Methods in enzymology最新文献

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.

Transketolase, a thiamine diphosphate-dependent enzyme, is widely distributed in nature and plays a crucial role in cellular metabolism. Its ability to synthesize α-hydroxyketones in a stereoselective manner, key precursors for high-value compounds like vicinal diols and amino alcohols, has garnered significant interest in synthetic chemistry. In this chapter, we review the engineering and applications of transketolase along with molecular docking studies, mutant library screening, and detailed experimental protocols. Engineering efforts have primarily focused on broadening substrate specificity for both donor and acceptor molecules, enhancing catalytic activity, improving stability, refining stereoselectivity, facilitating reverse cleavage reactions, and constructing novel covalent bonds. Advances in structural and computational analyses have deepened the understanding of the transketolase catalytic mechanism, guiding its engineering and significantly enhancing its industrial applicability. Current challenges in synthetic applications are also discussed to inform further optimization.

Therapeutic peptides have experienced significant growth over the past few decades, with several new candidates entering the market each year. A comprehensive overview of peptides currently in clinical trials is essential for understanding prevailing discovery strategies, key therapeutic targets, and areas where peptides have demonstrated the most promise. In this chapter, we systematically summarize and classify 287 peptides undergoing clinical evaluation, spanning a wide range of applications; from antimicrobial agents and cancer therapeutics to peptides used in guided surgeries. While the majority of these peptides are protein mimetics inspired by naturally occurring peptides and proteins, a notable portion also includes rationally designed peptides and those identified through phage display technologies. This analysis highlights the evolving landscape of peptide therapeutics and provides insights into emerging trends and opportunities in the field.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are versatile biocatalysts known for their ability to form diverse C-C, C-N, and C-S bonds. Despite this catalytic diversity, a PLP-dependent enzyme capable of promoting an intermolecular Mannich reaction to access α,β-diamino acids has not been described. Here, we report the engineering of LolT, a PLP-dependent Mannich cyclase from the loline alkaloid biosynthetic pathway, into a synthetically valuable Mannichase. Using iterative site-saturation mutagenesis and a double high-throughput screening platform, we identified mutations that significantly enhance LolT's non-native Mannichase activity. The best-performing variant, LolT^V4, exhibited a>60-fold improvement in catalytic turnover and enabled one-step, enantioselective synthesis of the unusual amino acid L-tambroline on a gram scale. This chapter provides a detailed experimental workflow for constructing mutant libraries, performing high-throughput functional screening, and validating hits through biochemical and analytical methods. Our work establishes a blueprint for repurposing PLP enzymes toward non-natural transformations, broadening the scope of biocatalysis for medicinal and synthetic chemistry applications.

Enzymatic reductive amination is now a green and selective method for the efficient conversion of ketones into chiral amines with high optical purity. Transaminases (TAs) have been widely employed at both laboratory and industrial scale for the synthesis of primary amines. Additionally, amine dehydrogenases (AmDHs), imine reductases (IREDs) and reductive aminases (RedAms) enable the stereoselective synthesis of primary, secondary and tertiary amines. Recent advancements in protein engineering have expanded the substrate scope and improved the stability of these biocatalysts, enabling broader applications. The use of immobilized enzymes and whole-cell systems further enhances the efficiency and sustainability of these methods. This chapter provides detailed protocols for enzymatic reductive amination for the synthesis of primary, secondary, and tertiary chiral amines using isolated or immobilized enzymes, or whole-cell biocatalysts.