Methods in enzymology最新文献

Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states. To detect A-to-I editing events and quantify them using Sanger sequencing, RNA samples are reverse transcribed, cDNA is amplified using gene-specific primers, and then sequenced. The chromatogram outputs are then compared to the genomic DNA sequence. As editing occurs in the context of dsRNA, the reverse transcription step is performed at a temperature as high as 65 °C, using thermostable reverse transcriptase to open double-stranded structures. However, this measure alone is insufficient for transcripts possessing long stems comprised of hundreds of nucleotide pairs. Consequently, the editing levels detected by Sanger sequencing are significantly lower than those obtained by HTS, and the amplification yield is low. We suggest that the reverse transcription is biased towards unedited transcripts, and the severity of the bias is dependent on the transcript's secondary structure. Here, we show how this bias can be significantly reduced to allow reliable detection of editing levels and sufficient product yield.

Group II introns are transposable elements that can propagate in host genomes through the "copy and paste" mechanism. They usually comprise RNA and protein components for effective propagation. Recently, we found that some bacterial GII-C introns without protein components had multiple copies in their resident genomes, implicating their potential transposition activity. We demonstrated that some of these systems are active for hydrolytic DNA cleavage and proved their DNA manipulation capability in bacterial or mammalian cells. These introns are therefore named HYdrolytic Endonucleolytic Ribozymes (HYERs). Here, we provide a detailed protocol for the systematic identification and characterization of HYERs and present our perspectives on its potential application in nucleic acid manipulation.

Selecting the appropriate mode of biocatalysis application is crucial for optimizing efficiency and sustainability. This chapter provides a comprehensive guide on key metrics to describe biocatalyst performance, including kinetic parameters such as reaction rates, cofactor requirements, dissociation constants (K_D), maximum velocities (V_max), turnover numbers (k_cat), and Michaelis constants (K_M). Additionally, it discusses biocatalysis metrics like turnover frequency (TOF), environmental factors (E-Factor), atom economy, productivities, and Life Cycle Assessment (LCA). The chapter also explores application types, focusing on whole-cell and cell-free enzyme applications, and offers a practical guide on selecting the most suitable mode of application based on specific project requirements. By integrating these considerations, researchers can effectively harness biocatalysis for innovative and sustainable solutions in various industrial processes.

Polyamines, spermidine (Spd) and Spermine (Spm), are polycations that serve a number of important biological functions. The tissue contents of polyamines are tightly regulated through their cellular import and export, as well as their metabolism (anabolism and catabolism). Polyamine catabolism in mediated via the spermidine/spermine N1-acetyltransferase (SAT1)/acetylpolyamine oxidase (APOX) cascade and oxidation of Spm by spermine oxidase (SMOX). The expression of SAT1 and SMOX increases in injured organs in response to trauma, ischemia/reperfusion, sepsis, and exposure to toxic compounds. Cisplatin is a highly effective chemotherapeutic agent that is used for the treatment of a variety of solid tumors. Its anti-tumor activity is mediated via its ability to form stable DNA adducts that inhibit the growth of actively proliferating cells. However, cisplatin also can lead to severe off-target deleterious effects (e.g., nephrotoxicity and ototoxicity), and because of such adverse effects the use of cisplatin has to be discontinued in many patients. Understanding and decoupling the therapeutic and toxic effects of cisplatin will lead to more effective use of this and other platinum-derived compounds in the treatment of cancer patients. Acute and chronic exposure to cisplatin in mice leads to severe renal tubular injuries and an increase in the expression of SAT1 and SMOX while the ablation of their genes in mice reduces the severity of nephrotoxic injuries caused by cisplatin. Furthermore, neutralization of the toxic by-products of polyamine degradation reduce the severity if cisplatin nephrotoxicity. These observations suggest that interventions targeting the adverse effects of enhanced polyamine catabolism may provide effective therapies by reducing the toxic effects of cisplatin without affecting its anti-neoplastic activity.

APOBEC cytidine deaminases guard cells in a variety of organisms from invading viruses and foreign nucleic acids. Recently, several human APOBECs have been implicated in mutating evolving cancer genomes. Expression of APOBEC3A and APOBEC3B in yeast allowed experimental derivation of the substitution patterns they cause in dividing cells, which provided critical links to these enzymes in the etiology of the COSMIC single base substitution (SBS) signatures 2 and 13 in human tumors. Additionally, the ability to scale yeast experiments to high-throughput screens allows use of this system to also investigate cellular pathways impacting the frequency of APOBEC-induced mutation. Here, we present validated methods utilizing yeast to determine APOBEC mutation signatures, genetic interactors, and chromosomal substrate preferences. These methods can be employed to assess the potential of other human APOBECs and APOBEC orthologs in different species to contribute to cancer genome evolution as well as define the pathways that protect the nuclear genome from inadvertent APOBEC activity during viral restriction.

Transketolase-like 1 (TKTL1) is one of the few proteins with a single amino acid substitution found in almost all present-day humans but absent from extinct archaic humans, Neandertals and Denisovans, and other primates. This amino acid substitution in TKTL1 is a lysine in archaic humans but an arginine in modern humans. Modern human TKTL1 (hTKTL1), but not archaic TKTL1 (aTKTL1), increases the abundance of basal radial glia (bRG), the subtype of neural progenitor cells that is most efficient to generate neurons. The techniques presented in this chapter have been pivotal to understand the implication of TKTL1 in the development of the neocortex. The techniques are the following: (i) Mouse and ferret in utero electroporation of plasmids to induce TKTL1 expression in the neocortex and study its implication in progenitor cell behaviour; (ii) incubation of electroporated mouse hemispheres with pharmacological inhibitors of metabolic pathways (ex-vivo rotation culture) to decipher the implication of TKTL1 in the pentose phosphate pathway; (iii) incubation of human foetal neocortical tissues with these inhibitors (free floating tissue culture) to confirm the physiological role of these metabolic pathways in human; (iv) knocking-out hTKTL1 in human foetal neocortical tissue using ex vivo electroporation and CRISPR/Cas9 to study the physiological role of hTKTL1 in neocortical development; and (v) ancestralization of the hTKTL1 sequence to aTKTL1 in human embryonic stem cells, used to generate cerebral organoids.

Transketolase (TK) is a pivotal enzyme of living systems metabolism, catalyzing the transfer of two-carbon units between substrates, like pentose phosphates in pentose phosphate pathway. Due to its central activity and involvement in biologically relevant routes, the inhibition of transketolase is object of interest for new therapeutics to contrast diabetes and cardiovascular diseases among the others, as well as due to its catalytic power for elongation/shortening carbon skeleton of molecules is of interest for production of chemicals. With atomistic details of TK's activity, therefore, faster steps forward can be done in a number fields and, for these reasons, the in-depth knowledge of TK activity is required. In the current chapter, the molecular description of H. Sapiens TK (hTK) catalytic reaction, which was gained in the framework of computational investigation, is presented. In particular, DFT-based studies applying quantum-chemical (QM) cluster approach and quantum mechanics/molecular mechanics (QM/MM) in its ONIOM scheme, on the conversion of d-xylulose-5-phosphate (X5P) and d-erythrose-4-phosphate (E4P) in d-fructose-6-phosphate (F6P) and d-glyceraldehyde-3-phosphate (G3P) are shown, presenting to the reader the main technical details of performing such calculations to study the reaction mechanism of the enzyme. Finally, focus on the effect of the distortion to the catalysis will be further discussed.

Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitous and versatile biocatalysts in living systems, catalyzing diverse C-C bond formation or cleavage reactions. Inspired by ThDP-dependent enzymes, chemists have developed biomimetic N-heterocyclic carbenes (NHCs) for organocatalysis, ligand design, as well as material synthesis. Inspired by the recent development in chemo-NHC-enabled radical catalysis, and based on the structural plasticity of ThDP enzymes-conserved cofactor-binding motifs coupled with highly evolvable active sites, our group has repurposed ThDP-dependent enzymes into efficient and stereoselective radical acyl transferases (RATs), three-component radical enzymes (3CREs), and C(sp³)-H bond radical acyl transferases (RAT_CH). Mechanistically, synergistic dual photo-/enzyme catalysis enabled the generation of an enzyme-bound ketyl radical and a prochiral carbon-centered radical. These two radicals then undergo stereocontrolled radical-radical cross-couplings within the active site, thus yielding a series of enantioenriched chiral ketones. This chapter outlines a detailed protocol for these photobiocatalytic reactions with engineered benzaldehyde lyases (PfBAL), catalogued by structure-guided semi-rational mutagenesis, protein expression and purification, photobiocatalytic reaction screening, and enantioselectivity determination. We hope this protocol can guide further work in expanding the catalytic repertoire of ThDP-dependent enzymes, particularly towards non-natural stereoselective radical transformations.

Biaryl coupling reactions are pivotal in the synthesis of complex therapeutic compounds, such as michelline B, vancomycin and arylomycin A2 derivatives. Synthesizing macrocycles, particularly the 2,2'-disubstituted biaryl-bridged peptide in arylomycin derivatives, present significant challenges, including low yields and the requirement for high transition metal loadings. Recent advances in DNA sequencing and enzyme engineering have facilitated the exploration of biocatalytic transformations. By leveraging enzyme engineering and substrate modifications, we report the development of a biocatalytic process using engineered cytochrome P450 enzymes for the oxidative carbon-carbon bond formation, yielding the biphenolic macrocycles present in arylomycin derivatives, at gram scale. This work underscores the transformative potential of P450 enzymes in synthetic organic chemistry, paving the way for novel pharmaceutical advancements.