ω-Transaminase in Biocatalysis
Methods, Reactions and Engineering
Karim Engelmark Cassimjee
Doctoral thesis
KTH Royal Institute of Technology
School of Biotechnology
Stockholm 2012
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Cover picture: The Enzyme at the Heart of the Green Chemical Factory
© David Karim Engelmark Cassimjee 2012
KTH Royal Institute of Technology School of Biotechnology AlbaNova University Centre SE-106 91 Stockholm Sweden
ISBN 978-91-7501-242-1 TRITA-BIO Report 2012:13 ISSN 1654-2312
Printed in Stockholm, March 2012 Universitetsservice US-AB Drottning Kristinas väg 53 B SE-100 44 Stockholm Sweden
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For Nadia, my one year old daughter, who I suspect will enjoy tearing out the pages (especially the ones with figures).
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Abstract
Biocatalysis offers an alternative to classic chemistry by using enzymes, the protein catalysts of Nature, for production of fine chemicals. Evolution has created enzymes capable of catalysis at moderate temperature of a specific reaction in the presence of a plethora of compounds in the aqueous cell environment. The focal point of biocatalysis is to utilise these traits in vitro, for creation of valuable molecules. The ω-transaminase is an enzyme capable of producing chiral amines, compounds used to great extent in pharmaceuticals. Much effort has in recent years been invested in the research and engineering of this enzyme type since the catalysed reaction offers an advantageous alternative to classical techniques. Nevertheless, there is a need for method development, adaptation of the enzyme and increased understanding of the catalytic mechanism for feasibility as an effective biocatalyst for unnatural substrates. This thesis addresses a chosen set of obstacles as a contribution to meeting the demands at hand. ω-Transaminase from Chromobacterium violaceum and Arthrobacter citreus was used. Many homologous ω-transaminases are available, which are also subject to engineering where variants are produced. To accurately compare their kinetic constants an active site quantification method is required but has not been available. Here such a method is presented (Paper 1) which encompasses a virtually irreversible half transamination reaction. In stereoselective synthesis the ω-transaminase catalysed equilibrium reaction inherently results in incomplete conversion. An equilibrium displacement system is presented (Paper II) where isopropylamine is the amino donor for transamination of acetophenone and derivatives thereof, coupled to an enzymatic cascade reaction. For many unnatural substrates the specificity and enantiospecificity is insufficient. Rationally redesigned variants were produced with improved properties for chosen substrates (Paper III and IV). The catalytic contributions of field and resonance of a variant compared to the wild type were investigated (Paper IV) for increased knowledge of the mechanism. For rational redesign of an enzyme the three-dimensional structure is required, of which only a few are available for the ω- transaminases. X-ray crystallographic structures of the holo and apo form of Chromobacterium violaceum ω-transaminase were made (Paper V) which revealed significant structural rearrangements upon coenzyme binding which may be of consequence for future engineering.
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Sammanfattning
Biokatalys utgör ett alternativ till klassisk kemi genom att finkemikalier produceras med hjälp av enzymer, naturens katalysatorer. Naturen har genom evolution skapat enzymer med förmåga att vid måttlig temperatur katalysera en specifik reaktion i närvaro av ett överflöd av föreningar i vattenmiljön hos den levande cellen. Huvudsyftet med biokatalys är att utnyttja dessa egenskaper in vitro, för att producera värdefulla molekyler. ω-Transaminaset är ett enzym med förmåga att producera kirala aminer, föreningar som används i läkemedelsutveckling i stor omfattning. Mycket forskning har under de senaste åren investerats i detta enzymslag eftersom den katalyserade reaktionen utgör ett fördelaktigt alternativ till klassiska tekniker. Dock finns det behov av metodutveckling, anpassning av enzymet och ökad förståelse för den katalytiska mekanismen för dess effektiva användning som biokatalysator för onaturliga substrat. Denna avhandling behandlar valda begränsande faktorer, ett bidrag till att möta de krav som ställs. ω-Transaminaser från Chromobacterium violaceum och Arthrobacter citreus användes. Många homologa ω-transaminaser finns tillgängliga, vilka också är föremål för utveckling av enzymvarianter. För att jämföra kinetiska data mellan dessa är en metod för kvantifiering av aktiva säten nödvändig. En sådan metod presenteras (Artikel I) som innefattar en synbart irreversibel halv-transamineringsreaktion. I stereoselektiv syntes med ω-transaminas resulterar den katalyserade jämviktsreaktionen i ofullständig omsättning. Ett jämviktsförskjutningssystem presenteras här (Artikel II) där iso- propylamin är aminodonator för transamineringen av acetofenon och dess derivat, kopplat till en enzymatisk kaskadreaktion. För många onaturliga substrat är specificiteten och enantio- specificiteten otillräckliga. Rationellt designade varianter produ- cerades med förbättrade egenskaper för utvalda substrat (Artikel III och IV). De katalytiska bidragen från fält- och resonanseffekter hos en variant jämfört med vildtypen undersöktes (Artikel IV) med syfte att öka kunskapen om reaktionsmekanismen. Rationell design av ett enzym fordrar dess tredimensionella struktur, endast ett fåtal sådana är tillgängliga för ω-transaminaser. Röntgenkristallografiska strukturer av holo- och apo-former av Chromobacterium violaceum ω-transaminas togs fram (Artikel V), som visade tydliga strukturella omorganiseringar vid koenzym- bindning som kan vara av betydelse för framtida utveckling.
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This thesis is based on the following scientific publications:
I Active Site Quantification of an ω-Transaminase by Performing a Half Transamination Reaction Karim Engelmark Cassimjee1, Maria Svedendahl Humble1, Valentina Miceli, Carla Granados Colomina, and Per Berglund ACS Catalysis 2011, 1, 1051-1055
II Transaminations with Isopropyl Amine: Equilibrium Displacement with Yeast Alcohol Dehydrogenase Coupled to in situ Cofactor Regeneration Karim Engelmark Cassimjee, Cecilia Branneby, Vahak Abedi, Andrew Wells and Per Berglund Chemical Communications 2010, 46, 5569-5571
III Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an ω-Transaminase Maria Svedendahl Humble1, Karim Engelmark Cassimjee1, Vahak Abedi, Hans-Jürgen Federsel and Per Berglund ChemCatChem 2012, In press, doi: 10.1002/cctc.201100487
IV Chromobacterium violaceum ω-Transaminase Variant Trp60Cys Shows Increased Specificity for (S)-1- Phenylethylamine and 4’-Substituted Acetophenones, and Follows Swain-Lupton Parameterisation Karim Engelmark Cassimjee, Maria Svedendahl Humble, Henrik Land, Vahak Abedi and Per Berglund Submitted manuscript
V Crystal Structures of the Chromobacterium violaceum ω- Transaminase Reveal Major Structural Rearrangements Upon Binding of Coenzyme PLP Maria Svedendahl Humble, Karim Engelmark Cassimjee, Maria Håkansson, Yengo Kimbung, Björn Walse, Vahak Abedi, Hans-Jürgen Federsel, Per Berglund and Derek T. Logan FEBS Journal 2012, 279, 779-792
The above publications will be referred to by their assigned roman numerals.
1 Shared first authorship
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Summary of the contributions of Karim E. Cassimjee (KC) to Papers I to V Paper I: KC supplied the idea of using a virtually irreversible half transamination reaction for active site quantification, designed and performed the active site quantification method, designed the molecular cloning, and performed the majority of the writing.
Paper II: KC supplied the idea of utilizing the selectivity of yeast alcohol dehydrogenase for equilibrium displacement, carried out the molecular cloning, reactions and analysis, and did the majority of the writing.
Paper III: KC designed the molecular cloning and the experimental procedures for enantioselectivity determination, and took part in the rational redesign and the writing.
Paper IV: KC performed the molecular cloning, reactions and analysis, reaction parameterisation, took part in the finding of the key point mutation, and performed the majority of the writing.
Paper V: KC designed the molecular cloning, performed stability experiments, and took part in the writing.
Publications by KC, not included in this thesis • One-Step Enzyme Extraction and Immobilization for Biocatalysis Applications Karim Engelmark Cassimjee, Robert Kourist, Diana Lindberg, Marianne Wittrup Larsen, Nguyen Hong Thanh, Mikael Widersten, Uwe T Bornscheuer and Per Berglund Biotechnology Journal 2011, 6, 463-469
• Silica-Immobilized His6-Tagged Enzyme: Alanine Racemase in Hydrophobic Solvent Karim Engelmark Cassimjee, Martin Trummer, Cecilia Branneby and Per Berglund Biotechnology and Bioengineering 2008, 99, 712-716
• Synthesis of Cyclic Polyamines by Enzymatic Generation of an Amino Aldehyde in situ Karim Engelmark Cassimjee, Sílvia Rodríguez Marín and Per Berglund Submitted manuscript
• Process for the Production of Polyamines Bernhard Hauer, Karim Engelmark Cassimjee, Per Berglund 2011 Patent publication number: WO2011051433
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List of Abbreviations
1-PEA 1-phenylethylamine Cv-ωTA Chromobacterium violaceum ω-transaminase E-value ratio of specificity constants (kcat/KM) of enantiomers ee enantiomeric excess Enz enzyme eq. equation GC gas chromatography HPLC high pressure liquid chromatography IMAC immobilised metal affinity chromatography IPA isopropylamine PLP pyridoxal-5’-phosphate PMP pyridoxamine-5’-phosphate RMSD root-mean-square deviation U enzyme activity unit, defined as (µmol/min)
Amino acid (residue) abbreviations Ala, A alanine Arg, R arginine Asn, N asparagine Asp, D aspartate Cys, C cysteine Glu, E glutamate Gln, Q glutamine Gly, G glycine His, H histidine Ile, I isoleucine Leu, L leucine Lys, K lysine Met, M methionine Phe, F phenylalanine Pro, P proline Ser, S serine Thr, T threonine Trp, W tryptophan Tyr, Y tyrosine Val, V valine
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”A tidy laboratory means a lazy chemist.”
Jöns Jacob Berzelius (1779-1848)
Note: If the above statement is valid in the opposite sense, i.e. a messy laboratory means a devoted chemist; my co-workers and I are possibly the most energetic chemists in the country.
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Contents
1 Introduction 1 1.1 Enzymes, proteins with catalytic ability 1 1.2 Enantiospecificity, an enzymatic trait 4 1.3 Enzyme kinetics 6 1.4 Biocatalysis 8 1.4.1 Enzyme engineering 9 1.5 ω-Transaminase, a PLP-dependent enzyme 10
2 Aims and Scope 15
3 Active Site Quantification of Cv-ωTA (Paper I) 17
4 Equilibrium Displacement of ω-Transaminase Reactions with Isopropylamine as Amino Donor (Paper II) 21
5 Rational Redesign of Cv-ωTA (Paper III and IV) 27 5.1 Improved enantioselectivity and reversed enantiomeric preference (Paper III) 29 5.2 Increased specificity (Paper IV) 32
6 Structure of Cv-ωTA (Paper V) 39
7 Concluding Discussion 44 7.1 Reactions and engineering with ω-transaminase, summarising discussion of Paper I-V 45 7.2 Future prospects 48 7.2.1 Dynamic kinetic resolution of an amine 48 7.2.2 Mechanistic studies 49
8 Acknowledgements 50
9 References 51
Appendix 57
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1 Introduction
This thesis is a contribution to the field of biocatalysis1 in which methods, reactions and engineering with ω-transaminase are presented. Biocatalysis is an integral part of industrial or white biotechnology.2,3
Fine chemicals, especially pharmaceuticals, are undoubtedly important products which have become essential for our society. Their fabrication is a continuously developing area where there is a demand for new and improved synthesis routes for target compounds. A frequent motif in pharmaceuticals is the chiral amine,4 a biofunctional group also present in natural metabolism.
In this section enzymology (with related concepts in chemistry), the field of biocatalysis and the ω-transaminase enzyme are briefly described. The concepts and observed traits of enzymes are put into the context of how they may be applied for engineering and synthesis of fine chemicals, chiral amines in particular.
1.1 Enzymes, proteins with catalytic ability In Nature, living organisms depend on a functioning metabolism. Cellular structures are created from simpler building blocks. Compounds are formed and broken down in what is described as controlled metabolic pathways in which energy is rearranged and utilised for vital processes. The lion’s share of the chemical reactions in these processes takes place in an aqueous environment and are catalysed by enzymes. The name, enzyme, originates from ancient Greek, meaning “in sourdough”. Enzymes are the foundation of chemical life; they uphold essentially all chemical reactions at a viable rate within a living organism.1,5
Proteins are polymers (polyamides) formed by the condensation of α- amino acids (molecules which contain an amino group and a carboxylic acid group with one methylene) which are found in all known forms of life. Generally, residues of twenty types of amino acid are present in proteins. The amino acids are joined in a defined
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Figure 1: Graphical representation of an α‐helix (left) and β‐sheets (right), common motifs in proteins order based on a gene sequence, in which each amino acid residue contributes with a distinct side chain with properties elegantly chosen by evolution. The proteins fold to form ordered vital structures, biologically functional units. The structures formed by the amino acid residues are many; the most common motifs are the α-helix and the β-sheet which are usually depicted in three- dimensional structures, shown in figure 1. When folded, the protein molecule can form a globular structure with a hydrophobic core, a result of minimization of hydrophobic exposure in an aqueous environment. The individual protein molecules may exist alone or form complexes with others. A protein which is capable of catalysing a chemical reaction is called an enzyme.
Observed at macro scale proteins behave in unexpected ways. E.g. with rising temperature a protein solution (such as egg white) will turn into a semi solid (an omelette6) whereas many other com- pounds or solutions thereof typically melt or lower in viscosity. This is a result of unfolding and subsequent aggregation of the protein chains. Looking at proteins in micro scale also yields fascinating findings. An enzyme is able to bind one or more distinct molecules or atoms, its substrates, and provide an environment which is more favourable for a chemical reaction of the substrate(s) than the surrounding solution. The location in the enzyme where this takes place is called the active site. According to the transition state theory,7 applicable for all types of catalysis, the reaction rate increase can be attributed to a lowering of the activation energy.
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The mechanisms vary between enzymes and remain a matter of debate. The theory of near attack conformers (NAC)8 presented by Bruice and co-workers attributes the lowering of the activation energy to the ability of an enzyme to place its substrate(s) in positions which are favourable for reaction. Another theory, presented by Warshel and co-workers, assigns the catalytic ability to the presence of favourable electrostatic interactions9 in the active site which stabilise the transition state. The enzyme macromolecule is a flexible structure, all known enzymes display this feature.10 The ability of conformational adaptation to favourable conformations during a catalytic process has been presented by Hammes-Schiffer and co-workers as being of paramount importance.10 In this view the substrate binding, transition state stabilisation, product release, and all transitions in-between, require different conformations of the enzyme for effective catalysis.
Some enzymatic reactions require a non-protein moiety, a specific molecule bound in the active site, for the catalysis to take place. Such moieties are called coenzymes.11 When the coenzyme is bound the complex is denoted as the holo enzyme, whereas the non- catalytic unit without coenzyme is called the apo enzyme. Coenzymes are distinguishable from substrates since they are regenerated, not consumed in the reaction. If the holo enzyme is stable and the coenzyme is not degraded no more than one equivalent of coenzyme to apo enzyme is needed for complete activation, i.e. complete formation of holo enzyme, in a given reaction. When non-natural conditions or non-natural substrates are applied, arguably, there is a possibility of increased instability of the enzyme-coenzyme complex; this should be taken into consideration when enzymes are used in chemical synthesis. When an enzyme catalyses an oxidation reaction a cosubstrate is used for a complementary reduction reaction, e.g. NAD+ can be reduced to NADH. The reversed reduction reaction will, naturally, yield NAD+ from NADH as a complementary oxidation.5 For in vitro synthesis applications the cosubstrate may be regenerated by an auxiliary reaction to circumvent the need for higher concentrations.
Inhibition of enzyme catalysis is a common observation, the reaction rate may drop in the presence of a substance (an inhibitor) or higher substrate or product concentrations.12,13 Inhibiting and activating
3 compounds are used by a living cell to control the flow of metabolism.5 When substrate concentrations are significantly higher than what is observed in the cell environment, arguably, inhibition arises as a consequence of the non-natural situation. The active site is in essence blocked by the presence of more than one substrate. In ordered multi substrate reactions high concentrations may lead to blockage by substrates binding in incorrect order. For in vitro applications catalysis at high concentrations may be desired, but in nature no evolutionary pressure existed to achieve this ability.
1.2 Enantiospecificity, an enzymatic trait A molecule is defined as chiral if it cannot be superimposed upon its mirror image, much alike a human hand. A molecule is not chiral (achiral) if it has symmetry elements of the second kind, i.e. a mirror plane, a centre of inversion, or a rotation-reflection axis. Chiral versions of otherwise identical compounds are termed enantiomers. In nature such molecules are predominantly existent as one of the possible versions. In chemistry, the common way of denoting the enantiomers are with an (S) (left handed) or (R) (right handed). Amino acids are usually classified under a different nomenclature which allows the majority of the in nature existing ones to be designated as L-enantiomers, as opposed to the corresponding D- enantiomers.14 An equal mix of enantiomers is referred to as a racemate.
An enzyme is composed only of L-amino acid residues and is thereby chiral and enantiopure. The interaction of an enzyme with chiral substrates can differ with the enantiomers. It may possess a high catalytic effect for one enantiomer of the substrate while the turnover of the other is low, sometimes virtually undetectable. The interaction of enantiomers with an enzyme can be envisioned as analogous to hands in gloves, depicted in Figure 2.
The enantiomers may be viewed upon as different substrates. Specificity is the term used to describe the ability of an enzyme to distinguish between substrates, enantiospecificity when the substrates are enantiomers. The term substrate specificity is generally used to describe different catalytic activity for similar substrates (not enantiomers). Enzymes also exhibit chemospecificity
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NH2 H2N
H H
Figure 2: A pair of human hands (the author’s) next to a right hand glove compared to the (S)‐ and (R)‐forms of 1‐phenylethylamine next to a ribbon representation of an ω‐transaminase enzyme. However rotated, either hand or molecule cannot be superimposed on the other. The right hand fits in the glove, analogous to how one of the enantiomers fits in the active site of the enzyme. for similar reactions, and regiospecificity for analogous reactions on different regions of a substrate. Figure 3 shows examples of substrates subject to specificity. An examination of the reactions and compounds of metabolic pathways reveals the necessity of these traits in order to uphold the order of metabolism. In essence, the enzyme is evolved to catalyse a reaction specifically enough for the remaining molecules in its environment to be sufficiently unperturbed for continued metabolism to function sufficiently. In this way a cell can produce one type of sugar, protein or lipid while consuming another.
NH2 NH2 regiospecificity alcohol dehydrogenase
substrate specificity ω‐transaminase OH OH O
N H chemospecificity O amidase or esterase O
Figure 3: Examples of substrates which are subject to chemo‐, regio‐ or substrate specificity with examples of enzyme types.
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1.3 Enzyme kinetics When steady-state conditions can be assumed, and the enzyme concentration is significantly lower than that of the substrate (the usual case) the reaction rate of an enzyme catalyzed reaction can be described by the Michaelis-Menten equation.15-19 The equation in its simplest form, for a reaction with one substrate without inhibition, gives: