Methods in enzymology最新文献

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.

Small RNA pathways, also known as RNA interference (RNAi), are dynamic and essential regulatory systems that robustly silence a wide range of target genes in a precise, temporal, and cell-specific manner. Preventing aberrant targeting of genes by RNAi requires checks and balances to maintain homeostasis within the RNAi pathways. Yet, at present, our understanding of the mechanisms governing these complex regulatory pathways remains rudimentary; despite knowing they are crucial to maintaining cell homeostasis. Here we describe how to use our paired small RNA and mRNA sequencing approach with our bioinformatic workflow to systematically perform comparative analyses on multi-'omics datasets to identify which factors exhibit differential expression driven by changes in RNAi-targeting to generate a list of putative feedback motifs within RNAi pathways. Our workflow has the flexibility to enable high-throughput detection of putative feedback motifs for any pathway of interest in any organism.

Understanding the mechanism and structure of transketolase is valuable across a range of disciplines, including enzymology, synthetic biology, drug development, and biocatalysis. Beyond offering insights into enzyme catalysis and thiamin-dependent chemistry, this knowledge enables the rational design of transketolase variants with altered substrate specificity and the creation of novel biosynthetic pathways to produce unusual sugars or chiral compounds. Transketolase is also a potential target for cancer treatment, as well as for metabolic or neurodegenerative diseases. This work presents protocols for analyzing transketolase activity, its catalytic mechanism, and structure. These include methods for steady-state kinetics, cofactor binding, detection of catalytic intermediates, and rapid kinetic studies using spectroscopic and biophysical techniques. Together, these protocols furnish a comprehensive toolkit for advancing both fundamental and applied transketolase research.

Transketolase (TK, EC 2.2.1.1) is an essential thiamine pyrophosphate (TPP)-dependent enzyme that plays a central role in carbohydrate metabolism, particularly in the pentose phosphate pathway (PPP) and the photosynthesis Calvin cycle. TK catalyzes a reversible transfer of a two-carbon ketol group (C2 moiety) between phosphorylated sugars, influencing metabolic flux in central carbon metabolism. In addition, TK has evolved specialized roles in sulfoglycolysis-a pathway critical for degrading the plant-derived sulfonated sugar sulfoquinovose (SQ) and sustaining global sulfur cycling. In anaerobes, the sulfoglycolytic transketolase-dependent (sulfo-TK) pathway uses SqwGH (EC 2.2.1.15), a TK encoded by a split-gene sqwG and sqwH, to catalyze two ketol transfers: first from 6-deoxy-6-sulfofructose (SF) to d-glyceraldehyde-3-phosphate (G3P), yielding 4-deoxy-4-sulfoerythrose (SE) which further undergoes aldose-ketose isomerization to generate 4-deoxy-4-sulfoerythrulose (SEu) for the second SqwGH-mediated transketolation. Here, we outline the identification, expression, purification, and activity assay of the split-gene encoded SqwGH. These approaches provide a comprehensive toolkit for researchers to dissect TK's evolutionary plasticity, and engineer its catalytic promiscuity for biocatalytic applications.

In recent years, mesophilic transketolases (TK) from S. cerevisiae and E. coli have been widely used for the synthesis of numerous chiral α-hydroxyketones preferentially polyhydroxylated. To improve the efficiency of these TKs, evolvability techniques have been applied but for biocatalytic applications, the stability against time, the resistance towards temperature and destabilizing mutagenesis factors are often provided by more robust and less flexible protein structures. To answer these criteria, the discovery of a thermostable TK from Geobacillus stearothermophilus (TK_gst) offers an efficient template for the construction of TK variants able to greatly extend the substrate scope while decreasing the reaction time and giving more resistance against unusual conditions. In this chapter, we describe a three-step workflow for the production of TK_gst variants designed for the synthesis of 1-deoxyketoses- or 1,2-dideoxyketoses from aliphatic α-ketoacids as donor substrates and their in situ generation by a d-amino acid oxidase coupled in one pot with the TK_gst variant. In a first step, TK_gst variant libraries are created on targeted positions identified in the active site by molecular modeling and are then submitted to site saturation mutagenesis. The second step consists in TK_gst variant library screening with qualitative and quantitative assays to select the best TK_gst variants which are used, in the third and final step, for the preparative-scale synthesis of the targeted 1-deoxyketoses or 1,2-dideoxyketoses. This approach can be applied to the synthesis of other α-hydroxyketones of biological interests by varying the donor and acceptor substrates.

This chapter describes methods of purification and biochemical characterization of plant transketolases. The methods have been developed initially for transketolase from the desiccation tolerant plant Craterostigma plantagineum. Like other plants, C. plantagineum encodes several isoforms of transketolase. The main isoform represents a key enzyme in the pentose phosphate cycle and in photosynthesis where it catalyzes the synthesis of sugar phosphates. Other isoforms synthesize rare sugar phosphates such as octulose-phosphate. This demonstrates that besides primary metabolism, transketolases in plants may be involved in the synthesis of species-specific sugar metabolites. If the identity of the sugar is not known, a combination of gas chromatography and mass spectrometry need to be applied for the identification. The different isoforms of transketolase can be localized in different cellular compartments, such as plastids and cytoplasm. Experimental strategies are described to demonstrate the subcellular localization of transketolases.