Expanding the repertoire of enzymatic C-C bond formation with one-carbon units

C-C bonds are the basis for virtually all organic molecules on Earth. In nature, hundreds of different enzymes catalyze reactions in which C-C bonds are formed. A major task of these enzymes is the fixation of carbon, i.e. a C-C bond formation with at least one-carbon (C1) molecule. Most of these enzymes utilize electrophilic C1 species to fix carbon, while only very few use nucleophilic or radical C1 species. In this work the repertoire of enzymatic C-C bond formation was expanded by 4 new examples, 3 of which are based on a C1 nucleophile and one on a C1 radical. The thiamine diphosphate (ThDP)-dependent enzyme oxalyl-CoA decarboxylase (OXC) generates a highly reactive carbanion/enamine intermediate that is protonated and released as formyl-CoA. Here it was shown that this intermediate can also undergo C-C bond formation with an electrophilic carbonyl center. This insight allowed to establish three novel C-C bond formation reactions. First, it was demonstrated that benzaldehyde serves as an excellent electrophile, giving rise to mandelyl-CoA. In combination with oxalyl-CoA synthetase and a thioesterase this enabled the one-pot synthesis of aromatic (S)-α-hydroxy acids with enantiomeric excess up to 99%. Second, it was found that OXC can also generate the carbanion/enamine intermediate from formyl-CoA. By coupling to exergonic reactions at high CO2 concentrations, OXC was shown to be reversible, that is, it can carboxylate formyl-CoA to oxalyl-CoA. Third, OXC was engineered to accept the C1 molecule formaldehyde as substrate, producing glycolyl-CoA. Through directed evolution the catalytic efficiency was improved by a factor of ~200 and the resulting variant was successfully employed in a whole-cell biocatalyst for the production of glycolate from formaldehyde. The glycyl radical enzyme pyruvate formate-lyase (PFL) can abstract a hydrogen atom from formate, thereby generating a highly reactive formyl radical that undergoes C-C bond formation with an acetyl moiety stemming from acetyl-CoA. Here it was shown that PFL exhibits promiscuous activity with glycolyl-CoA. Based on this activity, a pathway was established in vitro that converts glycolate and formate to glycerate. These additions to the toolbox of enzymatic C-C bond formation could contribute to achieve synthetic carbon fixation pathways in the future. Such pathways are thought to be instrumental in achieving a carbon neutral economy

Molecular Basis for Converting (2S)-Methylsuccinyl-CoA Dehydrogenase into an Oxidase

Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD)-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S)-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S)-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S)-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS) based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases

A Customized Bayesian Algorithm to Optimize Enzyme-Catalyzed Reactions

Design of experiments (DoE) plays an important role in optimizing the catalytic performance of chemical reactions. The most commonly used DoE relies on the response surface methodology (RSM) to model the variable space of experimental conditions with the fewest number of experiments. However, the RSM leads to an exponential increase in the number of required experiments as the number of variables increases. Herein we describe a Bayesian optimization algorithm (BOA) to optimize the continuous parameters (e.g., temperature, reaction time, reactant and enzyme concentrations, etc.) of enzyme-catalyzed reactions with the aim of maximizing performance. Compared to existing Bayesian optimization methods, we propose an improved algorithm that leads to better results under limited resources and time for experiments. To validate the versatility of the BOA, we benchmarked its performance with biocatalytic C–C bond formation and amination for the optimization of the turnover number. Gratifyingly, up to 80% improvement compared to RSM and up to 360% improvement vs previous Bayesian optimization algorithms were obtained. Importantly, this strategy enabled simultaneous optimization of both the enzyme’s activity and selectivity for cross-benzoin condensation.ISSN:2168-048

A customized Bayesian algorithm to optimize enzyme-catalyzed reactions

Design of experiments (DoE) plays an important role in optimizing the catalytic performance of chemical reactions. The most commonly-used DoE relies on the response surface methodology (RSM) to model the variable space of experimental condi-tions with a minimal number of experiments. However, the RSM leads to an exponential increase in the number of required experiments to be evaluated as the number of variables increases. Herein we describe a Bayesian optimization algorithm (BOA) to optimize the continuous parameters (e.g. temperature, reaction time, reactant and enzyme concentrations etc.) of enzyme-catalyzed reactions with the aim of maximizing performance. Compared to existing Bayesian optimization methods, we propose an improved algorithm that leads to better results under limited resources and time for experiments. To validate the versatility of BOA for the optimization of the turnover number in enzyme-catalyzed reactions, we benchmarked its per-formance for a biocatalytic C-C bond-forming reaction as well as an amination reaction. Gratifyingly, up to 80% improvement compared to RSM and up to 360% improvement vs. previous Bayesian optimization algorithms was obtained. Importantly, this strategy enabled the simultaneous optimization of both the enzyme’s activity and chemoselectivity for a cross-benzoin condensation

Engineering a Highly Efficient Carboligase for Synthetic One-Carbon Metabolism

One of the biggest challenges to realize a circular carbon economy is the synthesis of complex carbon compounds from one-carbon (C1) building blocks. Since the natural solution space of C1–C1 condensations is limited to highly complex enzymes, the development of more simple and robust biocatalysts may facilitate the engineering of C1 assimilation routes. Thiamine diphosphate-dependent enzymes harbor great potential for this task, due to their ability to create C–C bonds. Here, we employed structure-guided iterative saturation mutagenesis to convert oxalyl-CoA decarboxylase (OXC) from Methylobacterium extorquens into a glycolyl-CoA synthase (GCS) that allows for the direct condensation of the two C1 units formyl-CoA and formaldehyde. A quadruple variant MeOXC4 showed a 100 000-fold switch between OXC and GCS activities, a 200-fold increase in the GCS activity compared to the wild type, and formaldehyde affinity that is comparable to natural formaldehyde-converting enzymes. Notably, MeOCX4 outcompetes all other natural and engineered enzymes for C1–C1 condensations by more than 40-fold in catalytic efficiency and is highly soluble in Escherichia coli. In addition to the increased GCS activity, MeOXC4 showed up to 300-fold higher activity than the wild type toward a broad range of carbonyl acceptor substrates. When applied in vivo, MeOXC4 enables the production of glycolate from formaldehyde, overcoming the current bottleneck of C1–C1 condensation in whole-cell bioconversions and paving the way toward synthetic C1 assimilation routes in vivo

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy

The Role of New Morphological Parameters Provided by the BC 6800 Plus Analyzer in the Early Diagnosis of Sepsis

Background: Late diagnosis of sepsis is associated with adverse consequences and high mortality rate. The aim of this study was to evaluate the diagnostic value of hematologic research parameters, that reflect the cell morphology of blood cells, available on the BC 6800 plus automated analyzer (Mindray) for the early detection of sepsis. Materials and methods: A complete blood count (CBC) was performed by Mindray BC 6800 Plus Analyzer in 327 patients (223 with a confirmed diagnosis of sepsis following sepsis-3 criteria, 104 without sepsis), admitted at the Intensive Care Unit of the Novara's Hospital (Italy) and in 56 patients with localized infection. Results: In univariate logistic regression, age, Hb, RDW, MO#, NMR, NeuX, NeuY, NeuZ, LymX, MonX, MonY, MonZ were associated with sepsis (p < 0.005). In multivariate analysis, only RDW, NeuX, NeuY, NeuZ, MonX and MonZ were found to be independent predictors of sepsis (p < 0.005). Morphological research parameters are confirmed to be predictors of sepsis even when analyzing the group with localized infection. Conclusions: In addition to already established biomarkers and basic CBC parameters, new morphological cell parameters can be a valuable aid in the early diagnosis of sepsis at no additional cost

The effect of cell subset isolation method on gene expression in leukocytes

Multiple scientific disciplines require the isolation of specific subsets of blood cells from patient samples for gene expression analysis by microarray or RNA-sequencing, preserving disease- or treatment-related signatures. However, little is known with respect to the impact of different cell isolation methods on gene expression and the effects of positive selection, negative selection, and fluorescence activated cell sorting (FACS) have not previously been assessed in parallel. To address this knowledge gap, CD4+ T cells, CD8+ T cells, B cells, and monocytes were isolated from blood samples from five independent donors using positive immunomagnetic selection, negative immunomagnetic selection, and FACS. We hypothesized that positive selection and FACS would yield higher purity but may have an impact on gene expression since both methods utilize antibodies that bind surface receptors of the cell type of interest. Moreover, FACS might upregulate stress response genes due to passage of the cells through the sorter. Microarray gene expression data were generated and subjected to unsupervised clustering and differential gene expression analysis. Surprisingly, these analyses revealed that gene expression signatures were more similar between cells isolated by negative selection and FACS compared to cells isolated by positive selection. Moreover, genes that are involved in the response to stress generally had the highest expression in cells isolated by negative or positive selection and not FACS. Thus, FACS is the recommended method for isolation of leukocyte subsets for gene expression studies since this method results in the purest subset populations and does not appear to induce a stress response