Exploiting Enzyme Promiscuity for Rational Design

Exploiting Enzyme Promiscuity

for Rational Design

Cecilia Branneby

Royal Institute of Technology School of Biotechnology Department of Biochemistry Stockholm May 2005

Royal Institute of Technology AlbaNova University Center School of Biotechnology Department of Biochemistry SE-106 91 Stockholm Sweden

Printed at Universitetsservice US-AB Drottning Kristinas väg 53B SE-100 44 Stockholm Sweden

ISBN 91-7178-008-4

To my parents

Abstract

Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area. Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity. In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant. In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type.

Key words: Candida antarctica lipase B, alanine racemase, substrate specificity, reaction specificity, enzyme catalytic promiscuity

Sammanfattning

Enzymer har idag ett flertal industriella användningsområden. De ingår bland annat som viktiga komponenter i tvättmedel och katalyserar olika reaktioner vid framställning av jordbrukskemikalier, livsmedel, läkemedel och finkemikalier. Deras stora användbarhet beror till största del på att de uppvisar en hög selektivitet och har miljömässiga fördelar jämfört med traditionella katalysatorer. Olika metoder och tekniker inom molekylärbiologin har gjort det möjligt att hitta nya enzymaktiviteter vilket ytterligare ökat enzymernas användbarhet. Trots att enzymer katalyserar reaktioner med hög selektivitet, både med avseende på substrat och på reaktion, så är det idag känt att många enzymer även kan katalysera andra reaktioner. Dessa enzym sägs vara promiskuösa, dvs de visar en variation i de reaktioner de katalyserar. Det har föreslagits att detta är naturens eget sätt att utveckla mångsidighet. Genom att göra små förändringar i proteinsekvensen kan resultatet bli ett enzym som visar en ny reaktionsspecificitet. I den här avhandlingen kommer jag att presentera hur rationell design kan användas för att undersöka enzymers promiskuitet och för att ändra deras reaktionsspecificitet. Den första delen av arbetet beskriver hur Candida antarctica lipas B (CALB) genom en enpunktsmutation fick en ändrad reaktionsselektivitet och ökad aktivitet för aldoladditioner, Michaeladditioner och epoxideringar. Kvantmekaniska beräkningar gjordes för att förutsäga möjligheten att katalysera dessa reaktioner med den tänkta mutanten. Resultatet blev att detta borde vara möjligt. Den katalytiskt aktiva nukleofilen, en serin, muterades till en glycin eller alanine. Enzymerna uttrycktes i Pichia pastoris och analyser gjordes för de olika reaktionerna. Den bästa av mutanterna gav en initialhastighet fyra gånger över den vildtypskatalyserade vid aldoladdition av hexanal. Samma mutant visade även en ökad aktivitet för Mikaeladditioner och epoxideringar, jämfört med vildtypen. I den sista delen av avhandlingen beskrivs hur rationell design användes för att ändra reaktionsspecificiteten av alanine racemas från Geobacillus stearothermophilus. Aktivt protein uttrycktes i Escherichia coli. Den undersökta reaktionen var en transaminering av alanin till pyruvat och 2-butanon till 2- butylamin. En av mutanterna visade en ökad transamineringsaktivitet jämfört med vildtypsenzymet.

List of Publications

This thesis is based on the following publications, which in the text are referred to by their Roman numerals:

I. Carbon-Carbon Bonds by Hydrolytic Enzymes C. Branneby, P. Carlqvist, A. Magnusson, K. Hult, T. Brinck and P. Berglund Journal of the American Chemical Society 2003, 125: 874-875

II. Aldol Additions with Mutant Lipase: Analysis by Experiments and Theoretical Calculations C. Branneby*, P. Carlqvist*, K. Hult, T. Brinck and P. Berglund Journal of Molecular Catalysis B: Enzymatic 2004, 31: 123-128

III. Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions P. Carlqvist*, M. Svedendahl*, C. Branneby, K. Hult, T. Brinck and P. Berglund ChemBioChem 2005, 6: 331-336

IV. Investigation of Substrate Specificity of Geobacillus stearothermophilus Alanine Racemase C. Branneby, S. Park and P. Berglund Manuscript

V. Appendix: Enzyme Catalyzed Epoxidations C. Branneby

* Shared first authorship

Table of Contents

1 Introduction 1 1.1 Enzyme Specificity 2 1.2 Enantiomers 3 2 Techniques for Exploiting Enzyme Promiscuity 5 2.1 Enzyme Promiscuity 5 2.2 Protein Engineering 6 2.3 Site-Directed Mutagenesis 8 3 α/β-Hydrolase Fold Enzymes 11 3.1 Candida antarctica Lipase B 14 3.1.1 Protein Structure 14 3.1.2 Reaction Mechanism 15 3.2 Protein Engineering of Hydrolases 16 4 Rational Design of Candida antarctica Lipase B 17 4.1 Aldol Addition 18 4.1.1 Aldolases 18 4.1.2 Non-Enzymatic Catalysts 19 4.1.3 A CALB Mutant as Catalyst for Aldol Additions 20 4.2 Michael Addition 24 4.2.1 Catalysts for Michael Additions 25 4.2.2 A CALB Mutant as Catalyst for Michael Additions 25 4.3 Epoxidation 28 4.3.1 Catalysts for Epoxidations 28 4.3.2 A CALB Mutant as Catalyst for Epoxidations 29 4.4 Summary 31 5 PLP-Dependent Enzymes 33 5.1 PLP as Cofactor 33 5.2 Racemases and Transaminases 34 5.2.1 Reaction Mechanism 34 5.3 Alanine Racemases 35 5.3.1 Protein Structure 37 5.3.2 Reaction Mechanism 37 5.4 Protein Engineering of PLP-Dependent Enzymes 39

5.5 Activity and Stability of Mutants of an Alanine Racemase 40 Conclusions 45 Acknowledgements 47 References 49

Introduction

1 Introduction

The Cells own catalysts are, with a few exceptions (some RNAs and DNAs), proteins called enzymes. Enzymes are of highest importance in all living organisms and are involved in all reactions, from taking a breath to food digestion, making a flower bloom and also in the moldering of a fallen leaf. The effectiveness of enzymes is quite remarkable, which can be quite difficult to understand just by hearing that they increase the reaction rate 107-1019 times over that of the uncatalyzed reaction (Wolfenden & Snider, 2001). If it is instead explained by comparing the time that the enzyme needs to perform a reaction and the time that it takes for the uncatalyzed reaction to occur, it might be easier to understand. If an enzyme-catalyzed reaction takes 1s to occur, the same reaction would need up to 320 billion years without enzyme. It is now much easier to understand that a missing enzyme or one that does not have full activity can cause drastic effects. This is for instance seen in many diseases, such as lactose intolerance, and phenylketonuria. Enzymes do not only catalyze the reaction with a high reaction rate, they also show a very high substrate and reaction selectivity and almost all substrates and reaction steps have their own particular enzyme (Hudson, 1992). Besides their natural activity in every living organism, enzymes have also been used more specifically by man since the ancient China and Japan. Enzymes such as amylases and proteases have since then been used in the manufacturing of food and alcoholic drinks. Also Europeans have a long history with the use of enzymes. In the Iliad (about 400 B.C.) it is mentioned how cheese is produced in kid stomachs (Buchholz & Poulsen, 2000). The first examples of scientific development of enzyme catalysis are from the beginning of the 19th century. Spallanzani described how gastric juice caused meat liquefaction in 1783 and three years later, in 1786, starch hydrolysis was described by Scheele. The development that followed was slow and the real break-through in biotechnology did not come until the introduction of enzymes in detergents in the 1960’s (Buchholz & Poulsen, 2000). Today we can find enzymes as catalysts for example in detergents, in the agrochemical and food industry, and in the production of fine chemicals and pharmaceuticals (Schmid et al., 2001, Zaks, 2001, Houde et al., 2004).

1.1 Enzyme Specificity

The catalytic ability of an enzyme is found in the so-called active site, which is located in a cavity or cleft in the enzyme. This active site differs among enzymes, not only in size but it will also contain different catalytically active amino acids. Because of this, enzymes can stabilize different transition states and catalyze different reactions; they show reaction selectivity. It is often of interest to group enzymes in different classes. One way is to group them according to what type of reaction they catalyze and more specifically, which bonds they break and create. This system gives each enzyme a four-digit number, called the Enzymatic Commission Number (EC-number). The first number denotes the main reaction type and the next three are more and more specific to the individual enzyme. Candida antarctica lipase B has the EC-number 3.1.1.3 and alanine racemase with EC-number 5.1.1.1. The first digit here indicates that triacylglycerol lipases belong to the main group of hydrolases and alanine racemase to racemases. Both these groups will be further discussed later in this thesis, in chapter 3 and 5, respectively. As indicated with the EC numbers, different enzymes not only catalyze a specific kind of reaction, they can also discriminate between different substrates. This is called substrate selectivity. Even if two substrates are of the same size, they will show different reactivity depending on the nature of their functional groups. Not only because of difference in binding ability but also because they have different reactivity, chemoselectivity. The position of the functional group on the substrate is also important for the binding ability. The enzyme can for instance select between two hydroxyl groups on the same substrate, regioselectivity. Last but not least, enzymes can distinguish between stereoisomers, such as enantiomers (see chapter 1.2). As shown in figure 1.1 the active site gives a restricted area for the substrate to bind into. This means that one of the substrates will bind better than the other. This is called stereoselectivity.

HO H HO H

M M

L L

a) favored binding b) unfavored binding

Figure 1.1. Schematic picture of an active site consisting of one large (L) and one medium (M) sized pocket for the substrate to bind in. a) The fast reacting substrate places its large group in the large sized pocket, whereas b) the slow reacting enantiomer will have to place its large group in the medium sized pocket.

2 Introduction

The ability of enzymes to distinguish between enantiomers is used, for instance in the production of agrochemicals, food additives, and pharmaceuticals (Yagasaki & Ozaki, 1998, Zaks, 2001). Three of the methods that can be used to produce enantiomerically pure compounds are asymmetric synthesis, kinetic resolution, and dynamic kinetic resolution. An example of asymmetric synthesis is the stereoselective reduction of a ketone. Unless the ketone is symmetric the outcome will be an enantiomerically pure alcohol with a maximal yield of 100% (García-Urdiales et al., 2005). By kinetic resolution two enantiomers can be separated. If this is really effective, one of the enantiomers will turn into the product whereas the other is left almost unreacted. An example of a kinetic resolution is the acylation of secondary alcohols. The drawback with this method though, is the maximal yield of 50% (Faber, 2001, Pàmies & Bäckvall, 2003). There are ways to overcome this limitation, for example by including a racemization reaction to the kinetic resolution and performing a dynamic kinetic resolution, the maximal yield is increased up to 100%. A racemization reaction of the substrate will then be performed simultaneously with the resolution reaction, for example an acylation of a secondary alcohol, where the alcohol is racemized by oxidation/reduction. This means that the substrate will be kept racemic even though one enantiomer is converted to product. An important factor in a dynamic kinetic resolution is that only the substrate is allowed to racemize, as a racemization of the product would ruin the resolution effect. The limitation in the utility of this reaction is that it requires the two reactions to work under the same reaction conditions, which is not always possible. To be really effective it is also important that the racemization reaction goes much faster than the resolution reaction (Faber, 2001, Pàmies & Bäckvall, 2003).

1.2 Enantiomers

At a first look two compounds might seem identical, but when looking closer it is found that there is no way to turn them so that they get superimposable; they are each others mirror images/enantiomers (Figure 1.2). Enantiomers themselves have the same characteristics, such as melting point and polarity, but at their interaction with the chirality in Nature they become very different. Therefore it is of highest importance for the enzymes to synthesize only the right enantiomer.

Figure 1.2. These two substrates can at a first look seem to be identical, but a closer look shows that they are each others mirror image and can in no way be superimposed. They are enantiomers.

A few examples are shown below to illustrate the importance of having the pure enantiomers and the consequences that can follow if not. In the 1950’s, pregnant women were given Neurosedyn (Thalidomide, Figure 1.3.a) to treat their morning sickness. The drug was though a racemic mixture of the active substance, where one enantiomer had the desired effect whereas the other caused fetus malformations. Ketamine (Figure 1.3.b) is another example, where the S-enantiomer is an anesthetic drug and the R-enantiomer causes hallucination. There are also other areas than the pharmaceutical industry where we find differences between enantiomers and it is not always that one enantiomer is good and one is bad, they can both be good or one can be completely ineffective. Limonene, for example, is a compound where one enantiomer gives the lemon flavor while the other gives the flavor of orange (Figure 1.3.c). Amino acids (Figure 1.3.d) can also be found in both enantiomeric forms. Although most common in their L-enantiomeric form, there are also examples where the D-enantiomer is required. D-Alanine for example is an important compound in the bacterial cell wall structure, and D-serine has been found to have an important role in the human brain as a neurotransmitter.

O Cl

N O * NH * O O O HN a) Neurosedyn b) Ketamine

O + NH3 O * R * O- c) limonene d) amino acid

Figure 1.3. Some examples of compounds with highly different effects depending on their stereochemistry are a) Neurosedyn, b) Ketamine, c) limonene and d) amino acid. * Identifies the stereocenter.

4 Techniques for Exploiting Enzyme Promiscuity

2 Techniques for Exploiting Enzyme Promiscuity

2.1 Enzyme Promiscuity

In the introduction of this thesis enzymes are presented as catalysts with high substrate and reaction selectivity. This is true, but the selectivity is not absolute. There are constantly new examples of how enzymes are used as catalysts for reactions other than those considered their natural. This is called enzyme promiscuity (Bornscheuer & Kazlauskas, 2004). An example of enzyme promiscuity is proteases, which show amide hydrolysis as their main activity. These enzymes are also known to catalyze ester hydrolysis (Pogorevc & Faber, 2000). Enzyme promiscuity does not have to be seen as an unwanted side reaction in enzyme catalysis. The secondary activities are often far below what is considered to be useful, but with the help of mutations these activities can sometimes be reinforced (Penning & Jez, 2001). An example of this is papain, a protease, which also shows nitrile hydratase activity, although very low. A single point mutation in this enzyme gave a mutant, Gln19Glu, with a nitrile hydratase activity about 4×105 over that of the wild type. The wild type activity was at the same time decreased (Dufour et al., 1995). Another example is the engineering of cyclodextrin glycosyltransferase into a starch hydrolase. By comparing the structures of the two enzymes cyclodextrin glycosyltransferase and a maltogenic α-amylase two main differences could be identified in the substrate binding clefts. The introduction of these differences into cyclodextrin glycosyltransferase gave a mutant with increased hydrolysis activity. At the same time the cyclization activity was decreased to the extent that hydrolysis was the main activity (Leemhuis et al., 2003). Myoglobin, which is known to carry molecular oxygen in the muscles, can also catalyze a hydrogen peroxide supported peroxygenation of different substrates. A comparison of the protein structure with that of cytochrome c peroxidase suggested that a relocation of a histidine residue would give an enzyme with increased ability to transfer the ferryl oxygen to the substrate. The result was a highly stereospecific peroxygenase with an activity ten times that of the wild type in epoxidation of styrene (Ozaki et al., 1996).

It is not only in the laboratory that mutated enzymes are created to achieve proteins with new activities or other characteristics; this also happens in Nature. That is how the wide variety of proteins have developed from far fewer in ancient times and why we today have a large number of enzymes with different activities, but still with high structure similarity. This is called divergent evolution. One group of structurally related enzymes, the α/β- hydrolase fold family, will be discussed in chapter 3. Further examples of enzymatic promiscuity will be described in this thesis and yet others can be found in review articles (O’Brien & Herschlag, 1999, Bornscheuer &Kazlauskas, 2004, Berglund & Park, 2005). In addition to enzymatic promiscuity some enzymes have also been found to have different functionalities depending on their location in the cell, in which cell type they are expressed or if they are in a bound or in a free form. This has been called moonlighting functionality and the proteins showing this ability are thereby called moonlighting proteins (Jeffery, 1999, Copley, 2003). An example of this is phosphoglucose isomerase, which catalyzes the second step in the glycolysis in the cell. This enzyme is also secreted by the cell and found for example to stimulate the B cell maturation, although the regulating mechanism is not yet fully understood. Another example of how an enzyme can have different functions is cytochrome c. It is mainly known to be an electron- transporter in the mitochondria membrane, but can serve as a signal for apoptosis. When the cell is damaged or other signal cytochrome c is released into the intermembrane where it forms a protein complex that signals apoptosis (Yarnell, 2003). Dopa decarboxylase is yet another example. This enzyme plays an important role in the production of the neurotransmitters dopamine and serotonin. As many other pyridoxal 5´-phosphate dependent enzymes, it shows small side activities (see chapter 5), but the interesting finding here is that the enzyme seems to switch reaction specificity depending on the oxygen level in the cell. In the oxygen-deficient state the main activity is shifted from decarboxylation to a combined decarboxylation and transamination of aromatic L-amino acids. Kinetic and structural data have shown that this change in reaction specificity is caused by a change in the protein conformation in the absence of oxygen (Bertoldi & Votattorni, 2003).

2.2 Protein Engineering

With the technology available today it is possible to clone and overexpress proteins in high concentrations. In addition to this, the knowledge of how to mutate genes has made it possible to change the protein properties, such as reaction specificity or stability (Hult & Berglund, 2003). There are basically two main strategies to perform mutations, one random approach, and one more rational approach (Figure 2.1) (Penning, 2001).

6 Techniques for Exploiting Enzyme Promiscuity

Protein engineering

Random methods Rational methods a) Gene shuffling c) Domain swapping

Randomly recombined Selectively recombined b) Random mutagenesis d) Site-directed mutagenesis Random point mutations Single point mutation X X X X X X

Figure 2.1. The strategies to modify a protein-encoding gene can be divided in random and rational methods. Examples of random methods are a) gene shuffling and b) random mutagenesis. Rational methods can be exemplified by c) domain swapping and d) site-directed mutagenesis. Gene 1 is shown in grey, and gene 2 in black.

In those cases where no information about the protein structure is available or if no specific target for mutation has been identified a random method is used. Two examples of these are gene shuffling (Figure 2.1.a) and random mutagenesis (Figure 2.1.b). In gene shuffling a pool of homologous genes are partly digested and recombined to “new” full length genes (Stemmer, 1994). This can result in a large number of randomized genes, which have to be analyzed to find the one with the desired property. Random mutagenesis on the other hand, gives a large number of genes containing a varied number of point mutations. One method to achieve random mutagenesis is error-prone polymerase chain reaction (ep-PCR), where a polymerase lacking proofreading activity is used. This method will result in a gene with mutations, but further modifications of the reaction conditions are required as the error frequency from the beginning is still rather low (Ling and Robinson, 1997). A drawback with random methods is that they require a screening method, unless the created library is very small (Chen, 2001, García-Junceda et al., 2004). Random methods are also used in directed evolution, where mutations are made in more than one generation to change the protein characteristics. Rational methods include more specific methods, such as domain swapping (Figure 2.1.c) and site-directed mutagenesis (Figure 2.1.d). If the goal is to exchange a whole domain from one gene to another, domain swapping can be used. The last of the mentioned methods is site-directed mutagenesis, where one or a few specific amino acids are targeted (Chen, 2001). Specific primers are designed to introduce the wanted mutation. This will be discussed further in chapter 2.3. Rational methods can for instance be used to reinforce promiscuous

reactions, change substrate specificity and enzyme mechanism, and increase protein stability (Cedrone et al., 2000, Yokoigawa et al., 2003). Another benefit with rational design is that it does not require a screening method to find the mutant with the desired properties. To be able to use a rational method some knowledge about the protein sequence(s) and structure is required. Structures that have been solved either by x-ray crystallography or NMR can usually be found in the database RCSB protein databank (Berman et al., 2000). Today three-dimensional structures of 27663 proteins, peptides and viruses can be found in this database and if nucleic acids and carbohydrates are included, the total number of available structures is 30361 (April 5th, 2005). If the structure of the protein in question has not been solved it is possible to do a homology search of the protein sequence, in order to predict the structure of the full protein or a specific area of the protein. This requires that the protein structure of a homologous protein is known (Guex et al., 1999). The computer’s role in rational design is quite important. It first of all gives the possibility to study the protein structure to find the target for mutation. Further, it is possible to predict if a reaction that is to be studied by enzyme catalysis is possible. This is done by quantum chemical calculations, which gives the energy levels in the different steps of a reaction. An illustrative example on how different methods can be combined is the work done by Hellinga and coworkers. Here they present how modeling studies of both ribose-binding protein and triose phosphate isomerase could predict mutations of ribose-binding protein to introduce isomerase activity. The final mutant showed an activity 105-106 above background (Dwyer et al., 2004).

2.3 Site-Directed Mutagenesis

In site-directed mutagenesis a specific primer is designed to introduce a mutation (Figure 2.2.a). Two generally used methods to introduce mutations are overlap extension PCR (Ho et al., 1989), and QuickChange® site-directed mutagenesis (Stratagene). In overlap extension PCR (Figure 2.2.b), the mutation is introduced by using four primers and two separate PCR reactions. This gives two DNA fragments which have overlapping ends. Next, the original gene is degraded by an enzyme, DpnI, which recognizes methylation on the original strand. This leaves the mutated PCR product, which in the following PCR is extended to a full length gene. The overlapping ends of the first PCR product are here used as primers. The gene can then be cloned into a vector. QuickChange® site-directed mutagenesis is a more direct method and introduces the mutation on a gene that is already cloned into a vector (Figure 2.2c). By this method only the two primers containing the mutation are required. After the PCR the original sequence is, as before, degraded by DpnI, leaving only the new sequence, containing the mutation. Once the gene with the desired sequence has been cloned into a vector it is time to transform. The first host is almost exclusively Escherichia coli, even if the final goal is to express protein in a higher host. The reason is that E. coli does 8 Techniques for Exploiting Enzyme Promiscuity not incorporate the vector into its own genome, but carries the free vector (Figure 2.2.d). This offers an easy way to retain the gene for further work and analysis. Also in the case of protein expression E. coli is a first hand choice, as it only requires a short expression time. E. coli is easy to handle and is inexpensive compared to other hosts like Pichia pastoris. The limitation in the use of E. coli lies in that it is a prokaryote and can therefore not perform all post-translational modifications, such as glycosylation. If the expression in E. coli fails, P. pastoris is a commonly used host. Transformation into P. pastoris requires a linearized DNA, whereas when transforming to E.coli circular DNA can be used. P. pastoris will, as other eukaryotic hosts, incorporate the vector and gene into its own genome (Figure 2.2.d). The gene will thereby be more stable in the P. pastoris cell than the free vectors in E. coli, where it risks to be eliminated from the host. P. pastoris has the ability to glycosylate a protein, which for instance can be an important factor in the folding of an active protein. It also gives the possibility to excrete the protein extracellularly. This can be a large benefit in the purification of the protein, which can otherwise cause tedious work.

a 3´-....TGA ACG TAC AGA CTT AGC GAT ATA GAC GGG CTC AGC TAG….-5´ gene 5´-TGA ACG TAC AGA CTT AGA GAT ATA GAC GGG CTC AGC TAG-3´ primer

c b P1 3´ 5´ P2 Gene in plasmid with target site

P4 5´ 3´ P3 From first PCR Plasmid is denatured and 5´ primers containing the mutation 3´ are annealed 3´ 5´ Nonmutated, parental DNA template is digested a 3´ 3´ DNA polymerase catalyzed c From second PCR extension of the gene, resulting in a nicked full length template

5´ 3´ 3´ Final full length gene containing the mutation 5´ Clone gene into a vector

Nonmutated, parental DNA template is digested

Transform the nicked dsDNA, Transform the dsDNA which will be repaired in the cell

E. coli P. pastoris

Transformed Genome vector Transformed vector, incorporated into the genome

Figure 2.2. a) Original gene and designed primer, containing a single point mutation. The site directed mutagenesis can for instance be performed either by b) overlap extension PCR or c) QuickChange® site-directed mutagenesis. d) Once the gene is modified and cloned into a vector it is transformed into a host cell. Transformation into E. coli leaves the free vector within the cell, whereas P. pastoris and other eukaryotic cells will incorporate the vector into the host genome.

10 α/β-Hydrolase Fold Enzymes

3 α/β-Hydrolase Fold Enzymes

As mentioned earlier, enzymes can be classified according to the reactions they catalyze. Another way to classify enzymes is according to their fold. The α/β-hydrolase fold family is one of the largest fold families (Figure 3.1.a) (Ollis et al., 1992, Holmquist, 2000, Bugg, 2004). Enzymes within this family share the same three-dimensional fold and have a very well preserved active site, which consists of a catalytic triad, nucleophile/histidine/acid, and an oxyanion hole (Figure 3.1.b). The nucleophile can either be a serine, a cysteine or an aspartate and it is found on the so called “nucleophilic elbow”, a short sequence of Gly-X- Nuc-X-Gly that is commonly found within the α/β-hydrolases. By having the histidine and the acid (aspartate or glutamate) coordinated to the nucleophile, the pKa of the nucleophile is lowered which enables the nucleophilic attack. Another important structural part in the active site is the oxyanion hole. This usually consists of two backbone amide protons, but there are a few exceptions, where a side chain hydroxyl group from either a serine or a threonine has been found to hydrogen bind to the oxyanion. The purpose of the oxyanion hole is to stabilize the oxyanion formed in transition state, after the nucleophilic attack. None of the enzymes in this fold family requires a cofactor to be catalytically active. This has made these enzymes interesting biocatalysts as it is a large benefit not to need the sensitive and costly cofactors (Holmquist, 2000, Bugg, 2004). The proteins within the family of α/β-hydrolases show large homology, indicating that they have undergone divergent evolution from one enzyme and then developed different activities, sometimes by very small changes. This has resulted in a large group of enzymes that can catalyze a large variety of reactions. Examples of enzymes that are found here are lipases, esterases, dehalogenases, epoxide hydrolases and C-C hydrolases. Although the name indicates that all these enzymes catalyze hydrolysis, that is cleavage by reaction with water, there are also enzymes that catalyze other types of reactions within this family. One example of this is hydroxynitrile lyase, which catalyzes a C-C bond cleavage to form hydrogen cyanide and a ketone (Figure 3.2). Other examples are bromoperoxidase, chloroperoxidase and 2,4-dioxygenase (Holmquist, 2000).

Figure 3.1. a) Candida antarctica lipase B, CALB, is an example of an enzyme that is found within the α/β-hydrolase fold family. b) In CALB the catalytic triad, nucleophile/histidine/acid, consists of Ser/His/Asp, and the oxyanion hole consists of two backbone amide protons and a threonine hydroxyl. 12 α/β-Hydrolase Fold Enzymes

Active site

O Nuc oxyanion O hole Acid O- H H N N His

H2O H2O H2O O O O OH OH 2 CN 1 R 1 2 - 1 2 R O R R R CO2 R R

h y C d e r s - o C s a x l e y s o h r n a y i r d d t e y r t r i l h o e s e l e a l y d s i a e x s s o e p e

O HO OH O OH O R2 HCN 1 HO 1 - - 1 2 R OH R R2 R O CO2 R R

Figure 3.2. Four of the reaction types catalyzed by enzymes within the α/β- hydrolase folding class are esterification, epoxide hydrolysis, C-C hydrolysis and C-C bond cleavage. The catalytic triad and the oxyanion hole are found in all these enzymes although their reaction specificity differs.

Within the group of α/β-hydrolases we can for example find many enzymes that are involved in everyday functions in our bodies. One very important enzyme that is found within this fold class is acetylcholine esterase. Acetylcholine is a neurotransmitter that is released to mediate nerve impulses in the body. Once it is released from one nerve cell and has forwarded the signal to the next it is fast hydrolyzed to acetyl and choline, by acetylcholine esterase. The enzyme, acetylcholine esterase, is covalently bound to the postsynaptic membrane, the impulse receiver, and is an example of an enzyme that shows kinetic perfection, having a kcat/KM-value close to the diffusion limit (Stryer, 1995). Another enzyme that is found within this fold family is for instance pancreatic lipase, which is active in the digestion of dietary triglycerides in humans. Pancreatic lipase requires a colipase to be active in the pancreatic juice, as the colipase stabilizes the open, active, conformation of the enzyme (Holmquist, 2000).

3.1 Candida antarctica Lipase B

Candida antarctica is a yeast that was first isolated in Antarctic, with the aim to find enzymes with extreme properties (Kirk & Christensen, 2002). It produces two different kinds of lipases, named A (CALA) and B (CALB) (Patkar et al. 1993). Although CALA shows many interesting qualities, not least a highly uncommon substrate specificity, CALB has been studied more often and is now well characterized. Lipases are known to catalyze the hydrolysis of triacylglycerols as their natural reaction. They also show interfacial activation, which means that their activity increases drastically when they enter a water/lipid interface (Sarda & Desnuelle, 1958, Verger & De Haas, 1976, Martinelle et al., 1995). Lipases are often also found to be very stable and can be used even in organic solvents. This has highly increased their use and lipases can now be found in many industrial applications (Houde et al., 2004). Although CALB share many of these features it differs in that it does not show any interfacial activation. CALB (Figure 3.1) possesses a great regio- and stereoselectivity (see chapter 1.1), which is used in the resolution of racemic alcohols, amines and thiols (Chapman et al., 1996, Öhrner et al., 1996). It shows a high stability in organic solvents, which together with its high stereoselectivity gives it properties which are difficult for many other catalysts to reach. This has made it a very useful catalyst and it can now be found in many industrial processes (Kirk & Christensen, 2002). It is for instance found in industrial applications such as the pharmaceutical (Fishman et al., 2001, Popp et al., 2004) and cosmetic industry (Houde, 2004) and it is also a good catalyst for reactions such as polymerizations (Mei et al., 2003, Mahapatro et al., 2004), and resolutions to prepare pure enantiomers (Costa et al., 2004). Some more specific examples of the use of CALB are in the production of the bronchodilator (R,R)-formoterol (Campos et al., 2000) and an azole antifungal compound against Candida and pulmonary Aspergillus infections (Zaks & Dodds, 1997). CALB is also used in large scale production of the pure enantiomers of ethyl-3-hydroxybutyrate, which both are important intermediates in the pharmaceutical industry (Fishman et al., 2001).

3.1.1 Protein Structure The three-dimensional structure of CALB was solved in 1994 and not long thereafter another structure was solved, containing an inhibitor bound in the active site (Uppenberg et al., 1994 & 1995). These showed that CALB has an α/β-hydrolase fold (Figure 3.1.a). The active site lies buried in a cavity, but unlike most other lipases it does not have a lid to cover the active site and does therefore not show interfacial activation. From the crystal structures it was found that there are two pockets which the substrate (substrate 2 in Figure 3.3) can occupy (Figure 3.1.b). The medium sized pocket (M) cannot fit a group larger than ethyl, whereas the size restriction in the large pocket (L) is almost unlimited. This means that for 14 α/β-Hydrolase Fold Enzymes enantiomers with one group larger than ethyl, only one of the two enantiomers can fit comfortably into the active site. This gives CALB its stereoselectivity.

3.1.2 Reaction Mechanism In a hydrolysis of a carboxylic ester (Figure 3.3), catalyzed by CALB, substrate 1 binds in the active site and the carboxylic carbon is attacked by the nucleophilic serine. The tetrahedral intermediate is stabilized by the oxyanion hole. An acyl enzyme is then formed as the alcohol, product 1, is released. A water molecule or an alcohol, substrate 2, then enters the active site and attacks the ester whereby a new tetrahedral intermediate is formed. In the final step the product (product 2) leaves the active site.

Free enzyme Tetrahedral intermediate 1 Substrate 1 Ser Ser105 105 O Oxyanion O hole O R2 O R1 O H O H R1 O O H H N N 2 N N O R O Asp Asp187 187 His His224 224

O R3 O R2 OH Product 2 Product 1

Ser105 Oxyanion Ser105 Oxyanion hole hole O O O O 1 H R O O R1 O H H N N N N O R3 O Asp Asp187 R3 OH 187 His224 His224 Substrate 2

Tetrahedral intermediate 2 Acyl enzyme intermediate

Figure 3.3. Reaction mechanism of CALB. The reaction is catalyzed by the catalytic triad (Ser-His-Asp) and an oxyanion hole, consisting of three hydrogen bonds.

3.2 Protein Engineering of Hydrolases

Mutagenesis is an important tool to give information about enzymes. The role of the oxyanion hole has for example been demonstrated by mutation studies. A single point mutation of CALB, Thr40Ala/Val, gives a large decrease in kcat/KM for esters. By using substrates, containing a hydroxyl group the mutants partly regain their activity and show stereoselective hydrolysis of ethyl hydroxy esters (Magnusson et al., 2001). Another example of how an enzyme has been engineered to get increased enantioselectivity is epoxide hydrolase. This enzyme was subjected to directed evolution. One mutant could be detected with increased enantioselectivity, after only one round of error-prone PCR. The enantioselectivity in the resolution of glycidyl phenyl ether increased from 4.6 for the wild type catalyzed reaction to 10.8 for the mutant. The mutant was found to contain three mutations, but only one was found close to the active site (Reetz et al., 2003). There is also an example of a mutant that was created to increase the thermostability of CALB, or more specifically to increase its resistance towards irreversible thermal inactivation. The best mutant showed an over 20-fold increase in half-life time at 70 °C, and was retrieved after two rounds of error- prone PCR (Zhang et al., 2003).

A single-point mutation of CALB has also been shown to give an enzyme with increased activity towards aldol additions (paper I and II), Michael additions (paper III) and epoxidations (paper V). This is yet another example of enzymatic promiscuity and will be further presented in the following chapter.

16 Rational Design of Candida antarctica Lipase B

4 Rational Design of Candida antarctica Lipase B

Aldol additions, Michael additions and epoxidations are examples of reactions that are of high importance in organic synthesis, as they provide important intermediates for instance in the food and pharmaceutical industries (Schmid et al., 2001, Lee et al., 2003). Research is constantly ongoing to find new activities from Nature. We have chosen to use the stable protein structure of a lipase as a scaffold to harbor new reaction mechanisms. In this chapter I will present how a single point mutation of Candida antarctica lipase B (CALB) gave increased activity for three studied reactions, highly different from the natural hydrolysis activity. Initially, molecular modeling of CALB (Figure 4.1.a) suggested that this enzyme could also be a good catalyst for other reactions than those earlier examined with the wild type enzyme. By removing the nucleophilic serine and using the remaining catalytic dyad His/Asp and the oxyanion hole it was suggested to be a good catalyst for aldol additions, Michael additions, and epoxidations. One part of the molecular modeling was quantum mechanical (QM) calculations, which were used to calculate the activation energies for the reactions. Two mutants, Ser105Gly and Ser105Ala (Figure 4.1.b, c), were created by site-directed mutagenesis and expressed in Pichia pastoris. In the initial study, activity was found for aldol additions (paper I). The highest activity was found with the Ser105Ala mutant, which was thereby chosen for further investigation. Further studies were made on aldol additions (paper II) and also on Michael additions (paper III), and epoxidations (paper V).

N Gln40 H R1 Oxyanion hole Ser105 H O O O H O HN Thr40 H R2 O N H N O Asp187 His224 a) Catalytic triad Ser/His/Asp Hydrolysis, esterification

N Gln N Gln40 40 H Ala105 H Ala105 H 1 O H R1 O O R O H H Thr Thr40 HN 40 HN Nu 2 R2 R H N H O O N N H H N O O Asp187 Asp His 187 224 His224 b) Ser105Ala mutant c) Ser105Ala mutant Aldol addition Michael addition and epoxidation

Figure 4.1. Structure of the active site in CALB a) wild type with an ester bound in the transition state, after attack of the serine 105, b) Ser105Ala mutant with an enolate (first substrate in an aldol addition) and c) Ser105Ala mutant with the proposed transition state of a Michael addition (Nu= S, N) or epoxidation (Nu = O).

4.1 Aldol Addition

One of the most challenging and important tasks in organic synthesis is asymmetric C-C bond formation. Aldol addition is an example of such a reaction and is found in the synthesis and break down of carbohydrates in Nature. Catalysts for these reactions are found both among bio-, organo- and organometallic catalysts (Machajewski & Wong, 2000, Notz et al., 2004). In the following chapter aldolases, a biocatalyst, will be discussed more in detail together with my own results.

4.1.1 Aldolases Aldolases are the enzymes found in Nature as catalysts in the coupling between a donor (usually a ketone) and an acceptor (an aldehyde), in reactions which show both high stereoselectivity and works under mild reaction conditions. Mechanistically aldolases are divided in two classes (Gefflaut et al., 1995). 18 Rational Design of Candida antarctica Lipase B

Class I aldolases are found in all kinds of cells, prokaryotes as well as eukaryotes. These enzymes are characterized by the formation of an immonium intermediate in the active site, between the donor substrate and an active site lysine (Figure 4.2.a). Class II aldolases are only found in prokaryotes and lower eukaryotes such as yeast, algae and fungi. In these enzymes the donor substrate forms an enolate, which is stabilized by a zink ion (Figure 4.2.b). The most stable aldolases are generally found among the class II aldolases (Gijsen et al, 1996). In both cases the donor substrate binds in the active site and attacks the acceptor substrate to form the final product.

a) b) His His Ser271 Lys229 Zn2+ His HO O O- O- HN 2- P O3PO OH O O OH H O Tyr O Lys107 O + H3N OPO 2- 2- 3 OPO3 OH OH

Figure 4.2. Active site of a fructose-1,6-bisphosphate aldolase from a) class I, showing the formed immonium intermediate between the donor substrate and the active site lysine and b) class II, showing the donor substrate enolate, stabilized by a zink ion. The attack on the acceptor substrate is shown in both cases (Machajewski & Wong, 2000).

Aldolases show high substrate specificity for the donor substrate, which leads to four main groups, namely dihydroxyacetone phosphate, pyruvate (or phosphoenol pyruvate), acetaldehyde and glycine. Although highly limited in their acceptance of donor substrate they show a much broader acceptance of acceptor substrates. Deoxyriose-5-phosphate aldolase has for instance shown to catalyze the sequential aldol addition of acetaldehyde (De Sanits et al., 2003). Fructose-1,6-diphosphate has been used in the synthesis of iminosugars and deoxythiosugars (Takayama et al., 1997).

4.1.2 Non-Enzymatic Catalysts In addition to the aldolases there are a number of other chemical catalysts for aldol additions, which are outside of the scope of this thesis. One example is proline, which is usually classified as an organocatalyst, but has also been called “the simplest enzyme” (Movassaghi & Jacobsen, 2002). Proline and derivatives have shown high stereoselectivity in the aldolization of various aldehydes and ketones (Notz et al., 2004, Casas et al., 2005, Cobb et al., 2005). Also catalytic antibodies, called abzymes, are used as catalysts in aldol additions and retro-aldol additions. These antibodies show broad substrate

specificity and can today for example be found as catalysts in the activation of prodrugs. Some catalytic antibodies, with aldolase activity, are today commercially available (Machajewski & Wong, 2000). The active site of these catalytic antibodies contains a lysine residue which forms an imine intermediate with the donor substrate, resembling the class I aldolases (Figure 4.2.a). Another group of catalysts that can be used are metal complexes, which can be used to activate either the donor (such as Rh and Pd complexes) or the acceptor (such as Sn, Ti and Cu complexes) substrate in an aldol addition (Machajewski & Wong, 2000).

4.1.3 A CALB Mutant as Catalyst for Aldol Additions The initial idea to use the oxyanion hole in CALB to stabilize the reaction intermediate in an aldol addition was tested by quantum mechanical (QM) calculations. The overall activation energies from these calculations were found to be close to 15 kcal/mol for acetaldehyde and 17 kcal/mol for acetone, which suggested that the reactions were possible and would give reaction rate in the same order as natural aldolases (paper I). Two CALB mutants Ser105Gly and Ser105Ala were created by site-directed mutagenesis. The remaining active site consisted of the histidine, the aspartate and the oxyanion hole (Figure 4.1.b). The histidine was expected to function as a base in the deprotonation and formation of an enolate, which would then be stabilized by the oxyanion hole. This resembled the active site of a class II aldolase (Figure 4.2.b). In the initial studies the wild type enzyme and the two mutants, Ser105Gly and Ser105Ala, were explored as catalysts for aldol additions. The Ser105Ala mutant showed the highest activity, four times that of the wild type enzyme and about 300 times over the background reactions in the aldolization of hexanal. To confirm that the reaction took place in the active site reactions were also run with covalently inhibited enzyme and with albumin. The background reactions showed almost no activity, which confirms the importance of the active site in the catalysis (Table 4.1 and paper I).

Table 4.1. Aldolization of hexanal, catalyzed by CALB wild type and mutants. Control reactions were examined to verify that the reaction took place in the active site. Catalyst Activity [µmol prod × h-1 × g-1 enzyme] Albumin 0.2 Inhibited CALB 0.3 Wild type 19 Ser105Gly 57 Ser105Ala 80

20 Rational Design of Candida antarctica Lipase B

The catalytic antibodies that have been used in aldol additions usually have an activity between 15-150 day-1 (Tanaka et al., 2004). This can be compared with our CALB mutant, Ser105Ala, which showed an activity of 65 day-1, for hexanal (paper I). Further comparison between these catalysts shows that the catalytic antibodies react with high enantioselectivity, which was not found with our CALB mutant. Catalytic antibodies on the other hand do not seem to be as stable in organic solvent as our CALB mutant, which was used in pure cyclohexane (Hoffmann et al., 1998, Machajewski & Wong, 2000, Tanaka et al., 2004). The reaction rates in the initial studies were found to be much lower than what was predicted by the QM calculations. To find the reason for that, further experiments were performed. New substrates were used in an attempt to find one that had a better binding and showed a better reactivity, preferably with some stereoselectivity. In this part of the project only one of the mutants, Ser105Ala, was examined. Neither of the substrates showed any increase in reaction rate (paper II), which led to further modeling studies to find an explanation to this. From docking studies and molecular dynamics it could be concluded that the substrate bound in the right position, but that the distance between the α-proton of the substrate and the active site histidine was longer (4.2Å) than that of a hydrogen bond. The molecular dynamic studies that followed were performed to give a more accurate picture of how often the α-proton came within the right distance from the histidine. The results were that only about 0.5% of the structures were within the right distance and the average distance was found to be about 6.2 Å (Figure 4.3 and paper II). This meant that although the activation energy was low enough for the reaction to occur with a fairly good rate, the frequency for this to happen was too low for an overall high reaction rate.

N Gln40 Ala105 H Oxyanion hole R1 O H H O HN Thr40 H R2

O N H N O Asp187 His224

6.2 Å

Figure 4.3. a) The first substrate is bound in the active site and the substrate α-proton is attacked by the histidine. b) A structure of the active site of the CALB mutant, Ser105Ala, with the two substrates (hexanal) bound. The distance between the histidine and the substrate α-proton is marked, which is longer than that of a normal hydrogen bond.

In an attempt to find an explanation to the even lower reaction rates that were found with the new substrates compared to that of hexanal, their α-proton acidity were calculated (Scheme 4.1 and Table 4.2) (Hilal et al., 2003). The substrates with the lowest pKa-values were those that showed reactivity (1-6). The values for those substrates were quite similar. From this it could be expected that they would all show similar reaction rates, but then also factors like steric hindrance and binding affinity has to be taken in consideration.

22 Rational Design of Candida antarctica Lipase B

O O HO R2 O R2 O 1 + 1 1 1 R 2 R 2 R + R R R R2 R2 R1 R1

Scheme 4.1. General reaction scheme for aldol additions. R1 and R2 are defined in Table 4.2.

Table 4.2. Comparison of the pKa values and the relative reaction rates for aldolization of the respective substrate. Reaction scheme in Scheme 4.1. 1 2 a Substrate pKa for R and R Relative rate (substrate/hexanal) (1) R1=Bu, R2=H 15.5 100 (2) R1=Me, R2=H 15.2 65 (3) R1=i-Pr, R2=H 15.6 7 (4) R1=Bn, R2=H 11.7 5 (5) R1=Bn, R2=Me 15.0, 17.5 3 (6) R1=H, R2=Me 17.6 1 (7) R1=Et, R2=Me 19.1, 17.6 b (8) R1=Et, R2=Et 19.0, 18.9 b (9) R1=Pr, R2=Et 19.1, 18.9 b a Total product formation (aldol and α,β-unsaturated product), bthe reaction rate was calculated to be less than 0.03 relativ that of hexanal (1).

An unexpected result was that the reaction took place without any enantioselectivity. A shift in the diastereoisomeric ratio was detected compared to the uncatalyzed reaction, for hexanal (Figure 4.4). This shows that the enzyme catalyzed a stereoselective reaction, but suggests that two different binding modes are preferred. The substrates in the continuing study were chosen also to increase the chance to give stereoselective reactions (Table 4.2 and paper II). However, neither of these showed any enantioselectivity. No good explanation to this could be given, but a speculation was that the size of the active site allowed for the substrate to bind in more than one way.

TIC: C3XN304A.D 150000 140000 Products without enzyme Products without enzyme 130000 120000 On achiral column On chiral column 110000

100000

90000

80000

70000

60000

50000

40000

30000

20000

10000 36.50 36.60 36.7036.80 36.9037.00 37.10 37.2037.30 37.40 37.50 Time--> Products with enzyme Products with enzyme Abundance On achiralTIC: column C3XN306A.D On chiral column 280000

260000

240000

220000

200000

180000

160000

140000

120000

100000

80000

60000

40000

20000

30.2030.3030.4030.5030.6030.7030.8030.9031.0031.1031.2 Time-->

Figure 4.4. The aldol products were detected in enantiomeric pairs by analysis on GC, using both chiral and nonchiral column. The reaction was found to be stereoselective, with one enantiomeric pair formed faster by CALB-mutant catalysis.

In conclusion it can be said that the CALB mutant, Ser105Ala, gave an increased activity for aldol additions, compared to that of the wild-type enzyme. The low reaction rate was probably a consequence of lack of optimal geometry in the active site, while the general idea of the proposed reaction mechanism still holds.

4.2 Michael Addition

Michael addition was originally the name for conjugate additions of enolate ions to α,β-unsaturated carbonyl compounds. Nowadays Michael addition is used for reactions giving a 1,4-addition of any nucleophile to an α,β- unsaturated carbonyl compound (Scheme 4.2).

R O 3 Nu O R +NuH 3 R4 R1 R4 R1 R2 Nu= thiol, amine and alcohol R2

Scheme 4.2. General reaction scheme for Michael additions. The nucleophile is added to the α,β-unsaturated substrate in a 1,4-addition mode.

24 Rational Design of Candida antarctica Lipase B

This type of reaction is fundamental in organic synthesis, such as in the synthesis of compounds with a thioether linkage as well as in the formation of new carbon-carbon bonds (Wabnitz & Spencer, 2003, Yao et al., 2004). Michael additions are also of interest in the synthesis of N-substituted imidazoles (Cai et al., 2004 a,b).

4.2.1 Catalysts for Michael Additions Michael additions are not the most commonly found reactions in Nature but there are a few enzymes that show Michael addition activity as part of their natural activity, for instance some in baker’s yeast (Bertolli et al., 1981, Tomoya & Nobuo, 1984). More specific enzymes that have been used are O-acetylserine sulfhydrylase, a pyridoxal 5´-phosphate dependent enzyme (see chapter 5), which catalyzes a Michael addition in the synthesis of cysteine (Tai & Cook, 2001). Hydrolases have been used as catalysts in Michael additions (Kitazume et al., 1986, Cai et al., 2004 a,b, Torre et al., 2004, Yao et al., 2004) and thymidylate synthase is known to catalyzes the final step in the synthesis of the nucleotide dTMP (Ivanetich & Santi, 1992). Other catalysts, which in addition to aldol additions, catalyzes Michael additions are proline and derivatives thereof. These reactions proceed with high activity and in some cases also with high stereoselectivity (Notz et al., 2004, Cobb et al., 2005).

4.2.2 A CALB Mutant as Catalyst for Michael Additions Michael additions were the second reaction type to be investigated with the CALB mutant, Ser105Ala, next after aldol additions. From the initial QM calculations for Michael additions the activation energy was found to be about 12 kcal/mol, for methanethiol and acrolein. The experimental results that followed gave reaction rates in the range of 10-3 - 4 min-1 and the enzyme 7 proficiency ((kcat/KM)/knon) reached 10 , which is similar to natural enzymes (paper III). The reactions gave high yields, but no stereoselectivity could be detected. The reaction mechanism predicted for the Michael addition was that the histidine abstracts the proton from the nucleophile the nucleophile attacks the β-carbon on the α/β-unsaturated substrate, bound in the active site. This gives a transition state that is stabilized by the oxyanion hole (Figure 4.5.a), and next the final product is formed. In comparison to the long distance between the histidine and the substrate α- proton in an aldol addition (Figure 4.3), the distance to the thiol in a Michael addition in much shorter (Figure 4.5). This can partly explain for the higher reaction rate detected in the Michael addition (paper I and III).

N Gln40 Ala105 H H 1 O O R H HN Thr40 H Nu O N H N O Asp187 His224

4.5 Å

3.0 Å

Figure 4.5. The Michael addition takes place between the nucleophile and the carbonyl β-carbon, after activation by histidine 224. a) Shows the nucleophilic attack and b) shows the three-dimensional view of the substrates bound in the active site. The distance from the thiol to the histidine and the carbonyl β-carbon is shown.

In the Michael addition as well as the aldol addition the histidine, His224, had to be in its unprotonated state when the reaction started (Figure 4.5). A study of the reaction rates of Michael additions, catalyzed by enzyme that had been immobilized at pH 7.6 and 8.6, respectively, showed that the activity increased with a higher pH (paper III). The reaction rate was increased more than 10 times for the reaction between pentane-2-thiol and but-2-enal when the immobilization pH was increased from 7.6 to 8.6, for the Ser105Ala mutant (Figure 4.6). The reason for why this effect was not seen for the wild type could possibly be explained by that the nucleophilic serine, in the wild type, hydrogen binds to the histidine and thereby lowers its pKa. This hydrogen bond is not possible in the mutant. 26 Rational Design of Candida antarctica Lipase B

In conclusion the Michael additions were found to be successfully catalyzed by the CALB mutant, Ser105Ala, and the catalyst shows broad substrate specificity.

100 90 Ser105Ala, pH 8.6 80 Wild type, pH 8.6 70 Wild type, pH 7.6 60 Product 50 formation / % 40 Ser105Ala, pH 7.6 30 20 10 0 0102030 Time / days

Figure 4.6. Michael addition of 2-pentanethiol to 2-butenal in cyclohexane, catalyzed by C. antarctica lipase B Ser105Ala mutant (●,○) and wild-type (■,□). The bottom five curves show control experiments with imidazole (×), empty carrier (▲,∆), inhibited wild-type (◊) and the background reaction with substrates only (+). Filled and open symbols denote immobilization of enzyme or prewashed carrier with phosphate buffer of pH 8.6 or pH 7.6, respectively (paper III).

In the aldol addition one of the products obtained was the α,β-unsaturated carbonyl compound, the dehydrated aldol product. This compound can be a substrate in a Michael addition. To exploit this, aldol addition and Michael addition were run in a sequential reaction to form the Michael type product, starting with a saturated aldehyde and thiol (Scheme 4.3).

EtSH OH O O S O O abc CALB 1 molecular 2 CALB 3 Ser105Ala sieves Ser105Ala

2standard 13

Scheme 4.3. A sequential, one-pot, three step reaction of (a) aldol addition (b), dehydration and (c) Michael addition. The gas chromatogram identifies the products (1-3).

4.3 Epoxidation

Epoxides are compounds that contain a three-membered ring, including one oxygen atom. As three-membered rings are highly strained they are very reactive toward nucleophilic substitutions and can undergo stereospecific ring- opening to give bifunctional compounds. These compounds can in turn be used as intermediates in the preparation of enantiomerically pure bioactive compounds, for instance as intermediates for pharmaceuticals and fine chemicals (Wang et al., 2003). Epoxides can for example be formed from simple alkenes, α,β-unsaturated carbonyl compounds, and unsaturated fatty acids among others, using hydrogen peroxide (Scheme 4.4). Hydrogen peroxide is though a slow oxidant and it needs to be activated in order to be an efficient oxygen donor.

R2 R1 R2 + OH - H2O HO R1 catalyst O

Scheme 4.4. General reaction scheme for epoxidation of an alkene, using hydrogen peroxide.

4.3.1 Catalysts for Epoxidations There are today both chemical and biological catalysts available for epoxidations. Enzymes that naturally catalyze the epoxidation reactions are monooxygenases, which can catalyze epoxidation reactions of alkenes both with high regio- and stereospecificity (de Visser et al., 2002, Bernasconi et al., 2004). Also haloperoxidases, or more specifically chloroperoxidases, catalyze

28 Rational Design of Candida antarctica Lipase B stereoselective epoxidation of alkenes (Zaks & Dodds, 1995, Dembitsky, 2003). Epoxides can be synthesized in a reaction where hydrogen peroxide first reacts with a carboxylic acid to form a peroxy acid. This can be catalyzed by CALB (Björkling et al 1992, Lau, et al., 2000). In the following step, the peroxy acid reacts with an α,β-unsaturated compound, such as an alkene or an olefin to form an epoxide. This step is not reported to be enzyme catalyzed, but it is the rate limiting step in the reaction. Bicarbonate has shown to be an efficient activator of hydrogen peroxide and is used in epoxidations, resulting in high yields and small amounts of diols can be found only in a few cases. The benefit with bicarbonate, compared to other non-enzymatic catalysts, is that there are no toxic effects from the catalyst, but the drawback is that such system does not give any stereoselectivity (Yao & Richardson, 2000). The low enantiomeric purity in epoxidations is also a drawback with organic peroxy acids (Besse & Veschambre, 1994). Transition metal ligands, such as molybdenum and vanadium, are also used as catalysts for epoxidations and give good yield and high enantiomeric purity. Their substrate specificity is though narrow (Besse & Veschambre, 1994).

4.3.2 A CALB Mutant as Catalyst for Epoxidations Earlier reports show a sequential reaction where peroxide first reacts with a carboxylic acid in an enzyme–catalyzed peroxidation. The peroxy acid that is formed then reacts with an alkene to form an epoxide. The reaction mechanism proposed in figure 4.7 is on the other hand a direct lipase-catalyzed epoxidations.

H2O2 O Asp Asp187 R2 R1 187 O O Ala Ala105 105 R1 O O O Oxyanion H H N N N N H hole O His His224 224 O R2 Free enzyme H

O R2 R1 O H2O Asp187 O 1 Ala105 R O O Oxyanion H N NH hole R2 O His224 O H

Figure 4.7. Proposed reaction mechanism for epoxidation, catalyzed by a CALB mutant, Ser105Ala.

In this study CALB wild type and a mutant, CALB Ser105Ala, were used as catalysts in epoxidations of α,β-unsaturated carbonyl compounds. Hydrogen peroxide was used as oxygen donor, without extra addition of a carboxylic acid (paper V). The hypothesis behind this was that the α,β-unsaturated carbonyl compound would be activated by binding into the oxyanion hole, while the hydrogen peroxide would be activated by the active site histidine (Figure 4.7). The experimental work was in line with the hypothesis and the highest activity was found in the mutant catalyzed reaction, which further strengthened the hypothesis. It was concluded that the reaction mechanism in this case did not go via a peroxy acid as presented in earlier reports (Björkling et al., 1992, Skouridou et al, 2003), as the mutant, lacking the nucleophile, does not retain any hydrolysis activity. This led to the conclusion that the epoxidation was directly catalyzed by the lipase, according to the reaction mechanism presented in figure 4.7. 2-Butenal showed the highest reaction rate of the examined substrates, and also the highest yield 15 and 37%, after 620 h, for the wild-type- and Ser105Ala-enzyme, respectively. Also cinnamaldehyde and 2- cyclohexenone reacted (Table 4.3 and paper V). The rate enhancement was calculated to be 2.5×104 in the mutant catalyzed reaction of 2-butenal (Table 4.3). No stereoselectivity could be found here, as in the aldol- and Michael additions. This can possibly be explained by the relatively large active site that makes it possible for the nucleophilic attack to come from either side of the substrates.

Table 4.3. Calculations of the catalytic efficiency.

Substrate uncat WT Ser105Ala

[min-1M-1][min-1][[M] min-1] [M]

2-butenal 5.0×10-4 0.80 1.6×104 1.2 2.5×104 cinnamaldehyde 0.50 0.041 0.083 0.045 0.090 cyclohexanone 0.17 0.16 0.98 0.11 0.66 1-butene-3-one n.d. n.d. n.d. 2-methyl-2-pentenal n.d. n.d. n.d. n.d. = not detectable

30 Rational Design of Candida antarctica Lipase B

4.4 Summary

The CALB serine mutant Ser105Ala showed an increased activity for three promiscuous reactions, aldol addition, Michael addition and epoxidation. At the same time the natural activity, hydrolysis, was drastically decreased (Table 4.4).

Table 4.4. Data where the largest difference in activity between wild-type and mutant enzyme (paper I-III and V) was found. Reaction WT Ser105Ala

[h-1] [h-1]

hydrolysis 1.1×106 n.d. aldol addition 0.80 3.0 epoxidation 49 75 Michael addition 1.0×10-3 1.7 n.d. = not detectable

32 PLP-Dependent Enzymes

5 PLP-Dependent Enzymes

Vitamins and vitamin derivatives play important roles in all cells. Some vitamin derivatives work as cofactors in different enzymes. Pyridoxal 5’- phosphate (PLP) is the bioactive derivative of vitamin B6, pyridoxine. The biosynthesis of PLP occurs in plants and microorganisms, whereas humans must obtain it from dietary sources. PLP is found as cofactor in enzymes that catalyze various interconversions of α-amino acids, and in the synthesis of amino sugars (Eliot & Kirsch, 2004). It can also function as a catalyst itself, in its free form (Tsai et al., 1978, Pugnière et al., 1983, Jacob III, 1996). Within the group of PLP-dependent enzymes enzymatic promiscuity is very common; that is, the enzymes often show activity for more than one reaction (Percudani & Peracchi, 2003).

5.1 PLP as Cofactor

The cofactor, PLP, has been found as cofactor of enzymes in five of the six main enzyme classes and in over 140 enzymes (John, 1995, Toney, 2005). Thereby it is outstanding among all cofactors in the diversity of reactions it is involved in (Eliot & Kirsch, 2004). Some examples of PLP-dependent enzymes are racemases, transaminases, decarboxylases, synthases and aldolases. With only a few exceptions they all catalyze reactions involving amino acids and only the PLP-dependent phosphorylases do not directly involve a reaction between a substrate amine and a PLP carbonyl (John, 1995, Percudani & Peracchi, 2003, Eliot & Kirsch, 2004). All living cells contain PLP-dependent enzymes although their most pronounced abundance is found in prokaryotes where it is found in about 1.5% of all enzymes (Percudani & Peracchi, 2003). Being more common in prokaryotes, it has been said to be involved mainly in the fundamental reactions. It is also of high importance in eukaryotes, such as human cells. Decarboxylases are for instance involved in the biosynthesis of the neurotransmitters γ-aminobutyric acid (GABA) and serotonin and deficiency can cause depression and anxiety (McCarty, 2000). The PLP-dependent enzymes are also common drug targets, such as γ-aminobutyric acid aminotransferase in the treatment of epilepsy and serine hydroxymethyltransferase in cancer therapy (Eliot & Kirsch, 2004).

The PLP-dependent enzymes have been grouped in different ways. Christen and his coworkers grouped the enzymes in three families, α, β, γ, depending on at which atom the final reaction occurs (Alexander et al., 1994). Another way to group the enzymes is based on sequence and structure analysis. This has so far resulted in five fold-classes (Eliot & Kirsch, 2003).

5.2 Racemases and Transaminases

Among the amino acids the L-enantiomers are the most commonly occurring in Nature, but also the D-enantiomers of amino acids are known to have important functions. For instance, D-alanine and D-glutamate are found in the peptidoglycan layer of the bacterial cell wall. D-Serine is a signaling molecule in mammalian brains and D-aspartate is a mediator in the endocrine system (Yoshimura & Esaki, 2003). D-Amino acids are also used as intermediates in the production of pharmaceuticals, food additives and agrochemicals (Yagasaki & Ozaki, 1998). The interconversion between the two enantiomers of an amino acid to provide both enantiomers is catalyzed by racemases. Transaminases are important in the biosynthesis and metabolism of amino acids. Depending on the position of the reacting amine the transaminases are divided into α- and ω-transaminases (Shin et al., 2003). ω-Transaminases show a broad substrate acceptance and have in addition to amino acids shown activity for amines. They have therefore been used in the production of chiral amines (Shin & Kim, 1997).

5.2.1 Reaction Mechanism With the exception of the PLP-dependent phosphorylases all PLP-dependent enzymes share the same first reaction step. In the resting state PLP is found covalently bound to a lysine as an internal imine in the enzyme active site (Scheme 5.1.a). This makes it more reactive than the free PLP (John, 1995). During the reaction PLP is instead bound to the substrate amine group as an external imine (Scheme 5.1.b). From here the reactions will take different routes depending on the conformation of the formed imine complex. The bond to be broken will be in a plane perpendicular to the PLP-imine π-system, according to the Dunathan hypothesis (Dunathan, 1966 & 1971). Figure 5.1 shows the conformation for a racemization or transamination with the α-proton perpendicular to the PLP-imine π-system. As both racemizations and transaminations will show the same configuration at this stage there must be something else to determine which of the two reactions that is to take place and the protonation state of the pyridine nitrogen has shown to be an important factor. The protonation state is in turn dependent on which amino acid that is coordinated to the pyridine nitrogen (marked with an asterisk in Scheme 5.1.a). In transaminases there is either an aspartate or a glutamate, hydrogen bonding to the pyridine nitrogen, which will stabilize the quinonoid intermediate, and

34 PLP-Dependent Enzymes

the proton will be placed on the C4’ (Scheme 5.1.c). Hydrolysis of the structure that has gone through the proton transfer will result in transamination. Alanine racemase on the other hand has an arginine coordinated to the pyridine nitrogen which will therefore not be protonated and the quinonoid will not be stabilized (Sun & Toney, 1999, Yow et al, 2003, Eliot & Kirsch, 2004). It has been suggested that the racemization does not go through a quinonoid intermediate, which has also been shown by the missing absorbance at the region between 480-530 nm (Watanabe, et al., 2002, Spies & Toney, 2003). The reprotonation of the Cα can then come from either face of the imine and thereby form a racemate (Scheme 5.1.d).

-2 OPO3

HN CO2 N H H3C OH R α-carbon (stereocenter) planar

Figure 5.1. According to the Dunathan hypothesis (Dunathan, 1966 and 1971) the bond perpendicular to the coenzyme-imine π-system will be broken. In this figure the α-proton will be broken, resulting in racemization or transamination. The top figure shows the imine molecule, and the bottom figure shows the same molecule seen from the side.

5.3 Alanine Racemases

Alanine racemase was first identified in Streptococcus faecalis (Wood & Gunsalus, 1951). It’s first and for a long time only known function was to provide D-alanine to the peptidoglycan layer in the bacterial cell wall (Shaw et al., 1997, Nomura, et al., 2001). Therefore, alanine racemase has been selected as a target in the search for an inactivator that can be used as an antibacterial agent against for instance pneumonia, tuberculosis and osteomyelitis. Unfortunately the inhibitors found so far show unspecific inhibition also of other PLP- dependent enzymes (LeMagueres et al., 2003 and 2005). Alanine racemase has also been found in eukaryotes, such as fungi and in the animal kingdom. The first report of an alanine racemase from a non-bacterial source was from the fungus Tolypociadium niveum. Here it is a key enzyme in the biosynthesis of the cyclic undecapeptide cyclosporin A, which has immunosuppresive properties (Hoffmann et al., 1994). 35

Scheme 5.1. a) In the resting state PLP is bound to an active site lysine through an imine bond. b) The different possibilities of bond breaking to perform different reactions are shown. (I) In the case where the imine is deprotonated, the reaction route will be either transamination or racemization. c) Transamination is found when the pyridine-nitrogen is protonated and a quinonoid is formed, whereas in d) racemases the pyridine-nitrogen will not be protonated. Also the route for α-decarboxylation (II) and retro aldolization, here serine hydroxyl # methyltransferase (III) are shown. * Denotes the pyridine nitrogen (5.1.a) and marks the C4´ (5.1.c). 36 PLP-Dependent Enzymes

5.3.1 Protein Structure Alanine racemase in prokaryotes are found either as a monomer (Wasserman et al., 1984, Badet & Walsh, 1985, Esaki & Walsh, 1986) or a dimer (Inagaki et al., 1986, Tanizawa et al., 1988, Shaw et al., 1997). The eukaryotic enzymes on the other hand have been found to be trimeric or tetrameric (Hoffmann et al., 1994, Nomura et al., 2001). Although the structure of the active enzymes looks differently in different cells they are all built of relatively uniform monomers, about 40 kDa in size (Nomura et al. 2001). The dimeric alanine racemase from Geobacillus stearothermophilus belongs to fold type III, of the PLP-dependent enzyme folds. Each subunit consists of two domains, one eight stranded α/β barrel at the N-terminal and the other consists of three β-sheets at the C-terminal. In the dimer the N-terminal of one monomer interacts with the C-terminal of the other. Each dimer also contains two active sites, both located between the two monomers (Figure 5.2.a) (Shaw et al., 1997). Dissociation studies have shown that the monomer gives a non active protein (Toyama et al., 1991). Each active site has a PLP bound to a lysine in the resting state, the same lysine is involved in the proton transfer in the racemization reaction, together with tyrosine 265 (Figure 5.2.b). Water molecules have also been identified in the dimeric structure as important structure stabilizers (Mustata & Briggs, 2004).

5.3.2 Reaction Mechanism The structure and active site of alanine racemase has been well studied. In Geobacillus stearothermophilus Lys39, the PLP-binding residue, and Tyr265 are known to be the catalytically active amino acids. This has also been shown by mutations (Watanabe et al., 1999 a,b). A requirement to perform the racemization is that the proton can be transferred between the two catalytically active residues, Lys39 and Tyr265. There have been many suggestions for how this is carried out. One suggestion has been that water molecules in the active site are responsible for supplying the required protons to the catalytically active amino acids (Spies & Toney, 2003, Mustata & Briggs, 2004). Later studies have shown that no water molecule is at the right position to perform proton transfer. Another possible solution includes the substrate carboxylate in the proton transfer (Scheme 5.2) (Watanabe et al., 2002). This mechanism has not yet been fully accepted even though further experiments have been presented which agrees with this mechanism (Spies et al., 2004). By studying the crystal structure Esaki and coworkers suggested that the carboxylate can rotate freely in the active site to reach both bases (Watanabe et al., 2002), which disagrees with the suggestion that Arg136 participates in the substrate binding by hydrogen binding to the substrate carboxylate (Stamper et al., 1998).

Figure 5.2. Structural view of alanine racemase a) consisting of two monomers, shown in grey and purple, respectively. b) The active site shown in a close up. The catalytically active residues Lys39 and Tyr265 are shown, located on each side of the PLP-alanine complex. Arg219 is shown hydrogen bound to the pyridine nitrogen. 38 PLP-Dependent Enzymes

Lys Tyr´265 H 39 Lys39 O Tyr´265 H H2N O Tyr´ O H2N 265 H H O Lys39 O OH2 : O N N H H N O O H O O O O P O O P O O O O P O O O N N O : : N

O :

NH2 L-Ala O

H O

NH2 D-Ala Tyr´265 Tyr´265 O O O OH

H : O O H H H N N Lys N :N Lys39 H 39 H O H O H O O O P O O P O O O N

N :

Scheme 5.2. The latest suggestion to the reaction mechanism of alanine racemase involves the substrate carboxylate in the proton transfer between the two bases. The two bases are Lys39 and Tyr’265 and come from each of the two monomers. With this reaction mechanism there is no quinonoid intermediate formed (Watanabe et al., 2002).

5.4 Protein Engineering of PLP-Dependent Enzymes

There are many reports on how the PLP-dependent enzymes have been mutated to change substrate- or reaction specificity, and also to cause other changes to the protein, for instance to make them more thermostable. A few examples of all these reports will follow here, not least to illustrate that it really is fine tuning to give the proteins their high specificity and that minor changes can cause dramatic differences. Alanine racemase from Geobacillus stearothermophilus has been mutated to identify the catalytic residues (Kurokawa et al., 1998, Sun & Toney, 1999, Watanabe et al., 1999 a,b). A single point mutation of the catalytic base, Tyr265, is enough to turn the racemase into a transaminase by completely abolish its racemization activity whereas the transamination activity is increased at least four-fold (Watanabe et al., 1999a). In another report the same mutant, Tyr265, is shown to give a 2.3×105-fold increase in retroaldol activity, compared to that of the wild type enzyme (Seebeck & Hilvert, 2003). Alanine racemase shows very high substrate specificity and has in addition to alanine only shown minor activity for serine. Patrick et al (2002) have shown that tyrosine (Tyr354) plays an important role in this strict substrate specificity. A single point mutation of

Tyr354 for alanine, asparagine or glutamine gave an increased specific activity for racemization of serine with a factor of 50 to 80. Aspartate aminotransferase also shows promiscuity in that it catalyzes racemization as a side reaction. A single point mutation is enough to increase its racemization activity seven times and at the same time decrease the transamination activity six-fold (Vacca et al., 1995). With a triple mutation Christen and coworkers turned aspartate aminotransferase into an L-aspartate β-decarboxylase. The mutated amino acids are hydrogen bonding to the substrate’s α-carboxylate group, to the cofactor phosphate and the distal carboxylate of the substrate. kcat/KM for the decarboxylation is found to increase over 180 times with the best of the triple mutants, compared to the wild type. At the same time the transamination decreases with a factor of 1.3×105 (Graber et al., 1999). Ornithine decarboxylase is yet another example, which in addition to its decarboxylase activity catalyzes a decarboxylation-dependent transamination as a side reaction. A single point mutation of this enzyme increases the side reaction activity 200-fold, whereas the decarboxylation activity decreases 600-fold (Jackson et al., 2000). The psychrophilic (heat sensitive) alanine racemase from Bacillus psychrosaccharolyticus has a higher catalytic activity than the thermostable alanine racemase from Geobacillus stearothermophilus. In an attempt to increase the thermostability of the psychrophilic enzyme, a double point mutation was made to decrease the flexibility of the active site, which has been suggested to cause its naturally low thermostability. The mutant shows a higher activity up to 50 ºC and also the KM is markedly affected and decreased from 5.0 µM to 0.23 µM, despite that the mutation sites were located on the protein surface (Yokoigawa et al., 2003).

5.5 Activity and Stability of Mutants of an Alanine Racemase

The idea of this work was to change the substrate specificity of alanine racemase from Geobacillus stearothermophilus. The substrates of interest were amines, which differs from amino acids in that they do not contain an α- carboxylate. Considering the nature of amino acids and amines it was concluded that the active site of alanine racemase would need to be more hydrophobic to accept amines. From the suggested reaction mechanism (Figure 5.3) (Watanabe et al., 2002), it was also assumed that a group for proton transfer between the two bases was needed, in the absence of the substrate α- carboxylate. The protein crystal structure was studied to find the targets to mutate. Arginine 136 was identified to hydrogen bind to the substrate α- carboxylate and was therefore targeted and mutated for alanine, to increase the hydrophobicity in the active site. The best position to introduce a carboxylate, to facilitate the proton transfer, was found to be Cys311 (Figure 5.3), which was mutated for glutamate. A double mutant containing both these mutations, 40 PLP-Dependent Enzymes

Arg136Ala/Cys311Glu, was also made to create an enzyme which both had the ability to accept amines and could perform the proton transfer between the two bases in a racemization reaction. Finally a triple mutant was created, where also Tyr43 (Figure 5.3) was mutated for a histidine. This tyrosine hydrogen binds to the PLP-phosphate. A mutant where both this hydrogen bond and that between Arg136 and the PLP-hydroxyl was removed was considered, to study the effect on the protein thermostability.

Figure 5.3. Active site of alanine racemase, pointing out the targeted amino acids, Tyr43, Arg136, and Cys311. The catalytically active Lys39 and Tyr265 are shown in grey, together with the alanine-PLP complex, and Arg219 hydrogen bound to the pyridine nitrogen.

Alanine racemase was cloned from G. stearothermophilus and mutations were introduced into the host strain, using QuickChange® mutagenesis. Protein was expressed in E. coli and examined for activity. All proteins had activity for racemization of alanine, the single mutant Arg136Ala as much as 74%, compared to the wild-type enzyme (Figure 5.4 and paper IV). This alanine racemase is known to be thermostable and a frequently used purification step is to incubate the cell extract at 70 °C for 1 h, to denature other proteins (Inagaki et al., 1986). This procedure was performed for the expressed proteins, to examine the heat stability of the mutants, compared to the wild-type enzyme. Activity assays, after incubation, showed that all mutants had a decreased thermostability. Only the single mutant Cys311 retained activity, 8% of the initial activity (Figure 5.4 and paper IV).

4,0 3,5 3,0 n] i 2,5 e 2,0 g prot / 1,5 U [ 1,0 0,5 0,0 after incubation a l

WT before incubation A , u l la u , l a, G l is 1 G A 1 H 1 36A s311Glu 3 1 Arg136 4 g1 r ys3 r Cy ys3 C A Ty C Arg136

Figure 5.4. Specific activity was measured for alanine racemase wild type and mutants, before and after incubation at 70 °C for 1 h (paper IV).

In both racemization and transamination of amines and amino acids, a proton has to be abstracted (Scheme 5.1). The pKa of these protons on alanine and 2-butylamine are 34 and 54, respectively. To facilitate the deprotonation the acidity of these protons need to be decreased. Pyridoxal 5’-phosphate (PLP) has shown to be very efficient for this and is used by many enzymes. By forming the imine between PLP and the amino groups of alanine and 2-butylamine the pKa values decrease to 21 and 39, respectively. To further increase the possibility of deprotonation, the imine-nitrogen is found to be protonated, which further lowers the pKa. The enzyme also adds to this effect, by extra stabilization of the intermediate and the result is an efficient enzyme which catalyzes the deprotonation. The experimental work on racemization of amine, catalyzed by wild type and mutants, showed no activity. A possible conclusion from this could be that the acidity of the proton to be abstracted was not low enough, even when bound in the active site. Another explanation could be that the proton transfer between the two bases was not optimal, even with the introduced carboxylate in the active site. A known side reaction of alanine racemase is transamination, although this activity is low for the wild-type enzyme. Alanine racemase, wild type and mutants, were examined for transamination activity, not only for alanine, but also for 2-butanone. This resulted in a continuous system of transamination, which could be analyzed both on formation of pyruvate and amine (Scheme 5.3).

42 PLP-Dependent Enzymes

λ 340nm OH NADH NAD NH2 O Lactate CO2 CO 2 CO2 dehydrogenase

O NH2 O O OH OH P OH P OH O O O O

N N H H

NH 2 O analyzed on GC for stereoselective transamination

Scheme 5.3. Reaction scheme for the continuous transamination of alanine and 2- butanone. The reaction process can be analyzed both by detecting produced pyruvate (spectrophotometry) and 2-butylamine (GC) (paper IV).

From the experiments that were performed it was concluded that a single mutation, where a hydrogen bond donor to the substrate carboxylate was removed, was enough to increase the binding affinity for ketones. This could be detected by following the transamination of ketone and alanine and compare the different catalysts (Table 5.1 and paper IV). The formed amine was identified by GC-MS, although the concentrations were too low to identify any possible stereoselectivity. Transamination of 2-butanone is though not expected to take place with stereoselectivity, due to two main reasons. 2-Butanone is most likely able to bind in both directions in the active site and as the enzyme contains two bases, the proton abstraction and replacement could come from either side of the substrate (Figure 5.2.b). The final conclusion was that the alanine racemase mutant, Arg136Ala, had an increased affinity for ketones, which could bind into the active site and react in a continuous transamination together with alanine.

Table 5.1. Transamination of alanine and 2-butanone, catalyzed by alanine racemase wild type and mutants. The reactions were run with 100 mM substrate and at different temperatures. 37 °C 60 °C [µmol × min-1 × g-1 protein] [µmol × min-1 × g-1 protein]

WT 0.0050 0.0050 Arg136Ala 0.055 0.0047 Cys311Glu 0.0070 0.002 Arg136Ala/Cys311Glu 0.0030 n.d. Tyr43His/Arg136Ala/Cys311Glu n.d. n.d. n.d.= not detectable

Conclusions

Many enzymes are today known to catalyze also other reactions than their natural, although usually with much lower activity, called enzyme promiscuity. Throughout this thesis, several examples of enzyme promiscuity have been presented, from my research as well as from the literature. It has also been presented how enzymes with very small changes can be given new functionalities. Sometimes a single-point mutation can be enough to change the reaction specificity so a side reaction becomes the main activity. In Nature this is known as divergent evolution, and has resulted in a large number of enzymes with high homology. In this thesis I have presented how a yeast lipase, Candida antarctica lipase B, was targeted by site-directed mutagenesis to remove the catalytic serine, in order to increase its activity for three reactions that are found as side reactions in the wild type enzyme. The mutated enzyme catalyzed aldol additions, Michael additions and epoxidations of substrates that cannot be catalyzed by the natural enzymes. The second enzyme explored was alanine racemase from Geobacillus stearothermophilus. A single mutant, where the substrate carboxylate- binding arginine was mutated for an alanine, gave increased transamination activity for 2-butanone and alanine. There are today many examples where enzymes are used as catalysts in industry. Rational design is an established strategy to optimize the performance of such catalysts. With the increased interest in enzyme promiscuity and the challenge to imitate Nature’s own way to create diversity, enzyme catalysis will be of even larger use in the future.

Acknowledgements

Well, finally it’s time for the last page to be written. There are quite a few who I am very grateful to and who should have a special thanks on this page:

Professor Kalle Hult, for accepting me as a PhD student in the group being my supervisor. You have been great support, always full of ideas and ready to discuss.

Docent Per Berglund, my supervisor, always ready to help when I got stuck and good support. An extra thanks also for all the help now when I’ve been writing my thesis and needed extra help.

Park, thank you for all the help during my last year as a PhD student. Not only did you tell me all I know about molecular biology, but you always had a minute over to help out and discuss and come with good ideas.

Marianne for being a good friend, also outside the lab. Now I think I’m ready to go out running soon again and take a “fika”break. I’ll be just down the hill.

Linda, thank you for all your help with the protein pictures and for reading through big parts of my thesis.

I also want to thank my room mates Marianne, Magnus and Anders, who have made me look away from the computer to chat for a few minutes every now and then, even during the last couple of weeks. Cecilia M for nice chats, Åsa for always being nice and supportive, Maria and Peter C for collaboration, and everyone else in the biocat group, and here at the department. A special thanks to Anders and Valerie, for keeping me company many late nights at work these last few weeks, when we’ve been finishing up.

All “old” biocats, Jenny, Joke, Didier and Fredrik who helped me get started in the lab. I bet I tested you’re patience more than once with all my questions. Malin, my old roommate, you finished up first but now I’m there too. Thanks for the peptalk.

Anna and Totte, my supervisors during my diploma work and a big reason for why I wanted to continue with research. You were also my link to Kalle and a position as a PhD-student.

A thanks’ also to my collaborators at the Physical Chemistry Department and Organic Chemistry Department at Sundsvall Univeristy.

And I should not forget to thank all of you who have helped my by reading parts of this thesis.

I also want to thank all my friends, especially Annika, Erika, Åsa and Maria for being the best of friends and responsible for many good times during the last years since I’ve got to know you.

And now last but absolutely not least, my parents. Mom and dad, thanks for all support throughout all the years and for always believing in me.

48 References

References

Alexander, F.W., Sandmeier, E., Mehta, P.K., Christen, P. (1994) Evolutionary Relationships among Pyridoxal-5’-Phosphate-Dependent Enzymes. Regio- Specific α, β and γ Families. Eur. J. Biochem. 219: 953-960 Badet, B., Walsh, C. (1985) Purification of an Alanine Racemase from Streptococccus faecalis and Analysis of its Inactivation by (1- Aminoethyl)phosphonic Acid Enantiomers. Biochemistry 24: 1333-1341 Berglund, P., Park, S. (2005) Strategies for Altering Enzyme Reaction Specificity for Applied Biocatalysis. Curr. Org. Chem. 9: 325-336 Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., Shindyalov, I.N., Bourne, P.E. (2000) The Protein Data Bank. Nucleic Acids Res. 28: 235-242 (Program available at: http://www.rcsb.org/pdb/) Bernasconi, S., Orsini, F., Sello, G., Di Gennaro, P. (2004) Bacterial Monooxygenase Mediated Preparation of Nonracemic Chiral Oxiranes: Study of the Effects of Substituents Nature and Position. Tetrahedron: Asymmetry 15: 1603-1606 Bertoldi, M., Voltattorni, C.B. (2003) Reaction and Substrate Specificity of Recombinant Pig Kidney Dopa Decarboxylase under Aerobic and Anaerobic Conditions. Biochim. Biophys. Acta 1647: 42-47 Bertolli, G., Fronza, G., Fuganti, C., Grasselli, P., Majori, L., Spreafico, F. (1981) On the α-Ketols Obtained from α,β-Unsaturated Aromatic Aldehydes. Tetrahedron Lett. 22: 965-968 Besse, P., Veschambre, H. (1994) Chemical and Biological Synthesis of Chiral Epoxides. Tetrahedron 50: 8885-8927 Björkling, F., Frykman, H., Godtfredsen, S.E., Kirk, O. (1992) Lipase Catalyzed Synthesis of Peroxycarboxylic Acids and Lipase Mediated Oxidations. Tetrahedron 48: 4587-4592 Bornscheuer, U.T., Kazlauskas, R.J. (2004) Catalytic Promiscuity in Biocatalysis: Using Old Enzymes to Form New Bonds and Follow New Pathways. Angew. Chem. Int. Ed. 43: 6032-6040 Buchholz, K.B., Poulsen, P.B. (2000) in Applied Biocatalysis, 2nd ed., Straathof, A.J.J., Adlercreutz, P. (Eds.) Harwood Academic Publishers: Amsterdam, pp. 1-15 49

Bugg, T.D.H. (2004) Diverse Catalytic Activities in the αβ-Hydrolase Family of Enzymes: Activation of H2O, HCN, H2O2, and O2. Bioorg. Chem. 32: 367-375 Cai, Y., Yao, S.-P., Wu, Q., Lin, S.-F. (2004a) Michael Addition of Imidazole with Acrylates Catalyzed by Alkaline Protease from Bacillus subtilis in Organic Media. Biotechnol. Lett. 26: 525-528 Cai, Y., Sun, X.-F., Wang, N., Lin, X.-F. (2004b) Alkaline Protease from Bacillus subtilis Catalyzed Michael Addition of Pyrimidine Derivatives to α,β- Ethylenic Compounds in Organic Media. Synthesis 671-674 Campos, F., Bosch, M.P., Guerrero, A. (2000) An Efficient Enantioselective Synthesis of (R,R)-Formoterol, a Potent Bronchodilator, using Lipases. Tetrahedron: Asymmetry 11: 2705-2717 Casas, J., Engqvist, M., Ibrahem, I., Kaynak, B., Córdova, A. (2005) Direct Amino Acid Catalyzed Asymmetric Synthesis of Polyketide Sugars. Angew. Chem. Int. Ed. 44: 1343-1345 Cedrone, F., Ménez, A., Quéméneur, E. (2000) Tailoring New Enzyme Functions by Rational Redesign. Curr. Opin. Struct. Biol. 10: 405-410 Chapman, D.T., Crout, D.H.G., Mahmoudian, M., Scopes, D.I.C., Smith, P.W. (1996) Enantiomerically Pure Amines by a New Method: Biotransformation of Oxalamic Esters using the Lipase from Candida antarctica. Chem. Commun. (Cambridge, U. K.) 2415-2416 Chen, R. (2001) Enzyme Engineering: Rational Redesign Versus Directed Evolution. Trends Biotechnol. 19: 13-14 Cobb, A.J.A., Shaw, D.M., Longbottom, D.A., Gold, J.B., Ley, S.V. (2005) Organoctalysis with Proline Derivatives: Improved Catalysts for the Asymmetric Mannich, Nitro-Michael and Aldol Reactions. Org. Biomol. Chem. 3: 84-96 Copley, S.D. (2003) Enzymes with Extra Talents: Moonlighting Functions and Catalytic Promiscuity. Curr. Opin. Chem. Biol. 7: 265-272 Costa, C.E., Clososki, G.C., Barchesi, H.B., Zanotto, S.P., Nascimento, M.G., Comasseto, J.V. (2004) Enzymatic Resolution of (RS)-β-Hydroxy Selenides in Organic Media. Tetrahedron: Asymmetry 15: 3945-3954 Dembitsky, V.M. (2003) Oxidation, Epoxidation and Sufoxidation Rections Catlaysed by Haloperoxidases. Tetrahedron 59: 4701-4720 DeSanits, G., Liu, J., Clark, D.P., Heine, A., Wilson, I.A., Wong, C.-H. (2003) Structure-Based Mutagenesis Approaches toward Expanding the Substrate Specificity of D-2-Deoxyribose-5-Phosphate Aldolase. Bioorg. Med. Chem. 11: 43-52 Dufour, É., Storer, A.C., Ménard, R. (1995) Engineering Nitrile Hydratase Activity into a Cysteine Protease by a Single Mutation. Biochemistry 34: 16382- 16388

50 References

Dunathan, H.C. (1966) Conformation and Reaction Specificity in Pyridoxal Phosphate Enzymes. Proc. Natl. Acad. Sci. U.S.A. 55: 712-716 Dunathan, H.C. (1971) Stereochemical Aspects of Pyridoxal Phosphate Catalysis. Adv. Enzymol. 35: 79-134 Dwyer, M.A., Looger, L.L., Hellinga, H.W. (2004) Computational Design of a Biologically Active Enzyme. Science 304: 1967-1971 Eliot, A.C., Kirsch, J.F. (2003) Avoiding the Road Less Traveled: How the Topology of Enzyme-Substrate Complexes can Dictate Product Selection. Acc. Chem. Res. 36: 757-765 Eliot, A.C., Kirsch, J.F. (2004) Pyridoxal Phosphate Enzymes: Mechanistic, Structural, and Evolutionary Considerations. Annu. Rev. Biochem. 73: 383-415 Esaki, K., Walsh, C.T. (1986) Biosynthetic Alanine Racemase of Salmonella typhimurium: Purification and Characterization of the Enzyme Encoded by the Alr Gene. Biochemistry 25: 3261-3267 Faber, K. (2001) Non-Sequential Processes for the Transformation of a Racemate into a Single Stereoisomeric Product: Proposal for Stereochemical Classification. Chem. Eur. J. 7: 5005-5010 Fishman, A., Eroshov, M., Dee-Noor, S.S., van Mil, J., Cogan, U., Effenberger, R. (2001) A Two-Step Enzymatic Resolution Process for Large-Scale Production of (S)-and (R)-Ethyl-3-Hydroxybutyrate. Biotechnol. Bioeng. 74: 256-263 García-Junceda, E., García-García, J.F., Bastida, A., Fernández-Mayoralas, A. (2004) Enzymes in the Synthesis of Bioactive Compounds: the Prodigious Decades. Bioorg. Med. Chem. 12: 1817-1834 García-Urdiales, E., Alfonso, I., Gotor, V. (2005) Enantioselective Enzymatic Desymmetrizations in Organic Synthesis. Chem. Rev. 105: 313-354 Gefflaut, T., Blonski, C., Perie, J., Willson, M. (1995) Class I Aldolases: Substrate Specificity, Mechanism, Inhibitors and Structural Aspects. Prog. Biophys. Molec. Biol. 63: 301-340 Gijsen, H.J.M., Qiao, L., Fitz W., Wong, C.H. (1996) Recent Advances in the Chemoenzymatic Synthesis of Carbohydrates and Carbohydrate Mimetics. Chem. Rev. 96: 443-473 Graber, R., Kasper, P., Malashkevich, V.N., Strop, P., Gehring., Jansonius, J.N., Christen, P. (1999) Conversion of Aspartate Aminotransferase into an L- Aspartate β-Decarboxylase by a Tripe Active-Site Mutation. J. Biol. Chem. 274: 31203-31208 Guex, N., Diemand, A., Peitsch, M.C. (1999) Protein Modeling for All. TIBS 24: 364-367 (Program available at: http://swissmodel.expasy.org//SWISS- MODEL.html)

Hilal, S.H., Karickkoff, S.W., Carreira, L.A. (2003) Prediction of Chemical Reactivity Parameters and Physical Properties of Organic Compounds from Molecular Structure using SPARC, US EPA Report EPA/600/R-03/030, March (Program available at: http://ibmlc2.chem.uga.edu/sparc/) Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., Pease, L.R. (1989) Site-Directed Mutagenesis by Overlap Extension Using the Polymerase Chain Reaction. Gene 77: 51-59 Hoffmann, K., Schneider-Scherzer, E., Kleinkauf, H., Zocher, R. (1994) Purification and Characterization of Eucaryotic Alanine Racemase Acting as Key Enzyme in Cyclosporin Biosynthesis J. Biol. Chem. 269: 12710-12714 Hoffmann, T., Zhong, G., List, B., Shabat, D., Anderson, J., Gramatikova, S., Lerner, R.A., Barbas III, C.F. (1998) Aldolase Antibodies of Remarkable Scope. J.Am. Chem. Soc. 120: 2768-2779 Holmquist, M. (2000) Alpha/Beta-Hydrolase Fold Enzymes: Structures, Functions and Mechanisms. Curr. Protein Pept. Sci. 1: 209-235 Houde, A., Kademi, A., Leblanc, D. (2004) Lipases and Their Industrial Applications. Appl. Biochem. Biotechnol. 118: 155-170 Hult, K., Berglund, P. (2003) Engineered Enzymes for Improved Organic Synthesis. Curr. Opin. Biotechnol. 14: 395-400 Inagaki, K., Tanizawa, K., Badet, B., Walsh, C.T., Tanaka, H., Soda, K. (1986) Thermostable Alanine Racemase from Bacillus stearothermophilus: Molecular Alpha/Beta-Hydrolase Fold Enzymes: Structures, Functions and Mechanisms. Curr. Protein Pept. Sci. 1: 209-235 Ivanetich, K.M., Santi, D.V. (1992) 5,6-Dihydropyrimidine Adducts in the Reactions and Interactions of Pyrimidines with Proteins. Prog. Nucleic Acid Res. Mol. Biol. 42: 127-156 Jackson, L.K., Brooks, H.B., Osterman, A.L., Goldsmith, E.J., Philips, M.A. (2000) Altering the Reaction Specificity of Eukaryotic Ornithine Decarboxylase. Biochemistry 39: 11247-11257 Jacob III, P. (1996) Facile Pyridoxal-Catalyzed Racemization of Nornicotine and Related Compounds. J. Org. Chem. 61: 2916-2917 Jeffery, C.J. (1999) Moonlighting Proteins. Trends Biochem. Sci. 24: 8-11 John, R.A. (1995) Pyridoxal Phosphate-Dependent Enzymes. Biochim. Biophys. Acta 1248: 81-96 Kirk, O., Christensen, M.W. (2002) Lipases from Candida antarctica: Unique Biocatalysts from a Unique Origin. Org. Process Res. Dev. 6: 446-451 Kitazume, T., Ikeya, T., Murata, K. (1986) Synthesis of Optically Active Trifluorinated Compounds: Asymmetric Michael Addition with Hydrolytic Enzymes. J. Chem. Soc. Chem. Commun. 17: 1331-1333

52 References

Kurokawa, Y., Watanabe, A., Yoshimura, T., Esaki, N., Soda, K. (1998) Transamination as a Side-Reaction Catalyzed by Alanine Racemase of Bacillus stearothermophilus. J. Biochem.(Tokyo) 124: 1163-1169 Lau, R.M., van Rantwijk, F., Seddon, K.R., Sheldon, R.A. (2000) Lipase- Catalyzed Reactions in Ionic Liquids. Org. Lett. 2: 4189-4191 Lee, S.-J., Kang, H.-Y., Lee, Y. (2003) High-Throughput Screening Methods for Selecting L-Threonine Aldolases with Improved Activity. J. Mol. Cat. B: Enzym. 26: 265-272 Leemhuis, H., Kragh, K.M., Dijkstra, B.W., Dijkhuizen, L. (2003) Engineering Cyclodextrin Glycosyltransferase into Hydrolase with a High Exo-Specificity. J. Biotechnol. 103: 203-212 LeMagueres, P., Im, H., Dvorak, A., Strych, U., Benedik, M., Krause, K.L. (2003) Crystal Structure at 1.45 Å Resolution of Alanine Racemase from a Pathogenic Bacterium, Pseudomonas aeruginosa, Contains Both Internal and External Imine Forms. Biochemistry 42: 14752-14761 LeMagueres, P., Im, H., Ebalunode, J., Strych, U., Benedik, M., Briggs, J.M., Kohn, H., Krause, K.L. (2005) The 1.9 Å Crystal Structure of Alanine Racemase from Mycobacterium tuberculosis Contains a Conserved Entryway into the Active Site. Biochemistry 44: 1471-1481 Ling, M.M., Robinson, B.H. (1997) Approaches to DNA Mutagenesis An Overview. Anal. Biochem. 254: 157-178 Machajewski, T.D., Wong, C.-H. (2000) The Catalytic Asymmetric Aldol Reaction. Angew. Chem. Int. Ed. 39: 1352-1374 Magnusson, A., Hult, K., Holmquist, M. (2001) Creation of an Enantioselective Hydrolase by Engineered Substrate-Assisted Catalysis. J. Am. Chem. Soc. 123: 4353-4355 Mahapatro, A., Kumar, A., Kalra, B., Gross, R.A. (2004) Solvent-Free Adipic Acid/1,8-Octanediol Condensation Polymerizations Catalyzed by Candida antarctica Lipase B. Macromolecules 37: 35-40 Martinelle, M., Holmquist, M., Hult, K. (1995) On the Interfacial Activation of Candida antarctica Lipase A and B as Compared with Humicola lanuginosa Lipase. Biochim. Biophys. Acta 1258: 272-276 McCarty, M.F. (2000) High-Dose Pyridoxine as an ‘Anti-Stress’ Strategy. Medical Hypotheses 54: 803-807 Mei, Y., Kumar, A., Gross, R. (2003) Kinetics and Mechanism of Candida antarctica Lipase B Catalyzed Solution Polymerization of ε-Caprolactone. Macromolecules 36: 5530-5536 Movassaghi, M., Jacobsen, E.N. (2002) The Simplest “Enzyme”. Science 298: 1904-1905

Mustata, G., Briggs, J.M. (2004) Cluster Analysis of Water Molecules in Alanine Racemase and Their Putative Structural Role. Protein Eng., Des. Sel. 17: 223- 234 Nomura, T., Yamamoto, I., Morishita, F., Furukawa, Y., Matsushima, O. (2001) Purification and Some Properties of Alanine Racemase from a Bivalve Mollusc Corbicula japonica. J. Exp. Zool. 289: 1-9 Notz, W., Tanaka, F., Barbas III, C.F. (2004) Enamine-Based Organocatalysis with Proline and Diamines: The Development of Direct Catalytic Asymmetric Aldol, Mannich, Michael, and Diels-Alder Reactions. Acc. Chem. Res. 37: 580-591 O´Brien, P.J., Herschlag, D. (1999) Catalytic Promiscuity and the Evolution of New Enzymatic Activities. Chem. Biol. 6: R91-R105 Ollis, D.L., Cheah, E, Cygler, M., Dijkstra, B., Frolow, F., Franken, S.M., Harel, M., Remington, S.J., Silman, I., Schrag, J., Sussman, J.L., Verschueren, K.H.G., Goldman, A. (1992) The Alpha/Beta Hydrolase Fold. Prot. Eng. 5: 197-211 Ozaki, S.I., Matsui, T., Watanabe, Y. (1996) Conversion of Myoglobin into a Highly Stereospecific Peroxygenase by the L29H/H64L Mutation. J. Am. Chem. Soc. 118: 9784-9785 Pàmies, O., Bäckvall, J.-E. (2003) Combination of Enzymes and Metal Catalysts. A Powerful Approach in Asymmetric Catalysis. Chem. Rev. 103: 3247-3261 Patkar, S., Björkling, F., Zundel, M., Schulein, M., Svendsen, A., Heldt-Hansen, H.P., Gormsen, E. (1993) Purification of two Lipases from Candida antarctica and Their Inhibition by Various Inhibitors Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem. 32: 76-80 Patrick, W.M., Weisner, J., Blackburn, J. (2002) Site-Directed Mutagenesis of Tyr354 in Geobacillus stearothermophilus Alanine Racemase Identifies a Role in Controlling Substrate Specificity and a Possible Role in the Evolution of Antibiotic Resistance. ChemBioChem 8: 789-792 Penning, T.M., Jez, J.M. (2001) Enzyme Redesign. Chem. Rev. 101: 3027-3046 Percudani, R., Peracchi, A. (2003) A Genomic Overview of Pyridoxal- Phosphate-Dependent Enzymes. EMBO Rep. 4:850-854 Pogorevc, M., Faber, K. (2000) Biocatalytic Resolution of Sterically Hindered Alcohols, Carboxylic Acids and Esters Containing Fully Substituted Chiral Centers by Hydrolytic Enzymes. J. Mol. Cat. B: Enz. 10: 357-376 Popp, A., Gilch, A., Mersier, A.-L., Petersen, H., Rockinger-Mechlem, J., Stohrer, J. (2004) Enzymatic Kinetic Resolution of 1,3-Dioxolan-4-one and 1,3- Oxathiolan-5-one Derivatives: Synthesis of the Key Intermediate in the Industrial Synthesis of the Nucleoside Reverse Transcriptase Inhibitor AMDOXOVIR. Adv. Synth. Catal. 346: 682-690 Pugnière, M., Commeyras, A., Previero, A. (1983) Racemization of Amino Acid Esters Catalyzed by Pyridoxal 5’-Phosphate as a Step in the Production of L- Amino Acids. Biotechnol. Lett. 5: 447-452 54 References

Reetz, M.T., Torre, C., Eipper, A., Lohmer, R., Hermes, M., Brunner, B., Maichele, A., Bocola, M., Arand, M., Cronin, A., Genzel, Y., Archelas, A., Furstoss, R. (2004) Enhancing the Enantioselectivity of an Epoxide Hydrolase by Directed Evolution. Org. Lett. 6: 177-180 Sarda, L., Desnuelle, P. (1958) Action de la Lipase Pancréatique sur les Esters en Émulsion. Biochim. Biophys. Acta 30: 513-521 Schmid, A., Dordick, J.S., Hauer, B., Kiener, A., Wubbolts, M., Witholt, B. (2001) Industrial Biocatalysis Today and Tomorrow. Nature 409: 258-268 Seebeck, F.P., Hilvert, D. (2003) Conversion of a PLP-Dependent Racemase into an Aldolase by a Single Active Site Mutation. J. Am. Chem. Soc. 125: 10158- 10159 Shaw, J.P., Petsko, G.A., Ringe, D. (1997) Determination of the Structure of Alanine Racemase from Bacillus stearothermophilus at 1.9 Å Resolution. Biochemistry 36: 1329-1342 Shin, J.-S., Kim, B.-G. (1997) Kinetic Resolution of a-Methylbenzylamine with o- Transaminase Screeened from Soil Microorganisms: Application of a Biphasic System to Overcome Product Inhibition. Biotechnol. Bioeng. 55: 348- 358 Shin, J.-S., Jang, J.-W., Park, I., Kim, B.-G. (2003) Purification, Characterization, and Molecular Cloning of a Novel Amine:Pyruvate Transaminase from Vibrio fluvialis JS17. Appl. Microbiol. Biotechnol. 61: 463-471 Skouridou, V., Stamatis, H., Kolisis, F.N. (2003) Lipase-Mediated Epoxidation of α-Piene. J. Mol. Cat. B: Enz. 21: 67-69 Spies, M.A., Toney, M.D. (2003) Multiple Hydrogen Kinetic Isotope Effects for Enzymes Catalyzing Exchange with Solvent: Application to Alanine Racemase. Biochemistry 42: 5099-5107 Spies, M.A., Woodward, J.J., Watnik, M.R., Toney, M.D. (2004) Alanine Racemase Free Energy Profiles from Global Analyses of Progress Curves. J. Am. Chem. Soc. 126: 7464-7475 Stamper, G.F., Morollo, A.A., Ringe, D. (1998) Reaction of Alanine Racemase with 1-Aminoethylphosphonic Acid Forms a Stable External Imine. Biochemistry 37: 10438-10445 Stemmer, W.P.C. (1994) DNA Shuffling by Random Fragmentation and Reassembly: In vitro Recombination for Molecular Evolution. Proc. Natl. Acad. Sci. USA 91: 10747-10751 Stryer, L. (1995) Biochemistry, 4th ed., W.H. Freeman and Company: New York Sun, S., Toney, M.D. (1999) Evidence for a Two-Base Mechanism Involving Tyrosine-265 from Arginine-219 Mutants of Alanine Racemase. Biochemistry 38: 4058-4065 Tai, C.-H., Cook, P.F. (2001) Pyridoxal 5´-Dependent α,β-Elimination Reactions: Mechanism of O-Acetylserine. Acc. Chem. Res. 34: 49-59 55

Takayama, S., McGarvey, G.J., Wong, C.-H. (1997) Enzymes in Organic Synthesis: Recent Developments in Aldol Reactions and Glycosylations. Chem. Soc. Rev. 26: 407-415 Tanaka, F., Fuller, R., Shim, H., Lerner, R.A., Barbas III, C.F. (2004) Evolution of Aldolase Antibodies in Vitro: Correlation of Catalytic Activity and Reaction- Based Selection. J. Mol. Biol. 335: 1007-1018 Tanizawa, K., Ohshima, A., Scheidegger, A., Inagaki, K., Tanaka, H., Soda, K. (1988) Thermostable Alanine Racemase from Bacillus stearothermophilus: DNA and Protein Sequence Determination and Secondary Structure Prediction. Biochemistry 27: 1311-1316 Toney, M.D. (2005) Reaction Specificity in Pyridoxal Phosphate Enzyems. Arch. Biochem. Biophys. 433: 279-287 Torre, O., Alfonso, I., Gotor, V. (2004) Lipase Catalysed Michael Addition of Secondary Amines to Acrylonitrile. Chem. Commun. 1724-1725 Toyama, H., Esaki, N., Yoshimura, T., Tanizawa, K., Soda, K. (1991) Thermostable Alanine Racemase of Bacillus stearothermophilus: Subunit Dissociation and Unfolding. J. Biochem. 110: 279-283 Tsai, M.-D., Weintraub, H.J.R., Byrn, S.R., Chang, C.-J., Floss, H.G. (1978) Conformaiton-Reactivity Relationship for Pyridoxal Schiff’s Bases. Rates of Racemization and α-Hydrogen Exchange of the Pyridoxal Schiff’s Bases of Amino Acids. Biochemistry 17: 3183-3188 Uppenberg, J., Hansen, M.T., Patkar, S., Jones, T.A. (1994) The Sequence, Crystal Structure Determination and Refinement of two Crystal Forms of Lipase B from Candida antarctia. Structure (London, England) 2: 293-308 Uppenberg, J., Öhrner, N., Norin, M., Hult, K., Kleywegt, G.J., Patkar, S., Waagen, V., Anthonsen, T., Jones, T.A. (1995) Crystallographic and Molecular Modeling Studies of Lipase B from Candida antarctica Reveal a Stereospecificity Pocket for Secondary Alcohols. Biochemistry 34: 16838-16851 Vacca, R.A., Christen, P., Malashkevich, V.N., Jansonius, J.N., Sandmeier, E. (1995) Substitution of Apolar Residues in the Active Site of Aspartate Aminotransferase by Histidine: Effects on Reaction and Substrate Specificity. Eur. J. Biochem. 227: 481-487 Verger, R., De Haas, G.G. (1976) Interfacial Enzyme Kinetics of Lipolysis. Annu. Rev. Biophys. Bioeng. 5: 77-117 de Visser, S.P., Ogliaro, F., Sharma, P.K., Shaik, S. (2002) What Factors Affect the Regioselectivity of Oxidation by Cytochrome P450? A DFT Study of Allylic Hydroxylation and Double Bond Epoxidation in a Model Reaction. J. Am. Chem. Soc. 124: 11809-11826 Wabnitz, T.C., Spencer, J.B. (2003) A General, Brønsted Acid-Catalyzed Hetero- Michael Addition of Nitrogen, Oxygen, and Sulfur Nucleophiles. Org. Lett. 5: 2141-2144

56 References

Wasserman, S.A. Daub, E., Grisafi, P., Botstein, D., Walsh, C.T. (1984) Catabolic Alanine Racemase from Salmonella typhimurium: DNA Sequence, Enzyme Purification, and Characterization. Biochemistry 23: 5182-5187 Watanabe, A., Kurokawa, Y., Yoshimura, T., Esaki, N. (1999a) Role of Tyrosine 265 of Alanine Racemase from Bacillus stearothermophilus. J. Biochem. 125: 987- 990 Watanabe, A., Kurokawa, Y., Yoshimura, T., Kurihara, T., Soda, K., Esaki, N. (1999b) Role of Lysine 39 of Alanine Racemase from Bacillus stearothermophilus that Binds Pyridoxal 5’-Phosphate. J. Biol. Chem. 274: 4189- 4194 Watanabe, A. Yoshimura, T., Mikami, B., Hayashi, H., Kagamiyama, H., Esaki, N. (2002) Reaction Mechanism of Alanine Racemase from Bacillus stearothermophilus: X-ray Crystallographic Studies of the Enzyme Bound with N-(5´-Phosphopyridoxyl)alanine. J. Biol. Chem. 277: 19166-19172 Wolfenden, R., Snider, M.J. (2001) The Depth of Chemical Time and the Power of Enzymes as Catalysts. Acc. Chem. Res. 34: 938-945 Wood, W.A., Gunsalus, I.C. (1951) D-Alanine Formation: a Racemase in Streptococcus faecalis. J.Biol.Chem. 190: 403-416 Yagasaki, M., Ozaki, A. (1998) Industrial Biotransformations for the Production of D-Amino Acids. J. Mol. Cat. B: Enz. 4: 1-11 Yao, H., Richardson, D.E. (2000) Epoxidation of Alkenes with Bicarbonate- Activated Hydrogen Peroxide. J. Am. Chem. Soc. 122: 3220-3221 Yao, S.-P., Lu, D.-S., Wu, Q., Cai, Y., Xu, S.-H., Lin, X.-F. (2004) A Single- Enzyme, Two-Step, One-Pot Synthesis of N-Substituted Imidazole Derivatives Containing a Glucose Branch via Combined Acylation/Michael Addition Reaction. Chem. Commun. 17: 2006-2007 Yarnell, A. (2003) The Power of Promiscuity. Chem. Eng. News 8: 33-35 Yokoigawa, K., Okubo, Y., Soda, K., Misono, H. (2003) Improvement in Thermostability and Psychrophilicity of Psychrophilic Alanine Racemase by Site-Directed Mutagenesis. J. Mol. Cat. B: Enz. 23: 389-395 Yoshimura, T., Esaki, N. (2003) Amino Acid Racemases: Functions and Mechanisms. J. Biosci. Bioeng. 96: 103-109 Yow, G.Y., Watanabe, A., Yoshimura, T., Esaki, N. (2003) Conversion of the Catalytic Specificity of Alanine Racemase to a D-Amino Acid Aminotransferase Activity by a Double Active-Site Mutation. J. Mol. Cat. B: Enz. 23: 311-319 Zaks, A., Dodds, D.R. (1995) Chloroperoxidase-Catalyzed Asymmetric Oxidations: Substrate Specificity and Mechanistic Study. J. Am. Chem. Soc. 117: 10419-10424 Zaks, A., Dodds, D.R. (1997) Application of Biocatalysis and Biotransformations to the Synthesis of Pharmaceuticals. Drug Discovery Today 2: 513-531 57

Zaks, A. (2001) Industrial Biocatalysis. Curr. Opin. Chem. Biol. 5: 130-136 Zhang, N., Suen, W.-C., Widsor, W., Xiao, L., Madison, V., Zaks, A. (2003) Improving Tolerance of Candida antarctica Lipase B towards Irreversible Thermal Inactivation Through Directed Evolution. Prot. Eng. 16: 599-605 Öhrner, N., Orrenius, C., Mattson, A., Norin, T., Hult, K. (1996) Kinetic Resolutions of Amine and Thiol Analogues of Secondary Alcohols Catalyzed by the Candida antarctica Lipase B. Enzyme Microb. Technol. 19: 328-331