The hydrogen bonds are shown by dashes. Molecular graphics created with YASARA (www.yasara.org) and PovRay (www.povray.org).

19

2.2 Paper II: Fast carbon-carbon bond formation by a promiscuous lipase

The project continued by carbon-carbon bond forming Michael additions. The hypothesis was that Pseudozyma antarctica lipase B should be able to catalyze Michael additions if the enzyme was able to activate carbon nucleophiles. Just as in Paper I, the α,β-unsaturated carbonyl compound was supposed to be activated and stabilized by the oxyanion hole in Pseudozyma antarctica lipase B, while the carbon nucleophile was supposed to be activated by α-proton abstraction by His224 (Scheme 6).

To explore the Michael addition activity of Pseudozyma antarctica lipase B, 1,3-dicarbonyl compounds were selected as carbon nucleophiles for addition to various α,β-unsaturated carbonyl compounds. Enzyme-catalyzed reactions were performed without solvent, in organic solvent or water. Reactions using α,β-unsaturated carbonyl compounds longer than three carbons (like 2-butenal or 3-phenyl-2-propenal) were slow. The low reactivity of longer acceptor molecules could depend on steric hindrance. The most successful reactions are shown in Scheme 7, using 2-propenal, methyl vinyl ketone and methyl acrylate as Michael acceptors and acetyl acetone and dimethyl malonate as Michael donors.

20

O

NH

O

O

HN

OH

Gln106

Thr40NH

O

O

HN

O

HGln106

Thr40

NNH

O

NH

O

O

HN

OH

His224

Gln106

Thr40

O

O

Asp187

NNH

O

NH

O

O

HN

OH

His224

Gln106

Thr40

Asp187

O

O

H

O O

O

H

HHH

O

O

H

O

O

O

O

NNH

His224

O

O

Asp187

O

H

NNH

His224

O

O

Asp187

H

Ala105 Ala105

Ala105Ala105

Scheme 6. Proposed ordered bi uni reaction mechanism for Michael addition of acetylacetone to 2-propenal in Pseudozyma antarctica lipase B Ser105Ala.

Catalytic events per enzyme and time (indicated by v) were determined for reactions using the substrates in Scheme 7 under solvent free conditions at 20˚C. The substrate combinations are shown in Table 3. The conjugate addition of acetylacetone to 2-propenal was remarkably faster than the other reactions, 4000 s-1, which were 36 times faster compared to Pseudozyma antarctica lipase B wild-type and 108 times faster than the spontaneous background reaction. These are high values for a promiscuous reaction. The highest reaction rates were achieved using 2-propenal and lowest using methyl acrylate. Michael additions using acetylacetone generally performed better than when using dimethyl malonate. This could be due to the more acidic α-proton of acetyl acetone and by enzyme inhibition caused by acyl enzyme

21

formation using dimethyl malonate. The increased activity of Pseudozyma antarctica lipase B Ser105Ala compared to wild-type enzyme can be explained by decreased enzyme inhibition. In the Ser105Ala variant no hemiacetal or acyl formation is possible. Also, the Ser105Ala variant increased Michael donor activation due to a more basic His224.

O

R2

Enzyme

20 °C R2

O

R1

OO

R1

R1

O

O

R1

Scheme 7. Michael addition of 1,3-dicarbonyl compounds to α,β-unsaturated carbonyl compounds catalyzed by Pseudozyma antarctica lipase B Ser105Ala. The following substrates were used: (1) Acetylacetone, R1=Me; (2) Diethyl malonate, R1=OMe; (3) 2-Propenal, R2=H; (4) Methyl vinyl ketone, R2=Me and (5) Methyl acrylate, R2=OMe.

Table 3. Catalytic events per enzyme and time, indicated by v, for Pseudozyma antarctica lipase B Ser105Ala (mt) and Pseudozyma antarctica lipase B wild-type (wt) catalyzed solvent free Michael addition. The background reaction rates are shown for comparison. The acceptor and donor substrates are shown in Scheme 7.

No Acceptor Donor mtv [s-1]

wtv

[s-1] nonk

[s-1M-1] mtv / nonk

[M-1]

1 3 1 4000 110 2.6 × 10-5 1.5 × 108

2 4 1 1.2 0.9 4.8 × 10-9 2.4 × 108

3 5 1 0.081 0.0024 2.6 × 10-8 3.1 × 106

4 3 2 < 10-7 < 10-7 < 10-10 -

5 4 2 0.32 0.0058 4.4 × 10-7 7.2 × 105

6 5 2 0.009 1.1 × 10-5 1.5 × 10-11 5.9 × 108

Apparent kinetic constants were determined for a Michael

addition catalyzed by Pseudozyma antarctica lipase B Ser105Ala using methyl vinyl ketone and acetyl acetone in cyclohexane at 20˚C (Table 4). The concentration of methyl vinyl ketone was varied for three constant concentrations of acetylacetone. An apparent kinetic specificity constant ( )appMcat Kk was around 1 s-1M-1 in all three cases, but decreased with higher concentration of acetylacetone. This was explained by substrate inhibition of acetylacetone, which was also shown by docking simulations (Paper III and Figure 4). According to the ordered bi uni reaction mechanism for conjugate addition activity in

22

Pseudozyma antarctica lipase B Ser105Ala (Paper I), methyl vinyl ketone binds in the oxyanion hole while acetylacetone coordinates to His224. According to substrate dockings (Paper III), the preferred position of acetylacetone is in the oxyanion hole. Therefore, methyl vinyl ketone must enter the active-site before acetylacetone to prevent enzyme substrate inhibition. Finally, the catalytic proficiency, ( ) nonappMcat kKk , of the studied Michael addition was just as high as observed for conjugate addition of thiol nucleophiles using Pseudozyma antarctica lipase B Ser105Ala.

His224

Asp187

Thr40

Gln106 Acetylacetone

Ala105

Figure 4. Acetylacetone coordinated to the oxyanion hole of Pseudozyma antarctica lipase B Ser105Ala. The two carbonyl oxygens of acetylacetone are coordinated by three hydrogen bonds provided by Thr40 and Gln106. The catalytically active amino acid residues (Thr40, Asp187, Gln106 and His224) and Ala105 of Pseudozyma antarctica lipase B Ser105Ala and acetylacetone are shown in sticks. The hydrogen bonds are displayed by dashes. Molecular graphics created with YASARA (www.yasara.org) and PovRay (www.povray.org).

Table 4. Apparent kinetic constants for the Michael addition of acetylacetone to vinyl ketone catalyzed by Pseudozyma antarctica lipase B Ser105Ala in cyclohexane. The background reaction rate ( ) are shown for comparison. nonk

Concentration of acetylacetone 0.10 M 0.50 M 1.0 M

( )appMcat Kk [s-1M-1] 1.2 1.0 0.66 nonk [s-1M-1] 4.8 × 10-9 4.8 × 10-9 4.8 × 10-9

( ) nonappMcat kKk 2.4 × 108 2.2 × 108 1.4 × 108

To conclude, Paper II demonstrated carbon-carbon bond formation by Michael addition using Pseudozyma antarctica lipase B. Increased reaction rates were achieved using Pseudozyma antarctica lipase B Ser105Ala compared to Pseudozyma antarctica lipase B wild-type. The conjugate addition of acetylacetone to 2-propenal was

23

remarkably faster than the other reactions. The catalytic event per enzyme and minute for this reaction catalyzed by Pseudozyma antarctica lipase B Ser105Ala was 4000 s-1, which were 36 times faster compared to Pseudozyma antarctica lipase B wild-type.

24

2.3 Paper III: Suppressed native hydrolytic activity of a lipase to reveal promiscuous Michael addition activity in water

Pseudozyma antarctica lipase B normally catalyze hydrolysis in water solution. Paper III demonstrates a suppressed hydrolytic activity of Pseudozyma antarctica lipase B by the Ser105Ala mutation to reveal conjugate addition activity. This was displayed by a model system, shown in Scheme 8, consisting of a α,β-unsaturated ester (methyl acrylate) and a 1,3-dicarbonyl compound (acetylacetone) in buffer solution. Laboratory experiments were performed to distinguish the hydrolytic- and Michael addition activities of Pseudozyma antarctica lipase B wild-type and Ser105Ala variant. Computational experiments were implemented to provide a molecular understanding for the suppressed hydrolytic activity and the revealed Michael addition activity in Pseudozyma antarctica lipase B Ser105Ala.

O

O21 °C

O

OOO

O

O

20 mM NaOAc pH 5H2O

or

HO

O

Scheme 8. Model system to demonstrate the hydrolytic- and Michael addition activities in Pseudozyma antarctica lipase B wild-type and Ser105Ala variant. Methyl acrylate can be hydrolyzed to form acrylic acid or react with acetylacetone in a Michael addition to yield methyl 4-acetyl-5-oxo-hexanoate. The reaction was performed in water solution at 21˚C.

Laboratory experiments using the model system, displayed hydrolytic activity of Pseudozyma antarctica lipase B wild-type and conjugate addition activity of Pseudozyma antarctica lipase B Ser105Ala (Table 5). The wild-type enzyme catalyzed hydrolysis of methyl acrylate to acrylic acid according to a ping pong bi bi reaction mechanism (Martinelle and Hult, 1995 and Scheme 9). The nucleophilic Ser105 attacks the carbonyl carbon of methyl acrylate to form an acyl enzyme. The carbonyl oxygen of methyl acrylate is stabilized by the oxyanion hole, built up by hydrogen bonds from Thr40 and Gln106. A tetrahedral

25

intermediate forms and methanol leaves the active-site. Then, water attacks the carbonyl carbon of the acyl enzyme complex to form a second tetrahedral intermediate. Finally, acrylic acid leaves the active-site. The acyl enzyme can undergo nucleophilic attack by acetylacetone on the β-carbon, but this reaction is too slow to compete with the native hydrolysis activity.

Table 5. Catalytic events per enzyme and minute for the hydrolysis and Michael addition activities of Pseudozyma antarctica lipase B (PalB) wild-type or Pseudozyma antarctica lipase B (PalB) Ser105Ala.

appcatk [min-1]

Catalyst Hydrolysis Michael addition

PalB wild type 0.6 < 10-4 [a]

PalB Ser105Ala < 10-4 [b] 0.02

[a] Measured after 14 days. [b] Measured after 70 days.

No enzyme-catalyzed hydrolysis was detected for the model system using Pseudozyma antarctica lipase B Ser105Ala. In the Ser105Ala variant, the wild-type activity was suppressed while conjugate addition activity was displayed (Table 5). According to the proposed ordered bi uni reaction mechanism for conjugate addition activity in Pseudozyma antarctica lipase B Ser105Ala (Paper I), methyl acrylate is positioned and activated in the oxyanion hole. Acetylacetone should be positioned close to His224 for activation by α-proton abstraction. Then, nucleophilic attack by the activated acetylacetone on the β-carbon of methyl acrylate is possible. In the last step, the abstracted proton is added to the α-carbon of the Michael addition intermediate by His224. The conjugate addition is completed and the product, methyl 4-acetyl-5-oxo-hexanoate, leaves the active-site. The proposed reaction mechanism for Michael addition catalysis in Pseudozyma antarctica lipase B Ser105Ala is displayed in Scheme 10.

26

NNH

OH

O

O

NH

O

O

HN

OH

His224

Gln106

Thr40

MeOHOH

O

Asp187

H2O

Ser105

NNH

OH

O

O

NH

O

O

HN

OH

His224

Gln106

Thr40

O

O

Ser105 HN

NH

O

O

NH

O

O

HN

OH

His224

Gln106

Thr40

Ser105

O

O

Asp187

O

O

O

O

NNH

HO

NH

O

O

HN

OH

His224

Gln106

Thr40

Asp187

Ser105

O

O

Asp187

Scheme 9. Pseudozyma antarctica lipase B follows a ping pong bi bi reaction mechanism for hydrolysis of methyl acrylate to acrylic acid. An acyl enzyme is formed as the nucleophilic Ser105 attacks the carbonyl oxygen of methyl acrylate. Methanol leaves the active-site and is replaced by water. Acrylic acid is formed and leaves the enzyme active-site.

 

 

 

27

O

NH

O

O

HN

OH

Gln106

Thr40NH

O

O

HN

O

HGln106

Thr40

NNH

O

NH

O

O

HN

OH

His224

Gln106

Thr40

O

O

Asp187

NNH

O

NH

O

O

HN

OH

His224

Gln106

Thr40

Asp187

O

O

O

O O

O

H

HOO

O

O

O

O

O

O

O

NNH

His224

O

O

Asp187

O

O

NNH

His224

O

O

Asp187

H

Ala105 Ala105

Ala105Ala105

Scheme 10. Pseudozyma antarctica lipase B Ser105Ala follows a proposed ordered bi uni reaction mechanism for Michael addition of acetylacetone to methyl acrylate. Methyl acrylate is coordinated by hydrogen bonds to the oxyanion hole, while acetylacetone should be positioned close to His224. His224 should activate acetylacetone by abstraction of its α-proton before nucleophilic addition the β-carbon of methyl acrylate. Then, the proton is transferred to the α-carbon of the Michael addition intermediate. Finally, the Michael addition product is formed and released form the enzyme.

Apparent kinetic constants were determined for the model Michael addition catalyzed by Pseudozyma antarctica lipase B Ser105Ala (Table 6). The apparent turnover number, , and the value for the specificity constant,

appcatk

( )appMcat Kk , were low. This could be explained by enzyme inhibition by acetylacetone, as revealed by the kinetic study in Paper II and molecular modelling in Paper III. Dockings of

28

acetylacetone to Pseudozyma antarctica lipase B Ser105Ala display three hydrogen bonds to the oxyanion hole. Because of the low turnover number, the rate enhancement of the model Michael addition was only moderate, ~106 M-1.

Table 6. Apparent kinetic constants for Pseudozyma antarctica lipase B Ser105Ala catalyzed Michael addition of acetylacetone to methyl acrylate. The background reaction rate is shown for comparison.

appcatk

[min-1] MK

[M]

( )appMcat Kk [min-1 M-1]

nonk [min-1 M-1]

( ) nonappMcat kKk

0.08 0.1 0.7 8 × 10-7 9 × 105

Computer simulations were implemented to explore the model system on molecular level. The Michaelis complex and the transition state analogue for the Michael addition was explored by molecular docking simulations and molecular dynamics simulations. The simulations were evaluated according to a near attack conformer (NAC) definition (Lightstone and Bruice, 1996), also mentioned as productive mode and shown in Figure 5a for the Michaelis complex and Figure 5b for the transition state analogue for the Michael addition. The results from the docking simulations are summarized in Table 7, the results from the molecular dynamics simulations of the Michaelis complex are displayed in Table 8 and finally, the results from the molecular dynamics simulations for the transition state analogue is shown in Table 9.

29

O

Figure 5. a) Definition of near attack conformers (NACs) for the Michael addition of acetylacetone to methyl acrylate in Pseudozyma antarctica lipase B wild type and Pseudozyma antarctica lipase B Ser105Ala. Methyl acrylate must be coordinated to the oxyanion hole (Thr40 and Gln106) by at least two hydrogen bonds to stabilize the negatively charged oxygen of the enolate (1), acetylacetone should be activated by abstraction of one of its two α-protons by the Nε2 atom of His 224 and must therefore be within van der Waals distance 2.75 Å (2) and the distance between the β-carbon of methyl acrylate and the α-carbon of acetylacetone must be within van der Waals distance (3.4 Å) for nucleophilic attack (3). Distances in the oxyanion hole are shown in green letters a to c. b) Definition of near attack conformers (NACs) for the Michael addition transition-state analogue in both PalB variants. The Michael addition intermediate must be hydrogen bonded to the oxyanion hole to stabilize the enolate by at least two hydrogen bonds (1) and the α-carbon of the Michael addition intermediate must be within van der Waals distance (2.9 Å) to the H1 atom of His 224 to make the addition of the proton back to the α-carbon of the Michael addition intermediate possible (2). Distances in the oxyanion hole and to the proton on His224 are shown in green letters a to d.

Binding of Michaelis complex and transition state analogue for the Michael addition was explored by 250 independent molecular docking simulations of each substrate or transition state analogue using the AutoDock4 (Morris et al. 1998) program package in combination with AutoDock Tools (Sanner, 1999). Structures of Pseudozyma antarctica lipase B wild-type and Ser105Ala variant were prepared from the 1TCA crystal structure (Uppenberg et al. 1994) from the Protein Data Bank (www.rcsb.org). Molecular dynamics simulations were implemented using the YASARA software (Krieger et al. 2002), with all starting structures originating from the AutoDock4 simulations.

NNH

O NH

O OHN

H

His224Asp187

O

O

O

HH

O

O

3

1

2

Gln106

Thr40

a bc

NNH

ONH

OOO

HN H

His224

O

Asp187

O O

OO

1

2

His224 Thr40

H

a bc

d

30

Figure 6. Methyl acrylate coordinated to the oxyanion hole of Pseudozyma antarctica lipase B Ser105Ala by two hydrogen bonds. The catalytically active amino acid residues (Thr40, Asp187, Gln106 and His224) and Ala105 of Pseudozyma antarctica lipase B Ser105Ala and methyl acrylate are displayed in sticks. The hydrogen bonds are shown by dashes. Molecular graphics created with YASARA (www.yasara.org) and PovRay (www.povray.org).

Methyl acrylate is according to the proposed reaction mechanism supposed to bind into the oxyanion hole of Pseudozyma antarctica lipase B and therefore assumed to be the first substrate to enter the active-site. Methyl acrylate was docked into empty structures of both Pseudozyma antarctica lipase B wild-type and Pseudozyma antarctica lipase B Ser105Ala. All docking simulations to Pseudozyma antarctica lipase B wild-type ended in the active-site, but none of the docked structures showed a productive mode for reaction according to the NAC criteria. However, all dockings of methyl acrylate to Pseudozyma antarctica lipase B Ser105Ala displayed a productive mode according to the NAC criteria with two hydrogen bonds to Thr40 and Gln106 (Figure 6).

Acetylacetone was docked into both variants of Pseudozyma antarctica lipase B, with the oxyanion hole already occupied by methyl acrylate in a conformation closest to a productive mode. Dockings of acetylacetone to Pseudozyma antarctica lipase B wild-type displayed about 1/3 of the docking structures in the active-site. Using Pseudozyma antarctica lipase B Ser105Ala, almost all structures ended in the active-site. Acetylacetone found positions above methyl acrylate but not close to His224 as expected. This substrate contains two carbonyl functions that both want to coordinate to the oxyanion hole. The formation of a productive mode could not be displayed for the second substrate,

31

which was not surprising since there is no attraction between the α-carbon (C3) of acetylacetone and the Nε2 atom of His224 (Figure 7).

The transition state analogue, or Michael addition intermediate, was docked into both variants of Pseudozyma antarctica lipase B. All dockings to Pseudozyma antarctica lipase B wild-type ended in the active-site, but none of them fulfilled the NAC criteria. However, nearly all dockings of the transition state analogue to Pseudozyma antarctica lipase B Ser105Ala coordinated to the oxyanion hole, but only 1/10 of those were in a productive mode according to the NAC criteria (Figure 8).

C3

Nε2

Figure 7. Acetylacetone and methyl acrylate in Pseudozyma antarctica lipase B Ser105Ala. The catalytically active amino acid residues (Thr40, Asp187, Gln106 and His224) and Ala105 of Pseudozyma antarctica lipase B Ser105Ala, methyl acrylate and acetylacetone are displayed in sticks. The α-carbon of acetylacetone is marked by C3. The basic nitrogen of His224 is marked by Nε2. The hydrogen bonds are shown by dashes. Molecular graphics created with YASARA (www.yasara.org) and PovRay (www.povray.org).

Table 7. Fraction of correctly positioned structures fulfilling NAC conditions from docking simulations of methyl acrylate, acetylacetone and Michael addition intermediate to Pseudozyma antarctica lipase B (PalB) wild type and Ser105Ala.

Fraction of correctly positioned structures [%]

Simulation PalB wild type PalB Ser105Ala

Methyl acrylate 0 100

Acetylacetone[a] 96 97

Acetylacetone[b] 0 0

Intermediate 0 10

[a] Empty active-site. [b] Methyl acrylate already in the oxyanion hole.

32

Figure 8. Transition state analogue or Michael addition intermediate in Pseudozyma antarctica lipase B Ser105Ala. The catalytically active amino acid residues (Thr40, Asp187, Gln106 and His224) and Ala105 of Pseudozyma antarctica lipase B Ser105Ala and the Michael addition are displayed in sticks. The hydrogen bonds are shown by dashes. Molecular graphics created with YASARA (www.yasara.org) and PovRay (www.povray.org).

Table 8. Fraction of correctly positioned structures fulfilling NAC conditions from molecular dynamics simulations of the Michaelis complex to Pseudozyma antarctica lipase B (PalB) wild type and Ser105Ala. The average distances, D, are given for both enzyme variants.

Simulation Fraction [%]

Da[a]

[Å] Db[a]

[Å] Dc[a]

[Å]

PalB wild type 0 5.76 5.57 4.93

PalB Ser105Ala 0[b] 2.44 3.52 2.18

[a] See Figure 5a for definition of distances (D). [b] No coordination of acetylacetone to His224.

Table 9. Fraction of correctly positioned structures fulfilling NAC conditions from molecular dynamics simulations of the transition-state analogue to Pseudozyma antarctica lipase B (PalB) Ser105Ala. The average distances, D, are given.

Simulation Fraction [%]

Da[a]

[Å] Db[a]

[Å] Dc[a]

[Å] Dd[a]

[Å]

PalB Ser105Ala 97.5 2.34 1.77 2.04 2.25

[a] See Figure 5b for definition of distances (D).

33

The enzyme-substrate interactions were further explored by molecular dynamics simulations (Table 8 and 9). Using Pseudozyma antarctica lipase B wild-type, the molecular dynamics simulation of the Michaelis complex displayed no hydrogen bond coordination to the enzyme and the substrates started to drift away from the active-site. No further simulations were made to study the behaviour of the transition state analogue in the wild-type enzyme. However, the molecular dynamics simulations to Pseudozyma antarctica lipase B Ser105Ala showed hydrogen bond coordination to the oxyanion hole. The NAC criteria were never fulfilled, because acetylacetone was never close to His224 during the simulation. The transition state analogue fulfilled its NAC conditions in almost all of the studied simulation snapshots.

To conclude Paper III, the wild-type activity of Pseudozyma antarctica lipase B was suppressed more than 1000-fold by the removal of the catalytic Ser105. This demonstrates the possibility to suppress a native activity to expose a promiscuous activity. Further exploration of the model reaction on a molecular level revealed an increased capacity of Pseudozyma antarctica lipase B Ser105Ala to position the substrates for Michael addition close to a catalytically competent conformation, compared to Pseudozyma antarctica lipase B wild-type.

34

3. OUTLOOK AND FUTURE ASPECTS

In the last decade, the search for novel enzymes and the demand for new environmentally compatible processes have increased the research regarding multifunctional enzymes, in both academia and industry. An identified promiscuous enzyme activity can be increased by protein engineering to provide a novel enzyme to be used, for instance, in the pharmaceutical industry. Molecular modeling has become an important tool to predict and explore enzyme multifunctional behaviour, molecular restrictions and enzyme plasticity.

Enzyme catalytic promiscuity is only found if searched for, since these activities often are hidden behind a native enzyme activity. The idea of a promiscuous conjugate addition activity in Pseudozyma antarctica lipase B arose by combining general catalytic and structural knowledge about the enzyme together with chemical imagination. This approach may provide numerous new enzyme activities to be discovered. A solution to the current lack of enantioselectivity for the Pseudozyma antarctica lipase B catalyzed conjugate addition can make this approach valuable for industrial applications.

Today, industry uses a wide range of different enzymes for pharmaceutical production. Enzyme instability is a common problem when using enzymes in industrial applications. This can be solved either by protein engineering or by using a well-known stable enzyme which has the promiscuous activity of interest. Nature contains endless amounts of enzymes with undiscovered promiscuous activities. The discoveries of the future are only limited by our own chemical imagination.

35

4. ACKNOWLEDGEMENTS First of all, I want to thank my supervisor Professor Per Berglund for accepting me as a PhD student and for your guidance during these years. I am so grateful for your patience while I was on two maternity leaves. I also want to thank you for all the interesting travels, to conferences and meetings.

Professor Karl Hult, thank you, for being my co-supervisor, all interesting discussions and for your friendly and enthusiastic leadership of the biocatalysis group over the years.

Thank you, Professor Tore Brinck at the division of Physical Chemistry (KTH) for a great collaboration.

Thanks to all members of the Biocatalysis group, for the good times, meetings, discussions and help in the lab.

Thanks to all people on floor 2 for making a pleasant and friendly atmosphere in the lab as well as during lunches.

Thanks to my family: mother Anita, father Olof, my oldest brother Johan (Evelyn and their children Samuel, Vilgot and Evan) and my younger brother Mikael. I hope this thesis can help you to understand what I have been doing during all these years at KTH.

Lots of love and thanks to Jonas, Alva and Elin.

Finally, financial support from the Swedish Research Council (VR) for this project and from Vinnova for the “BIO-AMINES” project is gratefully acknowledged. EU-COST is acknowledged for the opportunity to join the COST Action CM0701 “CASCAT” Training School in Siena, Italy, in 2009.

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1. Introduction1.1 Multifunctional enzymes1.1.1 Moonlighting enzymes1.1.2 Promiscuous enzymes

1.2 Pseudozyma antarctica lipase B1.2.1 General1.2.2 Structure1.2.3 Reaction mechanism1.2.4 Pseudozyma antarctica lipase B promiscuity

1.3 Conjugate Additions1.3.1 General1.3.2 Chemical catalysts1.3.3 Enzyme catalysts

2. Present Investigation2.1 Paper I: Exploring the active-site of a rationally redesigned lipase for catalysis of Michael-type additions2.2 Paper II: Fast carbon-carbon bond formation by a promiscuous lipase2.3 Paper III: Suppressed native hydrolytic activity of a lipase to reveal promiscuous Michael addition activity in water

3. Outlook and Future Aspects4. Acknowledgements5. References