Control of reactive intermediates in enzymes and enzyme complexes

Enzymes are the catalysts of life. They accelerate the rate of chemical reactions that would otherwise take longer than an organism’s lifetime to take just millisecond. To achieve these remarkable rate enhancements enzymes arrange into a three dimensional fold that places its amino acids in a way, which binds the transition state of the reaction better than the substrates and products of the reaction, thereby lowering the activation energy of the reaction. Enzymes are also very specific and often only catalyze one specific chemical transformation without producing side products. They are able to achieve all this under ambient temperatures and in cells that contain over 2700 different metabolites. In this work we focus on the mechanisms enzyme use to control reactive intermediates both inside their active site and between enzymes of a metabolic pathway to avoid the formation of deleterious side products. In the first part we investigate the catalytic cycle of NAD(P)H dependent oxidoreductases. We show that the two enoyl-thioester reductases; Etr1p from Candida tropicalis of the MDR enzyme superfamily and InhA from Mycobacterium tuberculosis of the SDR enzyme superfamily form a covalent adduct between substrate and the C2 carbon of the cofactor. The observation of this reactive intermediate at the active site of enzymes from the two largest NAD(P)H dependent oxidoreductase superfamilies not only calls for a careful reconsideration of the canonical reaction mechanism of these enzymes, but also sets the basis for the development of novel tools to study, manipulate and inhibit their catalytic cycle. We demonstrate this by successfully changing the protonation specificity of Etr1p from re- to si- face. Using the molecular probe we show that a conserved threonine at the active site of Etr1p is mainly responsible for preventing the formation of a toxic side product and not for the stabilization of the wanted transition state along the reaction coordinate. This effect of destabilization of unwanted transition states, often termed ´negative catalysis´, poses a complementary mechanism of reaction control to the canonical transition state theory and is discussed in detail in this work. In the second part of this thesis we take a look at two enzyme complexes and the strategies they use to control the transfer of a reactive intermediate from one active site to the next one. The trifunctional propionyl-CoA synthase forms a closed reaction chamber to sequester the reactive acrylyl-CoA intermediate. This reaction chamber encloses all three active sites of the enzyme fusion protein, but does not show the directionality of a conventional tunnel, and the CoA ester intermediates are not covalently attached to the enzyme but freely diffuse within the compartment. The substrate channeling mechanism of the thiolase/HMG-CoA synthase complex of archaea most closely resembles the covalent swinging arm fatty acid and polyketide synthases use to channel their intermediates. In the thiolase/HMG-CoA synthase complex the intermediate is however not covalently attached, but instead tightly bound in a shared CoA binding site, enabling the pantothenyl-arm of CoA to swing from the thiolase active site to the HMG-CoA synthase active site. The two channeling systems we describe in this work therefore represent two alternative ways of channeling CoA ester intermediates in a non-covalent fashion

Control of reactive intermediates in enzymes and enzyme complexes

Enzymes are the catalysts of life. They accelerate the rate of chemical reactions that would otherwise take longer than an organism’s lifetime to take just millisecond. To achieve these remarkable rate enhancements enzymes arrange into a three dimensional fold that places its amino acids in a way, which binds the transition state of the reaction better than the substrates and products of the reaction, thereby lowering the activation energy of the reaction. Enzymes are also very specific and often only catalyze one specific chemical transformation without producing side products. They are able to achieve all this under ambient temperatures and in cells that contain over 2700 different metabolites. In this work we focus on the mechanisms enzyme use to control reactive intermediates both inside their active site and between enzymes of a metabolic pathway to avoid the formation of deleterious side products. In the first part we investigate the catalytic cycle of NAD(P)H dependent oxidoreductases. We show that the two enoyl-thioester reductases; Etr1p from Candida tropicalis of the MDR enzyme superfamily and InhA from Mycobacterium tuberculosis of the SDR enzyme superfamily form a covalent adduct between substrate and the C2 carbon of the cofactor. The observation of this reactive intermediate at the active site of enzymes from the two largest NAD(P)H dependent oxidoreductase superfamilies not only calls for a careful reconsideration of the canonical reaction mechanism of these enzymes, but also sets the basis for the development of novel tools to study, manipulate and inhibit their catalytic cycle. We demonstrate this by successfully changing the protonation specificity of Etr1p from re- to si- face. Using the molecular probe we show that a conserved threonine at the active site of Etr1p is mainly responsible for preventing the formation of a toxic side product and not for the stabilization of the wanted transition state along the reaction coordinate. This effect of destabilization of unwanted transition states, often termed ´negative catalysis´, poses a complementary mechanism of reaction control to the canonical transition state theory and is discussed in detail in this work. In the second part of this thesis we take a look at two enzyme complexes and the strategies they use to control the transfer of a reactive intermediate from one active site to the next one. The trifunctional propionyl-CoA synthase forms a closed reaction chamber to sequester the reactive acrylyl-CoA intermediate. This reaction chamber encloses all three active sites of the enzyme fusion protein, but does not show the directionality of a conventional tunnel, and the CoA ester intermediates are not covalently attached to the enzyme but freely diffuse within the compartment. The substrate channeling mechanism of the thiolase/HMG-CoA synthase complex of archaea most closely resembles the covalent swinging arm fatty acid and polyketide synthases use to channel their intermediates. In the thiolase/HMG-CoA synthase complex the intermediate is however not covalently attached, but instead tightly bound in a shared CoA binding site, enabling the pantothenyl-arm of CoA to swing from the thiolase active site to the HMG-CoA synthase active site. The two channeling systems we describe in this work therefore represent two alternative ways of channeling CoA ester intermediates in a non-covalent fashion

A common approach for absolute quantification of short chain CoA thioesters in prokaryotic and eukaryotic microbes

Background Thioesters of coenzyme A participate in 5% of all enzymatic reactions. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5–10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance of these metabolites explains the high interest to trace and quantify them in microbial cells. Results Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach assessed intracellular CoA thioesters down to the picomolar level and exhibited high precision and reproducibility for all microbes, as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA metabolism. A succinyl-CoA synthase defective mutant of C. glutamicum exhibited an unaffected level of succinyl-CoA that indicated a complete compensation by the L-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the actinomycete S. albus. The microbe revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum. Conclusions The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use one single protocol promises to facilitate automatized efforts, which rely on standardized workflows

A rapid cell-free expression and screening platform for antibody discovery

Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly

A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters

Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,β-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives), and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives). Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short- to long-chain, include branched and α,β-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s) and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories.ISSN:1420-304

Ultrastructural morphology is distinct among primary progenitor cell isolates from normal, inflamed, and cryopreserved equine hoof tissue and CD105K14 progenitor cells

The modern cell-free protein synthesis (CFPS) system is expanding the opportunity of cell-free biomanufacturing as a versatile platform for synthesizing various therapeutic proteins. However, synthesizing human protein in the bacterial CFPS system remains challenging due to the low expression level, protein misfolding, inactivity, and more. These challenges limit the use of a bacterial CFPS system for human therapeutic protein synthesis. In this study, we demonstrated the improved performance of a customized CFPS platform for human therapeutic protein production by investigating the factors that limit cell-free transcription-translation. The improvement of the CFPS platform has been made in three ways. First, the cell extract was prepared from the rare tRNA expressed host strain, and CFPS was performed with a codon-optimized gene for codon usage bias. The soluble protein yield was 15.2 times greater with the rare tRNA overexpressing host strain as cell extract and codon-optimized gene in the CFPS system. Next, we identify and prioritize the critical biomanufacturing factors for highly active crude cell lysate for human protein synthesis. Lastly, we engineer the CFPS reaction conditions to enhance protein yield. In this model, the therapeutic protein filaggrin expression was significantly improved by up to 23-fold, presenting 28 ± 5 μM of soluble protein yield. The customized CFPS system for filaggrin biomanufacturing described here demonstrates the potential of the CFPS system to be adapted for studying therapeutic proteins

Archaeal acetoacetyl-CoA thiolase/HMG-CoA synthase complex channels the intermediate via a fused CoA-binding site

International audienceMany reactions within a cell are thermodynamically unfavorable. To efficiently run some of those endergonic reactions, nature evolved intermediate-channeling enzyme complexes, in which the products of the first endergonic reactions are immediately consumed by the second exergonic reactions. Based on this concept, we studied how archaea overcome the unfavorable first reaction of isoprenoid biosynthesis-the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA catalyzed by acetoacetyl-CoA thiolases (thiolases). We natively isolated an enzyme complex comprising the thiolase and 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) from a fast-growing methanogenic archaeon,Methanothermococcus thermolithotrophicusHMGCS catalyzes the second reaction in the mevalonate pathway-the exergonic condensation of acetoacetyl-CoA and acetyl-CoA to HMG-CoA. The 380-kDa crystal structure revealed that both enzymes are held together by a third protein (DUF35) with so-far-unknown function. The active-site clefts of thiolase and HMGCS form a fused CoA-binding site, which allows for efficient coupling of the endergonic thiolase reaction with the exergonic HMGCS reaction. The tripartite complex is found in almost all archaeal genomes and in some bacterial ones. In addition, the DUF35 proteins are also important for polyhydroxyalkanoate (PHA) biosynthesis, most probably by functioning as a scaffold protein that connects thiolase with 3-ketoacyl-CoA reductase. This natural and highly conserved enzyme complex offers great potential to improve isoprenoid and PHA biosynthesis in biotechnologically relevant organisms

Awakening a latent carbon fixation cycle in Escherichia coli

Carbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. We identify the GED (Gnd–Entner–Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner–Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.ISSN:2041-172