Methods in enzymology最新文献

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.

While enzymes are potentially useful in various applications, their limited operational stability and production costs have led to an extensive search for stable catalytic agents that will retain the efficiency, specificity, and environmental-friendliness of natural enzymes. Despite extensive efforts, there is still an unmet need for improved enzyme mimics and novel concepts to discover and optimize such agents. Inspired by the catalytic activity of amyloids and the formation of amyloid-like assemblies by metabolites, our group pioneered the development of novel metabolite-metal co-assemblies (bio-nanozymes) that produce nanomaterials mimicking the catalytic function of common metalloenzymes that are being used for various technological applications. In addition to their notable activity, bio-nanozymes are remarkably safe as they are purely composed of amino acids and minerals that are harmless to the environment. The bio-nanozymes exhibit high efficiency and exceptional robustness, even under extreme conditions of temperature, pH, and salinity that are impractical for enzymes. Our group has recently also demonstrated the formation of ordered amino acid co-assemblies showing selective and preferential interactions comparable to the organization of residues in folded proteins. The identified bio-nanozymes can be used in various applications including environmental remediation, synthesis of new materials, and green energy.

Many self-assembling peptides can form amyloid like structures with different sizes and morphologies. Driven by non-covalent interactions, their aggregation can occur through distinct pathways. Additionally, they can bind metal ions to create enzyme like active sites that allow them to catalyze diverse reactions. Due to the non-crystalline nature of amyloids, it is quite challenging to elucidate their structures using experimental spectroscopic techniques. In this aspect, molecular dynamics (MD) simulations provide a useful tool to derive structures of these macromolecules in solution. They can be further validated by comparing with experimentally measured structural parameters. However, these simulations require a multi-step process starting from the selection of the initial structure to the analysis of MD trajectories. There are multiple force fields, parametrization protocols, equilibration processes, software and analysis tools available for this process. Therefore, it is complicated for non-experts to select the most relevant tools and perform these simulations effectively. In this chapter, a systematic methodology that covers all major aspects of modeling of catalytic peptides is provided in a user-friendly manner. It will be helpful for researchers in this critical area of research.

Plants are prolific producers of terpenoids. Terpenoid biosynthesis is initiated by terpene synthases (TPS). In plants, two types of terpenes synthase genes are recognized: typical plant TPS genes and microbial-terpene synthase like-genes (MTPSL). While TPS genes are ubiquitous in land plants, MTPSL genes appear to be restricted to non-seed land plants. Evolutionarily, TPS genes are specific to land plants, whereas MTPSL genes have related counterparts in other organisms, especially fungi and bacteria. The presence of microbial type TPS in plants, fungi and bacteria, with the latter two often being associated with plants, poses a challenge in accurately identifying bona fide MTPSL genes in plants. In this chapter, we present bioinformatic procedures designed to identify MTPSL genes in sequenced plant genomes and/or transcriptomes. Additionally, we outline validation methods for confirming the identified microbial-type TPS genes as genuine plant genes. The method described in this chapter can also be adopted to analyze microbial type TPS in organisms other than plants.

Structural biology research of terpene synthases (TSs) has provided a useful basis to understand their catalytic mechanisms in producing diverse terpene products with polycyclic ring systems and multiple chiral centers. However, compared to the large numbers of>95,000 terpenoids discovered to date, few structures of TSs have been solved and the understanding of their catalytic mechanisms is lagging. We here (i) introduce the basic catalytic logic, the structural architectures, and the metal-binding conserved motifs of TSs; (ii) provide detailed experimental procedures, in gene cloning and plasmid construction, protein purification, crystallization, X-ray diffraction data collection and structural elucidation, for structural biology research of TSs; and (iii) discuss the prospects of structure-based engineering and de novo design of TSs in generating valuable terpene molecules, which cannot be easily achieved by chemical synthesis.

Vanadium-dependent haloperoxidases (VHPOs) are a unique family of enzymes that utilize vanadate, an aqueous halide ion, and hydrogen peroxide to produce an electrophilic halogen species that can be incorporated into electron rich organic substrates. This halogen species can react with terpene substrates and trigger halonium-induced cyclization in a manner reminiscent of class II terpene synthases. While not all VHPOs act in this capacity, several notable examples from algal and actinobacterial species have been characterized to catalyze regio- and enantioselective reactions on terpene and meroterpenoid substrates, resulting in complex halogenated cyclic terpenes through the action of single enzyme. In this article, we describe the expression, purification, and chemical assays of NapH4, a difficult to express characterized VHPO that catalyzes the chloronium-induced cyclization of its meroterpenoid substrate.

The C-diazeniumdiolate (N-nitrosohydroxylamine) group in the amino acid graminine (Gra) is a newly discovered Fe(III) ligand in microbial siderophores. Graminine was first identified in the siderophore gramibactin, and since this discovery, other Gra-containing siderophores have been identified, including megapolibactins, plantaribactin, gladiobactin, trinickiabactin (gramibactin B), and tistrellabactins. The C-diazeniumdiolate is photoreactive in UV light which provides a convenient characterization tool for this type of siderophore. This report details the process of genomics-driven identification of bacteria producing Gra-containing siderophores based on selected biosynthetic enzymes, as well as bacterial culturing, isolation and characterization of the C-diazeniumdiolate siderophores containing Gra.

Siderophores are low-molecular-weight organic bacterial and fungal secondary metabolites that form high affinity complexes with Fe(III). These Fe(III)-siderophore complexes are part of the siderophore-mediated Fe(III) uptake mechanism, which is the most widespread strategy used by microbes to access sufficient iron for growth. Microbial competition for limited iron is met by biosynthetic gene clusters that encode for the biosynthesis of siderophores with variable molecular scaffolds and iron binding motifs. Some classes of siderophores have well understood biosynthetic pathways, which opens opportunities to further expand structural and property diversity using precursor-directed biosynthesis (PDB). PDB involves augmenting culture medium with non-native substrates to compete against native substrates during metabolite assembly. This chapter provides background information and technical details of conducting a PDB experiment towards producing a range of different analogues of the archetypal hydroxamic acid siderophore desferrioxamine B. This includes processes to semi-purify the culture supernatant and the use of liquid chromatography-tandem mass spectrometry for downstream analysis of analogues and groups of constitutional isomers.

In recent years, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become ever more apparent. This growing awareness and lack of inhibitory drugs has created a distinct need for biochemical tools that can be used to identify and characterize potential inhibitors of this family of enzymes. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. The RADD assay provides a rapid, real-time fluorescence readout of APOBEC3 DNA deamination and serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit. This method improves upon contemporary DNA deamination assays by offering a more rapid and quantifiable readout as well as providing a platform that is readily adaptable to a high-throughput format for inhibitor discovery. In this chapter we provide a detailed guide for the usage of the RADD assay for the characterization of APOBEC3 enzymes and potential inhibitors.

The NanoLuc split luciferase assay has proven to be a powerful tool for the analysis of protein translocation. Its flexibility has enabled in vivo, ex vivo, and in vitro studies-including systems reconstituting protein transport from pure components. The assay has been particularly useful in the characterization of bacterial secretion and mitochondrial protein import. In the latter case, MitoLuc has been developed for the investigation of the TIM23-pathway via import into the matrix of isolated yeast mitochondria. Subsequent analysis identified three distinct phases of import, rather than in a single continuous step. The assay has also been developed to monitor import into the mitochondrial matrix of intact cultured cells. This latter innovation has laid the foundations for further analysis of the import process in humans, including the consequences of interactions with cytosolic factors and neighboring organelles. The versatility of the MitoLuc assay is conducive for its adaptation to also monitor import into the inter-membrane space (MIA-pathway), and into the inner-membrane via the TIM22- and TIM23-complexes. Here, we present detailed protocols for the application of MitoLuc to mitochondria isolated from yeast and to those within cultured human cells.