Binding Interactions and Inhibition Mechanisms of Gold Complexes in Thiamine Diphosphate-Dependent Enzymes.

Thiamine diphosphate (ThDP)-dependent enzymes possess the unique ability to generate a carbene within their active site. In this study, we sought to harness this carbene to produce a Au(I) N-heterocyclic complex directly in the active site of ThDP enzymes, thereby establishing a novel platform for artificial metalloenzymes. Because direct metalation of ThDP proved challenging, we synthesized a ThDP mimic that acts as a competitive inhibitor with a high affinity (Ki = 1.5 µM). Upon metalation with Au(I), we observed that the complex became a more potent inhibitor (Ki = 0.7 µM). However, detailed analysis of the inhibition mode, native mass spectrometry, and size exclusion experiments revealed that the complex does not bind specifically to the active site of ThDP enzymes. Instead, it exhibits unspecific binding and exceeds the 1:1 stoichiometry. Similar binding patterns were observed for other Au(I) species. These findings prompt an important question regarding the inherent propensity of ThDP enzymes to bind strongly to Au. If this phenomenon holds true, it could pave the way for the development of Au-based drugs targeting these enzymes.

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.

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.

Underground isoleucine biosynthesis pathways in E. coli.

The promiscuous activities of enzymes provide fertile ground for the evolution of new metabolic pathways. Here, we systematically explore the ability of E. coli to harness underground metabolism to compensate for the deletion of an essential biosynthetic pathway. By deleting all threonine deaminases, we generated a strain in which isoleucine biosynthesis was interrupted at the level of 2-ketobutyrate. Incubation of this strain under aerobic conditions resulted in the emergence of a novel 2-ketobutyrate biosynthesis pathway based upon the promiscuous cleavage of O-succinyl-L-homoserine by cystathionine γ-synthase (MetB). Under anaerobic conditions, pyruvate formate-lyase enabled 2-ketobutyrate biosynthesis from propionyl-CoA and formate. Surprisingly, we found this anaerobic route to provide a substantial fraction of isoleucine in a wild-type strain when propionate is available in the medium. This study demonstrates the selective advantage underground metabolism offers, providing metabolic redundancy and flexibility which allow for the best use of environmental carbon sources.

Oxalyl-CoA Decarboxylase Enables Nucleophilic One-Carbon Extension of Aldehydes to Chiral α-Hydroxy Acids.

The synthesis of complex molecules from simple, renewable carbon units is the goal of a sustainable economy. Here we explored the biocatalytic potential of the thiamine-diphosphate-dependent (ThDP) oxalyl-CoA decarboxylase (OXC)/2-hydroxyacyl-CoA lyase (HACL) superfamily that naturally catalyzes the shortening of acyl-CoA thioester substrates through the release of the C1 -unit formyl-CoA. We show that the OXC/HACL superfamily contains promiscuous members that can be reversed to perform nucleophilic C1 -extensions of various aldehydes to yield the corresponding 2-hydroxyacyl-CoA thioesters. We improved the catalytic properties of Methylorubrum extorquens OXC by rational enzyme engineering and combined it with two newly described enzymes-a specific oxalyl-CoA synthetase and a 2-hydroxyacyl-CoA thioesterase. This enzymatic cascade enabled continuous conversion of oxalate and aromatic aldehydes into valuable (S)-α-hydroxy acids with enantiomeric excess up to 99 %.

A critical comparison of cellular and cell-free bioproduction systems.

Conversion of biological feedstocks into value-added chemicals is mostly performed via microbial fermentation. An emerging alternative approach is the use of cell-free systems, consisting of purified enzymes and cofactors. Unfortunately, the in vivo and in vitro research communities rarely interact, which leads to oversimplifications and exaggerations that do not permit fair comparison of the two strategies and impede synergistic interactions. Here, we provide a comprehensive account for the advantages and drawbacks associated with each strategy, and further discuss recent research efforts that aim to breach the limits of cellular and cell-free production. We also explore emerging hybrid solutions that integrate the benefits of both worlds and could expand the boundaries of biosynthesis.

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 synthetic pathway for the fixation of carbon dioxide in vitro.

Carbon dioxide (CO2) is an important carbon feedstock for a future green economy. This requires the development of efficient strategies for its conversion into multicarbon compounds. We describe a synthetic cycle for the continuous fixation of CO2 in vitro. The crotonyl-coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a reaction network of 17 enzymes that converts CO2 into organic molecules at a rate of 5 nanomoles of CO2 per minute per milligram of protein. The CETCH cycle was drafted by metabolic retrosynthesis, established with enzymes originating from nine different organisms of all three domains of life, and optimized in several rounds by enzyme engineering and metabolic proofreading. The CETCH cycle adds a seventh, synthetic alternative to the six naturally evolved CO2 fixation pathways, thereby opening the way for in vitro and in vivo applications.

High-speed, high-resolution, multielemental LA-ICP-TOFMS imaging: part II. critical evaluation of quantitative three-dimensional imaging of major, minor, and trace elements in geological samples.

Here we describe the capabilities of laser-ablation coupled to inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) for high-speed, high-resolution, quantitative three-dimensional (3D) multielemental imaging. The basic operating principles of this instrumental setup and a verification of 3D quantitative elemental imaging are provided. To demonstrate the potential of 3D LA-ICP-TOFMS imaging, high-resolution multielement images of a cesium-infiltrated Opalinus clay rock were recorded using LA with a laser-spot diameter of 5 µm coupled to ICP-TOFMS. Quantification of elements ablated from each individual laser pulse was carried out by 100% mass normalization, and the 3D elemental concentration images generated match well with the expected distribution of elements. After laser-ablation imaging, the sample surface morphology was investigated using confocal microscopy, which showed substantial surface roughness and evidence of matrix-dependent ablation yields. Depth assignment based on ablation yields from heterogeneous materials, such as Opalinus clay rock, will remain a challenge for 3D LA-ICPMS imaging. Nevertheless, this study demonstrates quantitative 3D multielemental imaging of geological samples at a considerably higher image-acquisition speed than previously reported, while also offering high spatial resolution and simultaneous multielemental detection.