Understanding Complex Mechanisms of Enzyme Reactivity:The Case of Limonene-1,2-Epoxide Hydrolases

Limonene-1,2-epoxide hydrolases (LEHs), a subset of the epoxide hydrolase family, present interesting opportunities for the mild, regio- and stereo- selective hydrolysis of epoxide substrates. However, moderate enantioselectivity for non-natural ligands, combined with narrow substrate specificity, has so far limited the use of LEHs as general biocatalytic tools. A detailed molecular understanding of the structural and dynamic determinants of activity may complement directed evolution approaches to expand the range of applicability of these enzymes. Herein, we have combined quantum mechanics/molecular mechanics (QM/MM) free energy calculations for the reaction with MD simulations of the enzyme internal dynamics, and the calculation of binding affinities (using the WaterSwap method) for various representatives of the enzyme conformational ensemble, to show that the presence of natural or non-natural substrates differentially modulates the dynamic and catalytic behavior of LEH. The cross-talk between the protein and the ligands favors the selection of specific substrate-dependent interactions in the binding site, priming reactive complexes to select different preferential reaction pathways. The knowledge gained via our combined approach provides a molecular rationale for LEH substrate preferences. The comprehensive strategy we present here is general and broadly applicable to other cases of enzyme–substrate selectivity and reactivity

Recent Developments in Flavin-Based Catalysis:Enzymatic Sulfoxidation

The synthesis of optically active sulfoxides, compounds due to their unique properties, has been a main target for synthetic organic chemistry. Recent efforts in the field of biocatalysis have allowed the preparation of enantiopure sulfoxides starting from the corresponding sulfides while using relatively mild conditions. In fact, several different types of redox biocatalysts have been found that can catalyze enantio- and/or regioselective sulfoxidations. The most promising group of enzymes able to perform selective sulfoxidations is the flavin-containing monooxygenases (FMOs). Enzymes containing a flavin cofactor have already been widely studied and used in organic synthesis, especially in reduction and/or oxidation processes. This chapter highlights the recent efforts in the preparation of chiral sulfoxides catalyzed by different types of flavoenzymes, with special attention to the parameters that can improve their catalytic properties. Novel approaches that allow performing selective sulfoxidations in which modified flavin systems are used are also discussed.</p

Preparation of enantiopure epoxides by biocatalytic kinetic resolution

Molecular chirality is of great importance for many processes in biological systems. Examples are interactions with enzymes and receptor systems for hormones, sensory recognition and drug metabolism. Activation of biological activity when initiated by interaction with bioactive compounds is highly based on complementary stereochemistry. Synthetic bioactive compounds, like agrochemicals and pharmaceuticals, are therefore now preferably produced as single enantiomers.Various bioactive compounds can be effectively prepared from enantiopure epoxides. These chiral building blocks can be used to introduce one or two adjacent chirality centers in a target molecule. In the present study, enzymatic kinetic resolution via direct epoxide ring-opening has been studied for the preparation of enantiopure epoxides. For this method, the biocatalytic activities of a bacterial epoxide carboxylase and a yeast epoxide hydrolase have been explored (Scheme 1).; Scheme 1. Kinetic resolution of cis - (R 1 = H, R 2 = alkyl) and trans - (R 1 = alkyl, R 2 = H) 2,3-disubstituted epoxides by enzymatic nucleophilic ring-opening. XH 2 represents NADPH or a reducing dithiol compound. (Ketone: R= alkyl).Bacterial epoxide carboxylaseIn alkene-utilizing bacteria, epoxides are generated by monooxygenases and subsequently further degraded. The epoxide-degrading enzyme system has been recently identified as an epoxide carboxylase. However, in the absence of CO 2 , the reaction catalyzed is actually an isomerization of the epoxide. The enzyme can therefore also be regarded as an epoxide isomerase.Epoxide carboxylase/isomerase from Xanthobacter Py2 was found enantioselective in the conversion of 2,3-disubstituted aliphatic epoxides (Scheme 1). Only (2 S )-enantiomers were converted by propene-grown cells of Xanthobacter Py2 and (2 R )-enantiomers were thus resolved from a racemic mixture with almost maximal feasible yield ( Chapter 2 ). Aliphatic 1,2-epoxides, being intermediates in 1-alkene metabolism, were converted without remarkable enantioselectivity.In the subsequent study, epoxide bioconversion was studied in more detail ( Chapter 3 ). Epoxide substrates were found to be converted to ketones via an hydroxy intermediate. The enzymatic reaction was dependent on NAD +and a reducing cofactor, which could be replaced by synthetic dithiol compounds. Based on these findings, a four-step reaction mechanism was proposed starting from nucleophilic ring-opening of the epoxide. Follow-up studies by various other research groups concentrated on the metabolism of the physiological substrate 1,2-epoxypropane. By these studies, the enzymatic steps involved in 1,2-epoxypropane metabolism were further elucidated.Yeast epoxide hydrolaseCofactor-independent microbial epoxide hydrolases are generally regarded as attractive biocatalytical tools. Epoxide hydrolase catalyzed ring-opening of epoxides can be exploited for the production of enantiopure epoxides and vicinal diols (Scheme 1). The biocatalytical potential of microbial epoxide hydrolases has been first recognized in studies using enzymes from fungal and bacterial origin. Epoxide hydrolase activities in yeasts have been subsequently explored.Yeast epoxide hydrolase (YEH) activity has been demonstrated for the hydrolysis of various structurally divergent epoxides by Rhodotorula glutinis ATCC 201718 ( Chapter 4 ). Very high enantioselectivities were determined in the hydrolysis of 2,3-disubstituted aryl and aliphatic epoxides (Scheme 1). Asymmetric hydrolysis of meso epoxides has been demonstrated and interestingly this property has been restricted to yeasts in particular.Typical other substrates for the yeast enzyme are monosubstituted aliphatic epoxides. Enantiomeric discrimination was expected to be difficult for these highly flexible and rather 'slim' molecules. Therefore, kinetic resolution of a homologous range of aliphatic 1,2-epoxides by Rhodotorula glutinis was studied in more detail ( Chapter 5 ). Activities as well as enantioselectivities were found to be strongly influenced by the chain length of the substrate used. Best results were obtained in the resolution of 1,2-epoxyhexane.Preparative-scale YEH-catalyzed resolution Preparative-scale kinetic resolutions were investigated with 1,2-epoxyhexane as a model substrate and cells of Rhodotorula glutinis as biocatalyst ( Chapter 6 ). Scaling-up was hampered by inhibition due to substrate toxicity, and to an even higher extend, by product inhibition of the formed diol. A critical inhibitory diol concentration was determined as 50 mM for 1,2-hexanediol. For protection against high epoxide concentrations, aqueous/organic two-phase reaction media were tested. Long-chain aliphatic alkanes were suitable biocompatible solvents and dodecane was selected for further applications. However, dodecane and other biocompatible solvents gave no protection towards the diol.Preparative-scale resolution of 1,2-epoxyhexane (22 g) was performed successfully in an aqueous/organic two-phase membrane bioreactor. A cascade configuration of two hollow-fiber membrane modules was used ( i ) to separate the biocatalyst from the organic solvent containing feed solution with concentrated epoxide (2 M) and ( ii ) to remove inhibitory amounts of diol.In a modified process design, the membrane bioreactor was used for continuous extractive kinetic resolution of 1,2-epoxyhexane (1 M in dodecane). Under these conditions, enantiopure ( S )-epoxide (13 g) was obtained in the effluent solvent phase. The process allowed long-term continuous production of enantiopure epoxide without the need for complete resolution of the racemic substrate in the feed reservoir. Optimization of this process will however be necessary for improvement of the productivity.</p

ENGINEERING OF RECOMBINANT WHOLE-CELL BIOCATALYSTS FOR GREEN AND EFFICIENT PRODUCTION OF CHIRAL CHEMICALS

Ph.DDOCTOR OF PHILOSOPH

Discovering novel hydrolases from hot environments

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this recordNovel hydrolases from hot and other extreme environments showing appropriate performance and/or novel functionalities and new approaches for their systematic screening are of great interest for developing new processes, for improving safety, health and environment issues. Existing processes could benefit as well from their properties. The workflow, based on the HotZyme project, describes a multitude of technologies and their integration from discovery to application, providing new tools for discovering, identifying and characterizing more novel thermostable hydrolases with desired functions from hot terrestrial and marine environments. To this end, hot springs worldwide were mined, resulting in hundreds of environmental samples and thousands of enrichment cultures growing on polymeric substrates of industrial interest. Using high-throughput sequencing and bioinformatics, 15 hot spring metagenomes, as well as several sequenced isolate genomes and transcriptomes were obtained. To facilitate the discovery of novel hydrolases, the annotation platform Anastasia and a whole-cell bioreporter-based functional screening method were developed. Sequence-based screening and functional screening together resulted in about 100 potentially new hydrolases of which more than a dozen have been characterized comprehensively from a biochemical and structural perspective. The characterized hydrolases include thermostable carboxylesterases, enol lactonases, quorum sensing lactonases, gluconolactonases, epoxide hydrolases, and cellulases. Apart from these novel thermostable hydrolases, the project generated an enormous amount of samples and data, thereby allowing the future discovery of even more novel enzymes.European CommissionEuropean Union FP

Oxidation of Ketones: A (Chemo-) Enzymatic Approach Using Oxygenases and Hydrolases

Oxidation reactions are important in organic chemistry as well as in nature. In industry, oxidations are commonly used for the synthesis of chemicals and pharmaceuticals, however such processes have a number of limitations, they use chlorinated solvents, stoichiometric oxidation reagents, and in some cases the reagents that have risks of explosion during transportation and storage. This has called for more environment-friendly alternative technologies for oxidation reactions. Baeyer-Villiger oxidation is a reaction in which a ketone is oxidized to an ester or a cyclic ketone to a lactone by treatment with peroxyacids. Lactones constitute an important group of chemicals used in flavors, fragrances, pharmaceutical intermediates and polymer building blocks. The work presented in this thesis concerns enzymes, including Baeyer-Villiger monooxygenases (BVMOs) that catalyse the Baeyer-Villiger oxidation using molecular oxygen as an oxidant, and perhydrolytic enzymes that can be used for in situ generation of peracid for oxidation of cyclic ketones. A simple colorimetric method was developed for detection of BVMO activity and was based on the formation of a purple colored product between an enolizable ketone and 3,5-dinitrobenzoic acid in an alkaline solution. The method was shown to have potential for screening of both wild type and recombinant microbial cells as well as for quantitative measurement of BVMO activity. Further, a recombinant BVMO from a strain of Dietzia was characterized. The sequence of the enzyme suggested that it is related to Ethionamide monooxygenases. The recombinant enzyme was active in whole cells and crude lysate but lost activity on purification. The enzyme was shown to have high activity towards several linear alkenes, and was also moderately active towards cyclobutanone, phenylacetone and thioanisole. Two perhydrolytic enzymes able to produce peracids from a carboxylic ester and hydrogen peroxide were studied for oxidation of cyclohexanone to caprolactone, a chemical of immense importance. The enzymes were immobilized as cross-linked enzyme aggregates (CLEAs). The well-studied lipase B from Candida antarctica (CaLB) gave a maximal caprolactone yield of 80% with ethyl acetate as acyl donor. The perhydrolase was able to produce peracids in an aqueous medium with ethylene glycol diacetate and hydrogen peroxide, and gave caprolactone yield of 70%. In both cases the formation as acetic acid as a coproduct showed to be an important factor for the deactivation of the enzyme. The use of monooxygenases, lipases and perhydrolases for the Baeyer-Villiger reaction constitutes a greener alternative to traditional chemical processes but the problem of enzyme stability remains to be solved

Biotransformation of alkenes by Rhodococcus OU

Epoxides are an important class of synthons, produced in large quantities (notably epoxyethane and epoxypropane) for the manufacture of polymers. Reaction of epoxides with nucleophiles is stereospecific, offering a route to homochiral pharmaceuticals and agrochemicals from homochiral epoxides. With few exceptions, production of homochiral epoxides is difficult to achieve by chemical syntheses alone. However, alkene epoxidation by monooxygenase enzymes has been shown to proceed with a high degree of stereoselectivity in many instances. The aims of this project were to isolate microorganisms capable of converting alkenes to epoxides and to select the most suitable isolate for further characterization. Two Gram positive bacteria were isolated using α-methylstyrene (αMeS-1) and octane (Rhodococcus OU). The latter isolate was subjected to a more detailed study. Rhodococcus OU were shown to convert a range of structurally diverse alkenes to their corresponding epoxides: aliphatic (1-alkenes from propene to 1-tetradecene and cis-2- butene), alicyclic (cyclopentene and cyclohexene) and aromatic (styrene, allylbenzene and allylphenylether) alkenes. Alcohols, aldehydes and ketones were produced from alkenes with sub-terminal double bonds, in addition to epoxides. The stereoselectivity of alkene epoxidation was investigated by chiral HPLC. Partial resolution of (±)-1,2-epoxy-3-phenoxypropane was achieved, although assignment of the two peaks was not possible. Biotransformation of allyl phenyl ether to 1,2-epoxy-3- phenoxypropane was shown to proceed in a stereoselective manner. Problems associated with the chiral analysis of styrene oxide were not overcome, but preliminary results suggest that Rhodococcus OU is completely stereoselective for (R)-(+)-styrene oxide. Alkene epoxidation was shown to occur by one or more monooxygenase enzymes, expression of which is inducible by growth on n-alkanes but not by growth on 1-hexanol or glucose. Catalytic activity was retained after freezing in liquid nitrogen and storage at -70°C, only diminishing after being stored in excess of two months. Optimization of 1-alkene epoxidation was investigated, with particular reference to 1-hexene epoxidation. The specific rate of 1-alkene epoxidation (qp) was shown to increase as chain length decreased, correlating with an increase in 1-alkene solubility in water. Increasing the biocatalyst concentration resulted in an increase in volumetric productivity, but a decrease in qp. Epoxidation of 1-hexene showed saturable kinetics, qp being maximal between 0.05% to 0.10% (v/v) 1-hexene, whilst the final concentration of 1,2-epoxyhexane attained was concentration-dependant up to 0.40% (v/v) 1-hexene (the maximum concentration tested). Addition of co-substrates was not shown to enhance qp

Synthetic and Biocatalytic Methods for the Chemoenzymatic Production of Novel Cryptophycin Anticancer Agents.

The cryptophycin family of cyanobacterial peptolides contains exceptionally potent antimitotic anticancer agents. Active at levels significantly lower than currently approved cancer therapies, synthetic cryptophycin 52 was also effective against multi-drug resistant cancers. Phase II clinical trials revealed minor peripheral neurotoxicity, however, making synthetic derivatization a priority for the development of safe, effective cryptophycins for the treatment of cancer. Specifically, incorporation of heterocycles on unit A of cryptophycin was proposed to increase the solubility and stability, as well as reduce toxicity of the parent drugs. To this end, an efficient and divergent synthetic route to unit A analogues was developed and optimized for the production of a diverse library of heterocyclic functionality. Incorporation with units B, C, and D yielded fully elaborated, SNAc-thioester bound seco-cryptophycins as substrates for macrocyclization. Cryptophycin thioesterase (CrpTE) activity was reconstituted in vitro and used to demonstrate impressive inherent flexibility for a suite of heterocyclic substrates. CrpTE was then optimized for activity and displayed little preference for reaction temperature, buffer pH, or DMSO concentration. Incredibly, CrpTE was active at up to 50% DMSO and in a variety of organic solvents. In fact, a novel cosolvent system of 20% diglyme with 1% MCD more than doubled CrpTE conversion with a natural substrate mimic and proved to be an effective strategy for the chemoenzymatic cyclization of the 2-pyridyl derivatized cryptophycin 500. Joined with the complementary heterocyclic substrate flexibility of cryptophycin epoxidase (CrpE), a powerful method now exists to produce unique cryptophycins in a campaign to access better anticancer agents. This chemoenzymatic method should also provide a means to construct affinity probes for mechanism of action studies and interrogation of CrpTE and CrpE active site architecture.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/102427/1/kbolduc_1.pd