Maria Svedendahl Humble

Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.

The bacterial ω‐transaminase from Chromobacterium violaceum (Cv‐ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5′‐phosphate (PLP). Here, we present four crystal structures of Cv‐ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo‐Cv‐ωTA structure reveals unique ‘relaxed’ conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered ‘roof ’ over the PLP‐binding site, whereas in the other apo form and the holo form the ‘roof’ is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the ‘roof’ structure may be associated with substrate binding in Cv‐ωTA and some other transaminases.

Revealing the Structural Basis of Promiscuous Amine Transaminase Activity

Enzymes are nature’s tool to speed up biochemical reactions that would otherwise hardly proceed. As a result of the complexity of the metabolic network of living cells, enzymes have to fulfill partially competing demands. On the one hand, a high precision in terms of reaction and substrate specificity is often required. This is achieved, for example, by a fine-tuned interaction between the active site structure and the substrate, which forces a defined positioning of the substrates’ reactive groups relative to the catalytic residues. On the other hand, some enzymes have evolved with a relaxed substrate specificity, which is useful and economic in several metabolic tasks: For example, a single enzyme can thus break down various chemically similar nutrients or transform different poisonous compounds for their detoxification. The ability to catalyze more than one chemical transformation within a single active site, called promiscuity, is not only an important property of some enzymes, but it is also recognized as a key mechanism of evolution and thus facilitates adaptation of enzymes to new metabolic needs. Furthermore, the enzymes’ ability to take over more than one physiological function renders metabolism more plastic, even if the promiscuous “underground activities” are very weak. Substrate d] and reaction promiscuity are also often the reasons why enzymes can be applied in biocatalysis. Many fine chemicals that are synthesized by biocatalytic transformations are not produced in nature, but nevertheless, enzymes that can be applied in their syntheses have been identified. For example, pyruvate decarboxylase can be used for the production of (R)-phenylacetylcarbinol. Esterases and lipases are successfully used in organic synthesis for the preparation of various optically active alcohols, acids, and amines. Their natural function, however, might be totally different, as shown for pig liver esterase, which plays a role in signal transduction by ester cleavage of methylated and prenylated proteins. Still, for most enzymes their promiscuous potential has not yet been revealed. One reason is that, after the main activity and possible function has been identified, further in-depth characterization is often not performed. Furthermore, if the promiscuous activity is unrelated to the natural one, a general activity screen is very laborious. Therefore, the understanding of the molecular principles of promiscuity is a challenging frontier in biocatalysis, as the knowledge obtained can be used as a starting point for protein engineering or for the discovery of other promiscuous enzymes. In a recent study, we identified four crystal structures [protein database (PDB) codes: 3HMU, 3I5T, 3FCR, and 3GJU] of transaminases with unknown function, and we could show that these enzymes interconvert small amino acids, for example, alanine (1) and succinic semialdehyde (2, Scheme 1). Additionally, chiral amines can be transaminated, but with very different efficiencies that range from 0.4 to 80 % relative activity, compared with the transamination of 1 + 2 (see values given in Scheme 1). From these observations, we concluded that the conversion of amines is a type of substrate promiscuity, which is pronounced to varying degrees in these four enzymes. Transaminases showing this type of catalytic activity are designated amine transaminases (ATAs): In contrast to the ubiquitous a-amino acid transaminases, they accept substrates lack-

Connecting Unexplored Protein Crystal Structures to Enzymatic Function

Biocatalysis has emerged as an important alternative to traditional chemical synthesis for the preparation of fine chemicals, as recently demonstrated by the biocatalytic manufacture of the drug sitagliptin by using an (R)-amine transaminase (ATA) created by intensive protein engineering. This process was superior with respect to optical purity, yield, and waste generation to the already established transition-metal-catalyzed production of sitagliptin. Process development, identification, and optimization of an appropriate enzyme represent inportant requirements to obtain a successful and efficient enzyme-catalyzed process. Nature has a rich reservoir from which a suitable enzyme can be found as a starting point. In contrast to classical approaches such as screening of strain collections, modern developments in the area of metagenomics offer enormous potential to screen for activity within nonculturable biodiversity. A major advancement was the development of next-generation sequencing techniques, which led to a substantial increase in the genetic information deposited in public databases; currently >20 million protein sequences are waiting to be explored. This development also led to the accumulation of protein sequences with unknown (or wrongly annotated) function, and thus a large part of this rich resource cannot be used reliably. We recently took advantage of this information and developed an in silico enzyme discovery strategy and identified 17 novel and unique (R)-selective ATAs in >5000 sequences deposited in public databases, although no gene or protein sequence was described in the literature ahead of our work. Furthermore, we could show that these (R)ATAs are synthetically useful, as demonstrated recently in the asymmetric synthesis of a set of 12 chiral amines by using 7 out of the 17 enzymes. Modern protein engineering methods rely on high-quality structural information as obtained by X-ray crystallography, which is then the basis for focused, directed evolution to improve the properties of the enzyme. Similar to developments in the genomics area, in the last years automated highthroughput methods were developed for crystallization and determination of crystal structures, which has resulted in a rapid increase in the number of solved protein structures. Still, for many enzymes no structure is available, as for example, (R)and (S)-selective ATAs, which have become very popular in the last years. Hence, structural information is highly desired to understand experimental results and to guide protein engineering. Interestingly, a trend similar to that pointed out above for the discovery of sequences exists: there is a growing number of crystal structures of enzymes that were never characterized with respect to substrate scope, enantioselectivity, or reaction specificity, and their physiological functions are also often unknown. Similar to the lack of experimental validation of annotated proteins in sequence databases, crystallized proteins with unknown function are only rarely characterized. Thus, many structures remain unexplored: For example, from 104 crystal structures of different transaminases found in the Brookhaven protein database (PDB), 46 structures do not have a literature citation that provides a description of the structure or the characterization data of the protein. Connecting the enzymatic function to unexplored proteins in the PDB could provide information that would be of diverse interest. These include protein engineering in the context of biotechnology or biochemical studies to explore enzyme mechanism. Herein we explore the crystal structures with unknown functions in the cluster of “ornithine-aminotransferase (OAT)-like proteins” (cd00610 of the NCBI conserved domain database) deposited in the PDB database. OAT are pyridoxal-5’-phosphate (PLP) dependent enzymes; they belong to PLP fold class I, which represent a very large and diverse superfamily. In the OAT subfamily, eight different enzyme activities are known (Table S1, Supporting Information). All 58 available 3D structures of this cluster show considerable similarity, but they are different in important residues in the active site that are obviously involved in substrate recognition. This search identified four structures (PDB codes: 3HMU, 3I5T, 3FCR, 3GJU), for which we could not find any information associated with their structures or functions. Additionally, these structures differed in im[a] F. Steffen-Munsberg, Dr. C. Vickers, A. Thontowi, Dr. S. Sch tzle, T. Tumlirsch, Prof. Dr. U. T. Bornscheuer, Prof. Dr. M. Hçhne Institute of Biochemistry Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany) Fax: (+ 49) 3834-86-794367 E-mail : matthias.hoehne@uni-greifswald.de uwe.bornscheuer@uni-greifswald.de [b] Dr. M. Svedendahl Humble Dept. of Organic Chemistry Arrhenius Laboratories Stockholm University, SE-106 91 Stockholm (Sweden) [c] H. Land, Prof. Dr. P. Berglund School of Biotechnology KTH Royal Institute of Technology AlbaNova University Center, 106 91 Stockholm (Sweden) Supporting information for this article contains the full experimental details and is available on the WWW under http://dx.doi.org/10.1002/ cctc.201200544.

Racemase Activity of B. cepacia Lipase Leads to Dual-Function Asymmetric Dynamic Kinetic Resolution of α-Aminonitriles†

The Diels-Alder reaction is one of the most powerful synthetic tools in organic chemistry, and asymmetric Diels-Alder catalysis allows for rapid construction of chiral carbon scaffolds. For this reason, considerable effort has been invested in developing efficient and stereoselective organo- and biocatalysts. However, Diels-Alder is a virtually unknown reaction in Nature, and to engineer an enzyme into a Diels-Alderase is therefore a challenging task. Despite several successful designs of catalytic antibodies since the 1980’s, their catalytic activities have remained low, and no true artificial ’Diels-Alderase’ enzyme was reported before 2010.In this thesis, we employ state-of-the-art computational tools to study the mechanism of organocatalyzed Diels-Alder in detail, and to redesign existing enzymes into intermolecular Diels-Alder catalysts. Papers I–IV explore the mechanistic variations when employing increasingly activated reactants and the effect of catalysis. In particular, the relation between the traditionally presumed concerted mechanism and a stepwise pathway, forming one bond at a time, is probed. Papers V–X deal with enzyme design and the computational aspects of predicting catalytic activity. Four novel, computationally designed Diels-Alderase candidates are presented in Papers VI–IX. In Paper X, a new parameterization of the Linear Interaction Energy model for predicting protein-ligand affinities is presented.A general finding in this thesis is that it is difficult to attain large transition state stabilization effects solely by hydrogen bond catalysis. In addition, water (the preferred solvent of enzymes) is well-known for catalyzing Diels- Alder by itself. Therefore, an efficient Diels-Alderase must rely on large binding affinities for the two substrates and preferential binding conformations close to the transition state geometry. In Papers VI–VIII, we co-designed the enzyme active site and substrates in order to achieve the best possible complementarity and maximize binding affinity and pre-organization. Even so, catalysis is limited by the maximum possible stabilization offered by hydrogen bonds, and by the inherently large energy barrier associated with the [4+2] cycloaddition.The stepwise Diels-Alder pathway, proceeding via a zwitterionic intermediate, may offer a productive alternative for enzyme catalysis, since an enzyme active site may be more differentiated towards stabilizing the high-energy states than for the standard mechanism. In Papers I and III, it is demonstrated that a hydrogen bond donor catalyst provides more stabilization of transition states having pronounced charge-transfer character, which shifts the preference towards a stepwise mechanism.Another alternative, explored in Paper IX, is to use an α,β -unsaturated ketone as a ’pro-diene’, and let the enzyme generate the diene in situ by general acid/base catalysis. The results show that the potential reduction in the reaction barrier with such a mechanism is much larger than for conventional Diels-Alder. Moreover, an acid/base-mediated pathway is a better mimic of how natural enzymes function, since remarkably few catalyze their reactions solely by non-covalent interactions.

Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an ω‐Transaminase

Transaminases inherently possess high enantiospecificity and are valuable tools for stereoselective synthesis of chiral amines in high yield from a ketone and a simple amino donor such as 2‐propylamine. Most known ω‐transaminases are (S)‐selective and there is, therefore, a need of (R)‐selective enzymes. We report the successful rational design of an (S)‐selective ω‐transaminase for reversed and improved enantioselectivity. Previously, engineering performed on this enzyme group was mainly based on directed evolution, with few exceptions. One reason for this is the current lack of 3D structures. We have explored the ω‐transaminase from Chromobacterium violaceum and have used a homology modeling/rational design approach to create enzyme variants for which the activity was increased and the enantioselectivity reversed. This work led to the identification of key amino acid residues that control the activity and enantiomeric preference. To increase the enantiospecificity of the C. violaceum ω‐transaminase, a possible single point mutation (W60C) in the active site was identified by homology modeling. By site‐directed mutagenesis this enzyme variant was created and it displayed an E value improved up to 15‐fold. In addition, to reverse the enantiomeric preference of the enzyme, two other point mutations (F88A/A231F) were identified. This double mutation created an enzyme variant, which displayed substrate dependent reversed enantiomeric preference with an E value shifted from 3.9 (S) to 63 (R) for 2‐aminotetralin.

Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec‐Alcohols in Organic Solvent

A method for determining lipase enantioselectivity in the transacylation of sec-alcohols in organic solvent was developed. The method was applied to a model library of Candida antarctica lipase A (CalA) variants for improved enantioselectivity (E values) in the kinetic resolution of 1-phenylethanol in isooctane. A focused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel-coated 96-well microtiter plates through a histidine tag (His6-tag), screened for transacylation of 1-phenylethanol in isooctane, and analyzed by GC. The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an E value of 100 (R), which is a dramatic improvement on the wild-type CalA (E=3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested.

7.18 C-X Bond Formation : Transaminases as Chiral Catalysts: Mechanism, Engineering, and Applications

Enantiomerically pure amines and amino acids are important building blocks in academic research as well as in industrial-scale chemical production. Transaminases are versatile enzymes providing access to such compounds of high enantiomeric excess. This chapter illustrates the available strategies with transaminases such as kinetic resolution or stereoselective synthesis and highlights many successful examples for amino acid and chiral amines synthesis. There are some known challenges linked to the use of transaminases, for example in terms of unfavorable equilibria and inhibition. Several successful examples to overcome these limitations are presented. Also, the classification of transaminases, mechanistic details, and various strategies for optimization are discussed.

YASARA: A Tool to Obtain Structural Guidance in Biocatalytic Investigations

In biocatalysis, structural knowledge regarding an enzyme and its substrate interactions complements and guides experimental investigations. Structural knowledge regarding an enzyme or a biocatalytic reaction system can be generated through computational techniques, such as homology- or molecular modeling. For this type of computational work, a computer program developed for molecular modeling of proteins is required. Here, we describe the use of the program YASARA Structure. Protocols for two specific biocatalytic applications, including both homology modeling and molecular modeling such as energy minimization, molecular docking simulations and molecular dynamics simulations, are shown. The applications are chosen to give realistic examples showing how structural knowledge through homology and molecular modeling is used to guide biocatalytic investigations and protein engineering studies.

Streamlined Preparation of Immobilized Candida antarctica Lipase B

Candida antarctica lipase B (CalB) was efficiently expressed (6.2 g L–1) in Escherichia coli by utilizing an N-terminal tag cassette and the XylS/Pm expression system in a fed-batch bioreactor; subsequent direct binding to EziG from crude extracts resulted in an immobilized catalyst with superior activity to Novozym 435.