Methods in enzymology最新文献

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.

Thiamine diphosphate (ThDP)-dependent enzymes are a versatile class of enzymes traditionally applied in biocatalysis for carbon-carbon bond forming or cleaving reactions via two electron chemistry. Recent advances have revealed their potential to catalyze radical-mediated transformations when combined with chemical hydrogen atom transfer (HAT) strategies, enabling the enantioselective functionalization of C(sp³)-H bonds. This approach allows for the direct acylation of benzylic C(sp³)-H sites across a broad range of organic substrates, affording enantioenriched ketones with good to high levels of enantioselectivity, reaching up to 96 % enantiomeric excess. Mechanistic studies have shown the involvement of radical intermediates derived from both the Breslow intermediate and the C-H substrate, and have highlighted the critical roles of the photocatalyst and hydrogen atom abstraction reagent in enabling efficient catalysis. These findings have expanded the reaction scope of ThDP-dependent enzymes to 'new-to-nature' transformations, opening new avenues for asymmetric synthesis with enzymes. This chapter reviews the expression and purification of the thiamine-dependent enzyme benzaldehyde lyase (PfBAL) as well as the setup and execution of photobiocatalytic C(sp³)-H acylation reactions using this enzyme.

Methionine aminopeptidases (MetAPs) and N-terminal acetyltransferases (NATs) function co-translationally at the ribosome to enzymatically modify the emerging nascent chain. Eukaryotes express two types of MetAPs, namely MetAP1 and MetAP2, which can both carry out N-terminal methionine excision (NME) at the ribosome during translation. Following NME, the most abundant NAT, NatA, can acetylate the penultimate amino acid of the nascent chain, under regulation of the NatA inhibitor HypK. Alternatively, NatA can accommodate a second enzyme, called NAA50, to form the NatE complex capable of acetylating the initiator methionine. The abundant N-terminal modifications facilitated by MetAP1/2 and NatA/E-HypK impinge on protein function, interactions, lifetime and overall proteostasis. Robust and reliable methods for the expression and purification of MetAPs and NATs set the stage for targeted functional and structural studies of these enzymes. Established methods for the production of pure ribosome-associated enzymes and stochiometric complexes will be delineated in this chapter.

Exactly two decades ago, the ability to use high-throughput RNA sequencing technology to identify sites of editing by ADARs was employed for the first time. Since that time, RNA sequencing has become a standard tool for researchers studying RNA biology and led to the discovery of RNA editing sites present in a multitude of organisms, across tissue types, and in disease. However, transcriptome-wide sequencing is not without limitations. Most notably, RNA sequencing depth of a given transcript is correlated with expression, and sequencing depth impacts the ability to robustly detect RNA editing events. This chapter focuses on a method for enrichment of low-abundance transcripts that can facilitate more efficient sequencing and detection of RNA editing events. An important note is that while we describe aspects of the protocol important for capturing intron-containing transcripts, this probe-based enrichment method could be easily modified to assess editing within any low-abundance transcript. We also provide some perspectives on the current limitations as well as important future directions for expanding this technology to gain more insights into how RNA editing can impact transcript diversity.

Assaying enzymes and microbes for activity for degradation of polymeric lignin is inherently challenging to do. This article describes several methods that our research group has developed for assay of lignin-oxidising enzymes and lignin-degrading microbes. The assay methods involve (1) colorimetric assays involving chemically nitrated lignin; (2) changes in molecular weight using gel filtration chromatography; (3) delignification of lignocellulose using Klason assay; (4) colorimetric assays for release of low molecular weight phenols and carbonyl compounds.

The broad substrate specificity of laccases makes these enzymes suitable for a wide range of applications. The use of protein engineering strategies to modulate the catalytic properties of these enzymes is a promising strategy to expand their use in the sustainable economy. Here we describe the construction of laccase-xylanase bifunctional enzyme by insertional fusion using a procedure based on the rational design starting with the analysis of the 3D-structure of laccase to select positions for the insertion of the xylanase domain, followed by the creation of the fusion construct by ligation of overlapping fragments generated by PCR. Finally, the heterologous expression and biochemical characterization of the laccase and xylanase activities of the fusion protein is described and demonstrate significant increase in the laccase activity. These protocols can be applied to the fusion of any pair of proteins.