The rising levels of atmospheric CO and its impact on climate change call for new methods to transform this greenhouse gas into beneficial compounds. Carboxylases have a significant role in the carbon cycle, converting gigatons of CO into biomass annually. One of the most effective and fastest carboxylases is crotonyl-CoA carboxylase/reductase (Ccr). To understand its underlying mechanism, we have developed computational methods and protocols based on all-atom molecular dynamics simulations. These methods provide the CO binding locations and free energy inside the active site, dependent on different conformations adopted by Ccr and the presence of the crotonyl-CoA substrate. Furthermore, the adaptive string method and quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations outline the CO fixation reaction via two different mechanisms. The direct mechanism involves a hydride transfer creating a reactive enolate, which then binds the electrophilic CO molecule, resulting in the carboxylated product. Alternatively, another mechanism involves the formation of a covalent adduct. Our simulations suggest that this adduct serves to store the enolate in a much more stable intermediate avoiding its reduction side reaction, explaining the enzyme's efficiency. Overall, this work presents computational methods for studying carboxylation reactions using Ccr as a model, providing general principles that can be applied to modeling other carboxylases.

Rubisco is the key enzyme in photosynthesis, catalyzing fixation of carbon dioxide from the atmosphere into energy storage molecules. Several inefficiencies in Rubisco limit the rate of photosynthesis, and, therefore, the growth of the plant. Rubisco is sensitive to light, making deactivation of the enzyme upon sampling likely. Moreover, the indirect methods often used to study its activity make obtaining reliable data difficult. In this Chapter, we describe an approach to generate reliable and repeatable data for Rubisco activities, activation state and abundance in plant leaves. We include methods to sample and extract proteins, minimizing Rubisco degradation and deactivation. We describe radiometric techniques to measure Rubisco activities and calculate its activation state at the time of sampling, and to quantify its abundance.

The primary role of acetyl-CoA carboxylases (ACCs) is to generate malonyl-CoA for use in fatty acid and lipid biosynthesis. However, malonyl-CoA is also used in other various metabolic processes such as secondary metabolite biosynthesis. The diverse utilization of malonyl-CoA makes ACCs targets for the development of inhibitors and also a target for engineering allosteric regulation for biofuel and secondary metabolite production. The ACC from Escherichia coli is representative most of bacterial systems, and is heteromeric, being comprised of four proteins encompassing three distinct subunits. Historically the purification of active E. coli ACC complexes has been problematic due to the reported facile dissociation into subunits. Most studies on heteromeric ACCs study the isolated subunits, which are active on their own. Nevertheless, in reconstituted systems, the subunits appear to have allosteric interactions. In this chapter, we provide methods to generate, purify and characterize these heteromeric ACCs complexes. We have used these methods to solve cryogenic electron microscopy structures of active E. coli ACC complexes. Purification of active ACC complexes represents a significant step forward in our ability to characterize how allosteric interactions and effectors alter catalytic activity. We expect future studies on the heteromeric ACC complexes will enable rational engineering of new antibiotics and biofuel production.

De novo purine biosynthesis is one of two pathways for the synthesis of purine nucleotides that are critical for numerous biological processes, most notably nucleic acid replication. Within the pathway, there is only one carbon-carbon bond formation which is the carboxylation of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). Interestingly, there are two unique pathways within purine biosynthesis to accomplish this transformation and this divergence is species specific. In humans and higher eukaryotes, CAIR is synthesized directly from AIR and carbon dioxide by the enzyme AIR carboxylase. In bacteria, yeast, fungi and plants, CAIR synthesis requires two steps. In the first, AIR is converted into the unstable carbamate, N-CAIR by the enzyme N-CAIR synthetase. N-CAIR is then converted into CAIR by transfer of the CO group from N5 to C4. This is catalyzed by the enzyme N-CAIR mutase. This divergence has provided a biochemical rationale for targeting CAIR synthesis in the development of antimicrobial agents, but recent studies have provided strong evidence that AIR carboxylase plays a critical role in several cancers. Given the significance of these enzymes as drug targets, methods to prepare and evaluate these enzymes is of interest. In this chapter, we have accumulated the most relevant assays and provided methods to synthesize the substrates and purify the enzymes.

Climate change due to anthropomorphic emissions will increase global temperature by at least 1.5 °C by the year 2030. One strategy to reduce the severity of the effects of climate change is to sequester carbon dioxide via natural biochemical cycles. Carbon monoxide dehydrogenase (CODH) has the remarkable ability to catalyze the reversible reduction of CO to CO without an overpotential and without reducing protons. It also is a key enzyme in the Wood-Ljungdahl pathway (WLP), which is the only known anaerobic carbon fixation pathway and fixes 10 % of carbon on earth every year. Characterization of this pathway is crucial because it may enable tools to mitigate climate change by using CO to produce biofuels, chemical feedstocks, and polymers. In the WLP, CODH associates with Acetyl-Coenzyme A synthase (ACS), which catalyzes the condensation of CO from CODH, a methyl group from a B-dependent methyltransferase, and CoA to form acetyl-CoA. In this complex, CO is shuttled through a 138 Å gas tunnel between the two enzymes. One valuable model for studying the CODH component of CODH/ACS is CODH-II from Carboxydothermus hydrogenoformans because it is stand-alone and is conducive to recombinant expression. Here we describe a detailed protocol for producing high-activity CODH-II in E. coli.

Dynamic structural biology enables studying biological events at the atomic scale from 10's of femtoseconds to a few seconds duration. With the advent of X-ray Free Electron Lasers (XFELs) and 4th generation synchrotrons, serial crystallography is becoming a major player for time-resolved experiments in structural biology. Despite significant progress, challenges such as obtaining sufficient amounts of protein to produce homogeneous microcrystal slurry, remain. Given this, it has been paramount to develop instrumentation that reduces the amount of microcrystal slurry required for experiments. Tape-drive systems use a conveyor belt made of X-ray transparent material as a motorized solid-support to steer deposited microcrystals into the beam. For efficient sample consumption on-demand ejectors can be synchronized with the X-ray pulses to expose crystals contained in droplets deposited on the tape. Reactions in the crystals can be triggered via various strategies, including pump-probe, substrate/ligand mixing, or gas incubation in the space between droplet ejection and X-ray illumination. Another challenge in time-resolved serial crystallography is interpreting the resulting electron density maps. This is especially difficult for metalloproteins where the active site metal is intimately involved in catalysis and often proceeds through multiple oxidation states during enzymatic catalysis. The unrestricted space around tape-drive systems can be used to accommodate complementary spectroscopic equipment. Here, we highlight tape-drive sample delivery systems for complementary and simultaneous X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) measurements. We describe how the combination of both XRD and XES is a powerful tool for time-resolved experiments at XFELs and synchrotrons.

Understanding the structures and dynamics of biomolecules and chemical compounds is crucial for deciphering their molecular functions and mechanisms. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) is a useful technique for determining structures at room temperature, while minimizing radiation damage. Time-resolved serial femtosecond crystallography (TR-SFX), which uses an optical laser or a mixing device, allows molecular dynamic visualization during a reaction at specific time points. Because the XFEL beamline has unique properties for beams and instruments, understanding the beamline system is essential to conduct TR-SFX experiments and develop related technologies. In this study, we introduce an experimental system for performing TR-SFX using a Nano Crystallography and Coherent Imaging (NCI) experimental hutch at the Pohang Accelerator Laboratory XFEL (PAL-XFEL). Specifically, we present the XFEL properties of the PAL-XFEL and the main instruments in the NCI experimental hutch. In addition, the characteristics and uses of the sample delivery methods for TR-SFX and the general sample preparation process are discussed. Furthermore, the general time schedule and experimental procedures for TR-SFX during the beam time are outlined, along with data analysis programs. This chapter contributes to understanding the performance of TR-SFX experiments conducted at the PAL-XFEL NCI experimental hutch.

The oxo-acid:ferredoxin oxidoreductase (OFOR) superfamily of enzymes are responsible for the reversible interconversion of CO and oxo-acids, using CoA-derivatives as co-substrates, and requiring redox equivalents in the form of a soluble redox-carrier protein ferredoxin (Fd). Ultimately, these enzymes are responsible for the reduction of CO to form pyruvate (in the case of PFOR) and oxo-glutarate (in the case of OGOR), by the reductive carboxylation reaction of acetyl-CoA and succinyl-CoA, respectively. The nature and kind of Fd that is the best redox-carrier to support the reductive reaction has been poorly studied to date. Most organisms that possess an OFOR contain multiple Fd redox-carriers (in addition to flavin-based flavodoxins). Here, we provide a guide for the comparison of various, similar, but non-identical Fd proteins that can interact with the PFOR from Chlorobaculum tepidum, as a model system. The conventional assay is presented, alongside an electrochemically detected assay, which demonstrates the inequivalence of Fd proteins in supporting either component of catalysis.

Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.

Analysis of protein dynamics is crucial for understanding the molecular mechanisms underlying protein function. To gain insights into the structural changes in proteins, time-resolved X-ray crystallography has been greatly advanced by the development of X-ray free-electron lasers. This tool has the potential to trace structural changes at atomic resolution; however, data interpretation and extrapolation to the solution state is often not straightforward as the in crystallo environment is not the same as it is in solution. On the other hand, time-resolved spectroscopy techniques, which have long been used for tracking protein dynamics, offer the advantage of being applicable irrespective of whether the target proteins are in crystalline or solution phase. Time-resolved IR spectroscopy is a particularly powerful technique, as it can be used on various proteins, including those that are colorless, and provides information on the chemical structures of functional sites of proteins and ligands which complements X-ray crystallography. This chapter presents the protocol for time-resolved IR microspectroscopic measurements of protein microcrystals. It includes an overview of the measurement system assembly, sample preparation, setting of experimental conditions, and time-resolved data analysis. It also describes, with examples, the usefulness of time-resolved IR measurements for comparing the dynamics between crystalline and solution conditions.

This chapter describes additions to the DIALS software package for processing serial still-shot crystallographic data, and the implementation of a pipeline, xia2.ssx, for processing and merging serial crystallography data using DIALS programs. To integrate partial still-shot diffraction data, a 3D gaussian profile model was developed that can describe anisotropic spot shapes. This model is optimised by maximum likelihood methods using the pixel-intensity distributions of strong diffraction spots, enabling simultaneous refinement of the profile model and Ewald-sphere offsets. We demonstrate the processing of an example SSX dataset where the improved partiality estimates lead to better model statistics compared with post-refined isotropic models. We also demonstrate some of the workflows available for merging SSX data, including processing time/dose resolved data series, where data can be separated at the point of merging after scaling and discuss the program outputs used to investigate the data throughout the pipeline.

CoSAXS is a state-of-the-art SAXS/WAXS beamline exploiting the high brilliance of the MAX IV 3 GeV synchrotron. By coupling advances in sample environment control with fast X-ray detectors, millisecond time-resolved scattering methods can follow structural dynamics of proteins in solution. In the present work, four sample environments are discussed. A sample environment for combined SAXS with UV-vis and fluorescence spectroscopy (SUrF) enables a comprehensive understanding of the time evolution of conformation in a model protein upon acid-driven denaturation. The use of microfluidic chips with SAXS allows the mapping of concentration with very small sample volumes. For highly reproducible sequences of mixing of components, it is possible using stopped-flow and SAXS to access the initial effects of mixing at 2 millisecond timescales with good signal to noise to allow structural interpretation. The intermediate structures in a protein are explored under light and temperature perturbations by using lasers to "pump" the protein and SAXS as the "probe". The methods described demonstrate that features at low q, corresponding to cooperative motions of the atoms in a protein, could be extracted at millisecond timescales, which results from CoSAXS being a highly-stable, low background, dedicated SAXS beamline.

In serial crystallography, large numbers of microcrystals are sequentially delivered to an X-ray beam and a diffraction pattern is obtained from each crystal. This serial approach was developed primarily for X-ray Free Electron Lasers (XFELs) where crystals are destroyed by the beam but is increasingly used in synchrotron experiments. The combination of XFEL and synchrotron-based serial crystallography enables time-resolved experiments over an extremely wide range of time domains - from femtoseconds to seconds - and allows intact or pristine structures free of the effects of radiation damage to be obtained. Several approaches have been developed for sample delivery with varying levels of sample efficiency and ease of use. In the fixed target approach, microcrystals are loaded onto a solid support which is then rastered through the X-ray beam. The key advantages of fixed targets are that every crystal loaded can be used for data collection, and that precise control of when crystals are moved into the beam allows for time-resolved experiments over a very wide range of time domains as well as multi-shot experiments characterising the effects of the X-ray beam on the sample. We describe the application of fixed targets for serial crystallography as implemented at beamline I24 at Diamond Light Source and at the SACLA XFEL. We discuss methodologies for time-resolved serial crystallography in fixed targets and describe best practices for obtaining high-quality structures covering sample preparation, data collection strategies and data analysis pipelines.

Biotin-dependent carboxylases have central roles in the metabolisms of fatty acids, amino acids and other compounds. Their functional importance is underscored by their strong conservation from bacteria to humans. These enzymes are large, multi-domain or multi-subunit complexes, and can have molecular weights of 500 to 750 kDa. Despite their large sizes, the first structures of most of these enzymes were determined using X-ray crystallography. This chapter presents various technical challenges that were overcome during their structure determination, which involves extensive optimization of the protein samples and their crystals. The cryo electron microscopy resolution revolution has made it easier to study these large complexes at the atomic level.

Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a valuable technique for time-resolved structural studies on enzymes. This method allows for the collection of high-resolution datasets of protein structures at various time points during a reaction initiated by light or mixing. Experiments are performed under non-cryogenic conditions and allow the collection of radiation damage free structures. At the European XFEL (EuXFEL), SFX experiments are mainly performed with liquid jets produced by gas dynamic virtual nozzles (GDVNs) and less frequent with a high viscous extruder (HVE). In this chapter we describe these delivery methods, with the focus on GDVNs. Instrumentation, sample requirements, and preparation steps for SFX beamtimes are discussed. Other sample delivery methods available at the EuXFEL are briefly introduced at the end of this chapter.

Carboxysomes are protein-based organelles that serve as the centerpiece of the bacterial CO concentration mechanism (CCM). They are present in all cyanobacteria and many chemoautotrophic proteobacteria and encapsulate the key enzymes for CO fixation, carbonic anhydrase and the carboxylase Rubisco, within a protein shell. The CCM actively accumulates bicarbonate in the cytosol, which diffuses into the carboxysome where carbonic anhydrase rapidly equilibrates it to CO. This creates a high CO concentration around Rubisco, ensuring efficient carboxylation. In this chapter, we present a general method for purifying α-carboxysomes and measuring carbonic anhydrase activity within these purified compartments. We exemplify this with α-carboxysomes purified from the chemoautotroph Halothiobacillus neapolitanus c2, a model organism for the α-carboxysome based CCM. However, this purification protocol can be adapted for other species, such as carboxysomes from α-cyanobacteria or carboxysomes expressed in heterologous hosts. Further, we describe the Khalifah/pH indicator assay for measuring steady-state kinetics of carbonic anhydrase catalyzed CO hydration. This method allows us to determine the kinetic parameters k, K and k/K for the purified α-carboxysomes. It uses a stopped-flow spectrometer for rapid mixing and detection, crucial for capturing the fast equilibrium between CO and bicarbonate. The reaction progress is monitored by absorbance via a pH indicator that changes color due to the proton release. While the method specifically focuses on measuring carbonic anhydrase activity on carboxysomes, it can be used to measure activity on carbonic anhydrases from other contexts as well.

The production of enzyme microcrystals for time resolved serial crystallography employing free electron laser or synchrotron radiation is a relatively new variation on traditional macromolecular crystallization for conventional single crystal X-ray analysis. While the fundamentals of macromolecular crystal growth are the same, some modifications and special considerations are in order if the objective is to produce uniform size, microcrystals in very large numbers for serial data collection. Presented here are the basic principles of protein crystal growth with particular attention to the approaches best employed to achieve the goal of microcrystals and some novel techniques, as well as old, that may be useful. Also discussed are the advantages of particular precipitants and certain methods of growing protein crystals that might be advantageous for serial data recording.

The molybdenum-containing CO dehydrogenase and the formate dehydrogenases catalyze important interconversions of one-carbon compounds, the former oxidizing CO to CO, and the latter the reversible interconversion of CO and formate. Methodologies to study these two enzymes are discussed.

Acetyl-CoA carboxylase catalyzes the first committed and regulated step in fatty acid synthesis in all animals, plants and bacteria. In most Gram-positive and Gram-negative bacteria, the enzyme is composed of three proteins: biotin carboxylase, biotin carboxyl carrier protein and carboxyltransferase. The reaction consists of two half-reactions. The first half reaction is catalyzed by biotin carboxylase and involves the carboxylation of the vitamin biotin which is covalently attached to the biotin carboxyl carrier protein. The second half reaction catalyzed by carboxyltransferase involves the transfer of the carboxyl group from biotin to acetyl-CoA to form malonyl-CoA. This chapter will describe the inhibitors of both the biotin carboxylase and carboxyltransferase components of bacterial acetyl-CoA carboxylase. Inhibitors that were used in the elucidation of the structure and mechanism of the enzyme will be discussed first. The second half will focus on inhibitors that also possess antibacterial activity.

Gamma-glutamyl carboxylase (GGCX), a polytopic membrane protein found in the endoplasmic reticulum, catalyzes the posttranslational modification of a variety of vitamin K-dependent (VKD) proteins to their functional forms. GGCX uses the free energy from the oxygenation of reduced vitamin K to remove the proton from the glutamate residue to drive VKD carboxylation. During the process of carboxylation, reduced vitamin K is oxidized to vitamin K epoxide. Therefore, GGCX is a dual-function enzyme that possesses both glutamate carboxylation and vitamin K epoxidation activities. Genetic variations in GGCX are mainly associated with bleeding disorders referred to as combined VKD coagulation factors deficiency. Comorbid non-bleeding phenotypes are also observed in patients carrying GGCX mutations. Our current knowledge concerning GGCX's function has been obtained mainly from in vitro experimentation under artificial conditions, which limits its use in interpreting the clinical phenotypes associated with GGCX genotypes. In this chapter, we describe the background, establishment, and application of mammalian cell-based assays for both the carboxylation and epoxidation activities of GGCX. We provide detailed procedures for making the reporter cell lines, creating CRISPR-Cas9-mediated gene-knockout reporter cell lines, and using these cell lines for functional studies of GGCX and its naturally occurring mutations. Combined with different reporter proteins, this cell-based strategy has been successfully used for the functional study of vitamin K-related enzymes, high-throughput screening of VKD carboxylation inhibitors, and genome-wide CRISPR-Cas9 knockout library screening of the unknown enzymes associated with vitamin K reduction.