Methods in enzymology最新文献

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.

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).

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.

Natural products are a fascinating source of chemical diversity and their biosynthetic pathways of biological complexity. The investigation and engineering of biosynthetic pathways towards polyketides in Actinomycetes provides challenges across all steps of the mutagenesis procedure. The typically GC-rich and long genes require robust PCR protocols. The resulting amplicons, often exceeding 10 kbp in length, require equally robust cloning procedures. Finally, the genetic manipulation of Actinomycetes, especially Streptomyces spp., calls for specialized procedures, in particular when the construction of several hundred variants is needed. This chapter will detail methods for all three steps of the process and have been previously used to generate numerous polyketide synthase variants in several Actinomycete species.

Although RNA-seq data are becoming more widely available for biomedical research, most datasets for short non-coding RNAs (sncRNAs) primarily focus on microRNA analysis using standard RNA-seq, which captures only sncRNAs with 5'-phosphate (5'-P) and 3'-hydroxyl (3'-OH) ends. Standard RNA-seq fails to sequence sncRNAs with different terminal phosphate states, including tRNA halves, the most abundant class of tRNA-derived sncRNAs that play diverse roles in various biological processes. tRNA halves are produced through the endoribonucleolytic cleavage of mature tRNA anticodon loops. The responsible endoribonucleases, such as Angiogenin, commonly leave a 2',3'-cyclic phosphate (cP) at the 3'-end of 5'-tRNA halves and forms a 5'-OH end of 3'-tRNA halves, making them incompatible with standard RNA-seq. We developed a method named "cP-RNA-seq" that selectively amplifies and sequences tRNA halves and other cP-containing sncRNAs. Here we describe a detailed and recently updated cP-RNA-seq protocol. In this method, the 3'-end of all sncRNAs, except those containing a cP, are cleaved through periodate treatment after phosphatase treatment. Consequently, adaptor ligation and cDNA amplification steps are exclusively applied to cP-containing sncRNAs. Our cP-RNA-seq only requires commercially available reagents and is broadly applicable for the global identification of tRNA halves and other cP-containing sncRNA repertoires in various transcriptomes.

Angiogenin (RNase 5) is an unusual member of the RNase A family with very weak RNase activity and a preference for tRNA. The tRNAs cleaved by angiogenin are thought to have a variety of roles in cellular processes including translation reprogramming, apoptosis, angiogenesis, and neuroprotection. We recently demonstrated that angiogenin is potently activated by the cytoplasmic 80S ribosome. Angiogenin's binding to the ribosome rearranges the C-terminus of the protein, opening the active site for the cleavage of tRNA delivered to the ribosomal A site which angiogenin occupies. Here, we describe the biochemical procedure to test angiogenin's activation by the ribosome using the assay termed the Ribosome Stimulated Angiogenin Nuclease Assay (RiSANA). RiSANA can be used to test the activity of wild-type or mutant angiogenin, or other RNases, against different tRNAs and with different ribosome complexes. For example, given that angiogenin has been implicated in anti-microbial activity, we tested the ability of bacterial 70S ribosomes to stimulate angiogenin activity and found that the E. coli ribosome does not stimulate angiogenin. We also assayed whether angiogenin's closest homolog, RNase 4, could be stimulated by the ribosome, but unlike angiogenin this enzyme was not further activated by the ribosome. The RiSANA assay promises to reveal new aspects of angiogenin mechanism and may aid in the development of new diagnostic tools and therapeutics.

Base editing and other precision editing agents have transformed the utility and therapeutic potential of CRISPR-based genome editing. While some native enzymes edit efficiently with their nature-derived function, many enzymes require rational engineering or directed evolution to enhance the compatibility with mammalian cell genome editing. While many methods of engineering and directed evolution exist, plate-based discrete evolution offers an ideal balance between ease of use and engineering power. Here, we describe a detailed method for the bacterial directed evolution of CRISPR base editors that compounds technical ease with flexibility of application.