Methods in enzymology最新文献

Modern advances in sequencing, "-omics," and bioinformatics have given rise to the field of genome mining, loosely defined as the use of genomic data to guide natural product (NP) discovery. This technique applies our understanding of biosynthetic logic to predict the genes encoding for production of novel compounds. The major steps include identification of these biosynthetic gene clusters (BGCs), their classification, and prioritization for subsequent experimentation. Despite decades of effort, determination of cluster boundaries without experimental validation remains one of the greatest challenges in genome mining. Genes encoded within a BGC are the foundation for all downstream analysis. Thus, accurate determination of gene cluster content is critical for effective prioritization of BGCs and prediction of their products. Synteny, or the conservation of homologous genes and their arrangement, provides an effective solution for predicting these borders. Over evolutionary time, transfer and rearrangement of genes results in variability surrounding BGCs, such that natural breaks in conservation underlie these functional units. In this chapter, we provide a comprehensive approach for using synteny to delineate BGC boundaries.

Chemically depolymerized low-molecular-weight lignin can be converted into polymer building blocks using bacterial convergent metabolic systems called biological funneling. Various bacterial enzyme genes involved in the catabolism of lignin-derived aromatic compounds have been identified and characterized in detail. This information is essential for developing the bioproduction of high-value-added chemicals from lignin. Transporters responsible for the first step in catabolism mediate the transport of substrates across biological membranes. Since substrate uptake in biological membranes can be an obstacle or a rate-limiting process in the bacterial production of value-added compounds, it is vital to understand not only enzyme functions but also uptake systems. In this chapter, we focus on the bacterial transporters for lignin-derived aromatic compounds that have been reported and introduce methods for the characterization of transporters, primarily through in vivo analyses. In addition, we will present an antibody-based analysis of the cellular localization of transporters.

Several bacterial dye-decolorising peroxidases have been identified, that have activity for oxidation of lignin model compounds and polymeric lignin. This article describes biochemical methods for lignin-oxidising peroxidase DyP1B from Pseudomonas fluorescens Pf-5. The article presents methods for: (1) enzyme purification and heme reconstitution; (2) steady-state kinetic enzyme assays; (3) pre-steady state kinetic analysis; (4) testing of the enzyme against polymeric lignin substrates.

The class IIb histone deacetylase HDAC10 is responsible for the deacetylation of intracellular polyamines, in particular N⁸-acetylspermidine. HDAC10 is emerging as an attractive target for drug design owing to its role as an inducer of autophagy, and high-resolution crystal structures enable structure-based drug design efforts. The only crystal structure available to date is that of HDAC10 from Danio rerio (zebrafish), but a construct containing the A24E and D94A substitutions yields an active site contour that more closely resembles that of human HDAC10. The use of this "humanized" construct has advanced our understanding of HDAC10-inhibitor structure-activity relationships. Here, we outline the preparation, purification, assay, and crystallization of humanized zebrafish HDAC10-inhibitor complexes. The plasmid containing the humanized zebrafish HDAC10 construct for heterologous expression in Escherichia coli is available through Addgene (#225542).

Multinuclear non-heme iron-dependent oxidative enzymes (MNIOs) are a family of diiron/triiron enzymes that install post-translational modifications (PTMs) on ribosomally produced peptides. These modifications include oxazolone-thioamide formation, carbon excision, thiooxazole formation, α-keto acid formation, and N-Cα bond cleavage, demonstrating the high functional diversity of MNIOs. Many MNIOs function together with a partner protein that helps recruit the substrate peptide. This review outlines experimental methods for the expression and purification of a representative MNIO (TglH) and its peptide substrate (TglA), as well as the characterization of the resulting PTM using various spectroscopic methods and isotope labeling. These protocols can be applied to study other MNIO-encoding pathways, with case-by-case adaptations and differences highlighted. Continued genome mining of MNIOs is likely to reveal more novel enzymatic functions, advancing our understanding of their catalytic mechanisms and their roles in natural product biosynthesis.

The versatility of heme proteins in nature stems from the intricate control exerted by their protein scaffolds. De novo protein design offers a powerful means to dissect and recreate these structure-function relationships, enabling construction of novel metalloproteins with tailored functionalities. Here, we describe the computational design and characterization MPP1, a four-helix bundle protein designed to bind an abiological Mn-diphenylporphyrin (MnDPP) cofactor. Using parameterized coiled-coil backbones, flexible backbone sequence design in Rosetta, and structure-guided loop building, MPP1 was designed to accommodate the cofactor with precise positioning of axial ligands and second-shell interactions, as well as purposeful accessibility for oxidants and substrates. The resulting protein was the first crystallographically characterized de novo designed porphyrin-binding protein. MPP1 demonstrated the ability to stabilize a high-valent Mn(V)-oxo species and mediate thioether oxidation. This chapter details the computational strategies, cofactor incorporation, and solution characterization necessary to design and evaluate four-helix bundle proteins capable of binding porphyrin and porphyrin-like cofactors with atomic-level precision. Keywords: de novo design, protein design, bioinorganic chemistry, metalloporphyrins, heme proteins.

Natural and artificial metalloproteins play a critical role in biochemistry, with the first X-ray crystal structures ever solved belonging to heme proteins Due to their ability to carry out a diverse array of challenging reactions at ambient temperature, effective metalloenzyme design and isolation strategies are highly desirable. Control of active site geometry is often the key requirement for catalysis and its mutagenesis helps probe a wide variety of biological and abiological reactions. In the case of small-molecule activation, introduction of new metal-binding sites to non-native heme scaffolds can unlock new chemistry. In this chapter, we will provide methods used in our lab for the design and experimental preparation of artificial metalloenzymes containing a heme-copper center to mimic and understand heme-copper oxidases. The methods can be applied to design other heterobinuclear centers containing heme, such as the heme-nonheme iron center in nitric oxidase reductases.

Substituting the native metal of metalloenzymes can significantly alter the enzymes' reactivity and spectroscopic properties. Cobalt is especially attractive as a substitute for the native iron center in hemoproteins, as it generates metal variants with complementary spectroscopic properties and could enable new modes of reactivity. Here, we describe a detailed protocol for the biosynthesis and incorporation of cobalt protoporphyrin IX (CoPPIX) into hemoproteins, replacing the native heme b cofactor during expression in the common laboratory strain Escherichia coli BL21(DE3). This protocol is described using the model hemoprotein Physeter macrocephalus (sperm whale) myoglobin. Because of cobalt's unique electronic and geometric properties, cobalt-substituted hemoproteins offer a valuable handle for spectroscopic characterization and structural studies. We describe analytic methods of assessing cofactor identity and purity, including electronic absorption spectroscopy, liquid-chromatography/mass-spectrometry, inductively coupled plasma-mass spectrometry, and electron paramagnetic resonance spectroscopy. This method for generating artificial metalloenzymes is effective, easy to implement, and can produce useful quantities of Co-substituted hemoproteins.

Multidrug efflux transporters are critical contributors to antimicrobial resistance. This chapter details methodologies for identifying and functionally characterizing efflux pumps in halophilic archaea, expanding upon recent work that provided the first experimental evidence of active antibiotic efflux in an archaeon. Using Halorubrum amylolyticum CSM52 as a model, we describe genomic identification of putative efflux pump genes via whole-genome sequencing and RAST annotation, followed by phylogenetic analyses and comparative genomics to establish evolutionary context. We then outline functional assays in the native archaeal strain, including a fluorescence-based Hoechst 33342 accumulation assay to detect active efflux and antibiotic susceptibility testing in the presence of efflux pump inhibitors (EPIs) to reveal efflux-mediated resistance. To overcome challenges of manipulating extremophiles, we detail cloning of archaeal MATE (Multidrug and Toxin Extrusion) transporter genes into Escherichia coli and heterologous expression under inducible conditions, enabling characterization of pump activity in a model bacterial system. Fluorometric efflux assays in these E. coli clones confirmed transporter function and inhibitor specificity. We also describe integrative structural approaches: homology modeling of the archaeal MATE pumps (using YASARA and AlphaFold3) and molecular docking of substrates and inhibitors to elucidate mechanistic interactions. The relevance of archaeal efflux pumps to antimicrobial resistance (AMR) is discussed, including their potential to harbor and disseminate novel resistance determinants. Finally, we address the interpretation and limitations of using heterologous systems. This comprehensive methodological framework provides a roadmap for exploring efflux-mediated drug resistance in archaea, an emerging and important aspect of AMR research.

Antibiotic resistance is a growing threat in the modern world. In Gram-negative bacteria, one factor contributing to antibiotic resistance is the tripartite efflux pumps which push antibiotics out of the cell against their concentration gradient. These pumps consist of three main protein complexes: an outer membrane protein, an inner membrane protein, and the periplasmic adapter protein, which connects the two membrane proteins. Multiple efflux pumps in resistant strains use the same outer membrane protein, TolC. This protein is a homotrimeric transmembrane membrane beta barrel with a periplasmic homotrimeric alpha-helical barrel. Until recently, isolation of folded TolC from the outer membrane was quite difficult, leading to low yields. Our lab developed an inclusion body isolation and refolding protocol to increase the yield of trimeric TolC. We identified two crucial factors that support TolC refolding: detergent choice and protein concentration and found that this method is also successful for some TolC homologs (V. cholerae VceC and C. jejuni CmeC). This chapter seeks to provide an in-depth guide for investigators wanting to refold TolC or its homologs, by giving insight into common pitfalls and other issues we have noted in our work.