Rigorous Model-Based Design and Experimental Verification of Enzyme-Catalyzed Carboligation under Enzyme Inactivation

Enzyme catalyzed reactions are complex reactions due to the interplay of the enzyme, the reactants, and the operating conditions. To handle this complexity systematically and make use of a design space without technical restrictions, we apply the model based approach of elementary process functions (EPF) for selecting the best process design for enzyme catalysis problems. As a representative case study, we consider the carboligation of propanal and benzaldehyde catalyzed by benzaldehyde lyase from Pseudomonas fluorescens (PfBAL) to produce (R)-2-hydroxy-1-phenylbutan-1-one, because of the substrate dependent reaction rates and the challenging substrate dependent PfBAL inactivation. The apparatus independent EPF concept optimizes the material fluxes influencing the enzyme catalyzed reaction for the given process intensification scenarios. The final product concentration is improved by 13% with the optimized feeding rates, and the optimization results are verified experimentally. In general, the rigorous model driven approach could lead to selecting the best existing reactor, designing novel reactors for enzyme catalysis, and combining protein engineering and process systems engineering concept

Synthetic in vitro transcriptional oscillators

The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system's robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells

Combining Genomics, Metabolome Analysis, and Biochemical Modelling to Understand Metabolic Networks

Now that complete genome sequences are available for a variety of organisms, the elucidation of gene functions involved in metabolism necessarily includes a better understanding of cellular responses upon mutations on all levels of gene products, mRNA, proteins, and metabolites. Such progress is essential since the observable properties of organisms – the phenotypes – are produced by the genotype in juxtaposition with the environment. Whereas much has been done to make mRNA and protein profiling possible, considerably less effort has been put into profiling the end products of gene expression, metabolites. To date, analytical approaches have been aimed primarily at the accurate quantification of a number of pre-defined target metabolites, or at producing fingerprints of metabolic changes without individually determining metabolite identities. Neither of these approaches allows the formation of an in-depth understanding of the biochemical behaviour within metabolic networks. Yet, by carefully choosing protocols for sample preparation and analytical techniques, a number of chemically different classes of compounds can be quantified simultaneously to enable such understanding. In this review, the terms describing various metabolite-oriented approaches are given, and the differences among these approaches are outlined. Metabolite target analysis, metabolite profiling, metabolomics, and metabolic fingerprinting are considered. For each approach, a number of examples are given, and potential applications are discussed

The effect of phenytoin, phenobarbitone, dexamethasone and flurbiprofen on misonidazole neurotoxicity in mice.

Using a quantitative cytochemical technique for measuring beta-glucuronidase activity in the peripheral nerves of mice, we have investigated the effectiveness of four potential adjuncts for reducing the dose limiting neurotoxicity of misonidazole (MISO) in the clinic. Under the conditions used, the most effective adjunct was the steroid anti-inflammatory agent dexamethasone. When given over the week previous to MISO treatment, this agent almost completely eliminated the MISO neurotoxicity as determined at week 4 after commencement of MISO dosing. The second most effective adjunct was phenytoin, the third flurbiprofen and the last adjunct, phenobarbitone, was ineffective. Dexamethasone, phenytoin and phenobarbitone all reduced the clearance half-life of MISO and hence the drug exposure dose calculated as the area under the curve of MISO tissue concentration against time. However, no correlation was evident with these parameters and MISO neurotoxicity in the mouse. Dexamethasone, whilst affording protection against MISO toxicity, did not alter the radiosensitivity of the anaplastic MT tumour

Vitamin K1 pharmacokinetics in a clinical study and VKORC1 enzyme kinetics using HPLC methodology

The main objective of the present work by means of a phase I clinical study was to determine inter-individual variance in pharmacokinetics of intravenous and oral phylloquinone (vitamin K1) mixed micelles formulation in humans as well as to explore a possible effect of the VKORC1 promoter polymorphism c.-1639 G>A on the metabolism of phylloquinone. The pharmacokinetics of phylloquinone mixed micelles formulation (Konakion® MM 2 mg) were evaluated, in healthy human adult volunteers (n=30; 15 m, 15 f) using an open phase I design protocol upon oral and intravenous administration. The probands were subjected equally distributed (n=10; 5 m, 5 f) to three genotype-specific groups regarding VKORC1 promoter polymorphism c.-1639 G>A (GG, AG and AA) to explore their relationship to specific pharmacokinetic parameters. Phylloquinone serum levels were determined by reversed phase HPLC with fluorometric detection after post-column zinc reduction. The method proved to be highly accurate, robust and reliable and showed a limit of detection and quantification of 0.015 ng mL-1 and 0.15 ng mL-1, respectively. Measured phylloquinone serum concentrations were subjected to pharmacokinetic evaluation using a non-compartment analysis. Pharmacokinetic analysis of serum phylloquinone concentration versus time profiles revealed significant differences in main pharmacokinetic parameters. Significant inter-individual pharmacokinetic variance of vitamin K fate in the human body could be indicated. Further, an influence of the VKORC1 promoter polymorphism c.-1639 G>A on the pharmacokinetic properties of phylloquinone in humans was shown. Significant differences in main pharmacokinetic parameters such as bioavailability and terminal half-life between groups suggest corresponding differences in processing of vitamin K in the human body. The relevance of polymorphisms in CYP4F2 and ABCC6 in this regard must be further elucidated in an enlarged sampling. The clinical importance of potential genetic determinants of vitamin K status should be further investigated with respect to effects on absorption, distribution, metabolism and elimination of vitamin K. Furthermore, the enzymatic characteristics of the VKORC1 were examined by studying its enzyme kinetics. Comparing vitamin K1 and K2 as substrates and their apparent kinetic constants Km and Vmax, the binding affinity of vitamin K2 epoxide to the VKORC1 appears to be higher while vitamin K1 epoxide seems to bind in a weaker manner to the enzyme

Multiple nucleophilic elbows leading to multiple active sites in a single module esterase from Sorangium cellulosum

The catalytic residues in carbohydrate esterase enzyme families constitute a highly conserved triad: serine, histidine and aspartic acid. This catalytic triad is generally located in a very sharp turn of the protein backbone structure, called the nucleophilic elbow and identified by the consensus sequence GXSXG. An esterase from Sorangium cellulosum Soce56 that contains five nucleophilic elbows was cloned and expressed in Escherichia coli and the function of each nucleophilic elbowed site was characterized. In order to elucidate the function of each nucleophilic elbow, site directed mutagenesis was used to generate variants with deactivated nucleophilic elbows and the functional promiscuity was analyzed. In silico analysis together with enzymological characterization interestingly showed that each nucleophilic elbow formed a local active site with varied substrate specificities and affinities. To our knowledge, this is the first report presenting the role of multiple nucleophilic elbows in the catalytic promiscuity of an esterase. Further structural analysis at protein unit level indicates the new evolutionary trajectories in emerging promiscuous esterases. NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Structural Biology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Structural Biology, 2015. http://dx.doi.org/10.1016/j.jsb.2015.04.00

Mechanistic characterization of acetic acid resistance enzymes of Acetobacer aceti

Acetobacter aceti (A. aceti) is a Gram-negative, acidophilic bacterium that is used for the industrial production of acetic acid from ethanol. Oxidation of ethanol by membrane-bound oxidoreductases provides energy for A. aceti and the production of high concentrations of acetic acid is an effective defense mechanism. Acetic acid diffuses through cell membranes at low pH and effectively kills many bacteria, including E. coli, at low millimolar concentrations. The ability of A. aceti to thrive in molar concentrations of acetic acid is partially due to the twin subjects of this thesis, the acetic acid resistance factors AarA (citrate synthase, AaCS) and AarC (succinyl-CoA:acetate CoA-transferase). AarC and CS exploit the distinct properties of the thioester moiety in acetyl-CoA (AcCoA) to catalyze different reversible reactions. AarC takes advantage of the relatively high leaving group potential of the CoA thiolate (anionic or RS– form) to transfer the acetyl moiety of AcCoA to an active site glutamate. In contrast, CS uses the relatively acidic carbon adjacent to the thioester moiety to catalyze a Claisen/aldol condensation reaction that forms a new carbon-carbon bond. Class I CoA-transferases such as AarC produce acylglutamyl anhydride intermediates that undergo attack by the CoA thiolate on one of its two carbonyl carbon atoms, forming distinct internal or external tetrahedral intermediates less than 3 Å apart. In this study, crystal structures were used to examine the role of the internal oxyanion hole residue Asn347 and the highly conserved elements of the external oxyanion hole. First, a structure of the active mutant AarC-N347A bound to CoA revealed both solvent substitution for the deleted carboxamide and displacement of the adjacent Glu294. This indicates that Asn347 both polarizes and orients the essential glutamate Glu294. Second, AarC was crystallized with the nonhydrolyzable AcCoA analogue dethiaacetyl-CoA (AcMX) in an attempt to trap a closed enzyme complex containing a stable analogue of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and a truncated AcMX, an unexpected result hinting at what would have been an unprecedented cleavage of the ketone moiety into an acetyl group and the CoA analogue MX. Solution studies confirmed that AcMX decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. Authentic MX was synthesized to evaluate the hypothesis that it is derived from AcMX. A crystal structure of AarC bound to MX showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even for crystals grown in the presence of exogenous acetate. These findings imply that AcMX degradation results in the production of an activated acetyl donor; a working hypothesis involving ketone oxidation is offered. Moreover, the ability of MX to induce full active site closure suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. The remainder of this thesis concerns the enzyme CS, an essential part of central metabolism in aerobes and many other organisms. The CS reaction comprises two successive reactions: a Claisen/aldol condensation of AcCoA and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that close the active site assemble a catalytically competent condensation active site. The 2.2 Å resolution crystal structure of CS from the thermoacidophile Thermoplasma acidophilum (TpCS) fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an open structure that, when compared with several liganded TpCS structures, helps to define a complete path for active site closure. (Abstract shortened by ProQuest.