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Functional analysis of transmembrane terpene cyclases involved in fungal meroterpenoid biosynthesis. 参与真菌经萜生物合成的跨膜萜环酶的功能分析。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-24 DOI: 10.1016/bs.mie.2024.02.007
Jia Tang, Yudai Matsuda
{"title":"Functional analysis of transmembrane terpene cyclases involved in fungal meroterpenoid biosynthesis.","authors":"Jia Tang, Yudai Matsuda","doi":"10.1016/bs.mie.2024.02.007","DOIUrl":"10.1016/bs.mie.2024.02.007","url":null,"abstract":"<p><p>Pyr4-family terpene cyclases are noncanonical transmembrane class II terpene cyclases that catalyze a variety of cyclization reactions in the biosynthesis of microbial terpenoids, such as meroterpenoids. However, although these cyclases are widely distributed in microorganisms, their three-dimensional structures have not been determined, possibly due to the transmembrane locations of these enzymes. In this chapter, we describe procedures for the functional analysis of transmembrane terpene cyclases based on their model structures generated using AlphaFold2. We used AdrI, the Pyr4-family terpene cyclase required for the biosynthesis of andrastin A and its homologs, as an example.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"419-445"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469489","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Characterization of UbiA terpene synthases with a precursor overproduction system in Escherichia coli. 大肠杆菌中带有前体过量生产系统的 UbiA 萜烯合成酶的特征。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-21 DOI: 10.1016/bs.mie.2024.02.001
Tyler A Alsup, Melvin Osei Opoku, Jeffrey D Rudolf
{"title":"Characterization of UbiA terpene synthases with a precursor overproduction system in Escherichia coli.","authors":"Tyler A Alsup, Melvin Osei Opoku, Jeffrey D Rudolf","doi":"10.1016/bs.mie.2024.02.001","DOIUrl":"10.1016/bs.mie.2024.02.001","url":null,"abstract":"<p><p>Expression and purification of membrane-bound proteins remains a challenge and limits enzymology efforts, contributing to a substantial knowledge gap in the biochemical functions of many proteins found in nature. Accordingly, the study of bacterial UbiA terpene synthases (TSs) has been limited due to the experimental hurdles required to purify active enzymes for characterization in vitro. Previous work employed the use of microsomes or crude membrane fractions to test enzyme activity; however, these methods can be labor intensive, require access to an ultracentrifuge, or may not be suitable for all membrane-bound TSs. We detail here an alternative strategy for the in vivo expression and biochemical characterization of the membrane associated UbiA TSs by employing a precursor overproduction system in Escherichia coli.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"395-417"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11216710/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469486","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Methods for the discovery and characterization of octocoral terpene cyclases. 章鱼萜环化酶的发现和表征方法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-05 DOI: 10.1016/bs.mie.2024.02.011
Immo Burkhardt, Lara Dürr, Natalie E Grayson, Bradley S Moore
{"title":"Methods for the discovery and characterization of octocoral terpene cyclases.","authors":"Immo Burkhardt, Lara Dürr, Natalie E Grayson, Bradley S Moore","doi":"10.1016/bs.mie.2024.02.011","DOIUrl":"10.1016/bs.mie.2024.02.011","url":null,"abstract":"<p><p>Octocorals are the most prolific source of terpenoids in the marine environment, with more than 4000 different compounds known from the phylum to date. However, the biochemical and genetic origin of their production remained elusive until recent studies showed that octocorals encode genes responsible for the biosynthesis of terpenoids in their own chromosomal DNA rather than from microbial symbionts as originally proposed. The identified coral genes include those encoding a new group of class I terpene cyclases (TCs) clustered among other candidate classes of tailoring enzymes. Phylogenetic analyses established octocoral TCs as a monophyletic clade, distinct from TCs of plants, bacteria, and other organisms. The newly discovered group of TCs appears to be ubiquitous in octocorals and is evolutionarily ancient. Given the recent discovery of octocoral terpenoid biochemistry and only limited genomic data presently available, there is substantial potential for discovering new biosynthetic pathways from octocorals for terpene production. The following chapter outlines practical experimental procedures for octocoral DNA and RNA extraction, genome and transcriptome assembly and mining, TC cloning and gene expression, protein purification, and in vitro analyses.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"343-371"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11590171/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Robust magnetic tweezers for membrane protein folding studies. 用于膜蛋白折叠研究的强力磁镊。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-16 DOI: 10.1016/bs.mie.2023.12.014
Seoyoon Kim, Duyoung Min
{"title":"Robust magnetic tweezers for membrane protein folding studies.","authors":"Seoyoon Kim, Duyoung Min","doi":"10.1016/bs.mie.2023.12.014","DOIUrl":"10.1016/bs.mie.2023.12.014","url":null,"abstract":"<p><p>Single-molecule magnetic tweezers have recently been adapted for monitoring the interactions between transmembrane helices of membrane proteins within lipid bilayers. In this chapter, we describe the procedures of conducting studies on membrane protein folding using a robust magnetic tweezer method. This tweezer method is capable of observing thousands of (un)folding transitions over extended periods of several to tens of hours. Using this approach, we can dissect the folding pathways of membrane proteins, determine their folding time scales, and map the folding energy landscapes, with a higher statistical reliability. Our robust magnetic tweezers also allow for estimating the folding speed limit of helical membrane proteins, which serves as a link between the kinetics and barrier energies.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"694 ","pages":"285-301"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Identification of bona fide RNA G-quadruplex binding proteins. 鉴定真正的 RNA G-四叠体结合蛋白。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2023-12-21 DOI: 10.1016/bs.mie.2023.12.001
Prakash Kharel, Pavel Ivanov
{"title":"Identification of bona fide RNA G-quadruplex binding proteins.","authors":"Prakash Kharel, Pavel Ivanov","doi":"10.1016/bs.mie.2023.12.001","DOIUrl":"10.1016/bs.mie.2023.12.001","url":null,"abstract":"<p><p>RNAs often accomplish their diverse functions through direct interactions with RNA-binding proteins (RBPs) in a sequence- and/or structure-dependent manner. RNA G-quadruplexes (rG4s) are unique secondary structures formed by guanine-rich RNA sequences which impact RNA function independently and in combination with RBPs. Efforts from several labs have identified dozens of rG4 specific RBPs (rG4BPs), although the research is still in the growing phase. Here we present methods for the systematic identification of rG4BPs using a pull-down approach that takes advantage of the chemical modification of guanine bases. This allows abolishing the rG4 structures while still maintaining the base composition intact, and hence helps in recognizing true rG4BPS (in contrast to G-rich motif binders). In combination with other biochemical assays, such an approach can be efficiently used for the identification and characterization of bona fide rG4BPs.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"695 ","pages":"255-274"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140194171","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Lanthanum-fluoride electrode-based methods to monitor fluoride transport in cells and reconstituted lipid vesicles. 基于氟化镧电极的方法，用于监测细胞和重组脂质囊泡中的氟迁移。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-09 DOI: 10.1016/bs.mie.2024.01.012
Chia-Yu Kang, Minjun An, Randy B Stockbridge
{"title":"Lanthanum-fluoride electrode-based methods to monitor fluoride transport in cells and reconstituted lipid vesicles.","authors":"Chia-Yu Kang, Minjun An, Randy B Stockbridge","doi":"10.1016/bs.mie.2024.01.012","DOIUrl":"10.1016/bs.mie.2024.01.012","url":null,"abstract":"<p><p>Fluoride (F<sup>-</sup>) export proteins, including F<sup>-</sup> channels and F<sup>-</sup> transporters, are widespread in biology. They contribute to cellular resistance against fluoride ion, which has relevance as an ancient xenobiotic, and in more modern contexts like organofluorine biosynthesis and degradation or dental medicine. This chapter summarizes quantitative methods to measure fluoride transport across membranes using fluoride-specific lanthanum-fluoride electrodes. Electrode-based measurements can be used to measure unitary fluoride transport rates by membrane proteins that have been purified and reconstituted into lipid vesicles, or to monitor fluoride efflux into living microbial cells. Thus, fluoride electrode-based measurements yield quantitative mechanistic insight into one of the major determinants of fluoride resistance in microorganisms, fungi, yeasts, and plants.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"696 ","pages":"43-63"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140851424","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Biotransformation of fluorinated drugs and xenobiotics by the model fungus Cunninghamella elegans. 模式真菌 Cunninghamella elegans 对含氟药物和异种生物的生物转化。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-16 DOI: 10.1016/bs.mie.2023.12.016
Mohd Faheem Khan, Carina Hof, Patricie Niemcova, Cormac D Murphy
{"title":"Biotransformation of fluorinated drugs and xenobiotics by the model fungus Cunninghamella elegans.","authors":"Mohd Faheem Khan, Carina Hof, Patricie Niemcova, Cormac D Murphy","doi":"10.1016/bs.mie.2023.12.016","DOIUrl":"10.1016/bs.mie.2023.12.016","url":null,"abstract":"<p><p>Some species of the genus Cunninghamella (C. elegans, C. echinulata and C. blaskesleeana) produce the same phase I and phase II metabolites when incubated with xenobiotics as mammals, and thus are considered microbial models of mammalian metabolism. This had made these fungi attractive for metabolism studies with drugs, pesticides and environmental pollutants. As a substantial proportion of pharmaceuticals and agrochemicals are fluorinated, their biotransformation has been studied in Cunninghamella fungi and C. elegans in particular. This article details the methods employed for cultivating the fungi in planktonic and biofilm cultures, and extraction and analysis of fluorinated metabolites. Furthermore, protocols for the heterologous expression of Cunninghamella cytochromes P450 (CYPs), which are the enzymes associated with phase I metabolism, are described.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"696 ","pages":"251-285"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140859737","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Sample delivery for structural biology at the European XFEL.
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-10-22 DOI: 10.1016/bs.mie.2024.10.007
Katerina Dörner, Peter Smyth, Joachim Schulz
{"title":"Sample delivery for structural biology at the European XFEL.","authors":"Katerina Dörner, Peter Smyth, Joachim Schulz","doi":"10.1016/bs.mie.2024.10.007","DOIUrl":"10.1016/bs.mie.2024.10.007","url":null,"abstract":"<p><p>Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a valuable technique for time-resolved structural studies on enzymes. This method allows for the collection of high-resolution datasets of protein structures at various time points during a reaction initiated by light or mixing. Experiments are performed under non-cryogenic conditions and allow the collection of radiation damage free structures. At the European XFEL (EuXFEL), SFX experiments are mainly performed with liquid jets produced by gas dynamic virtual nozzles (GDVNs) and less frequent with a high viscous extruder (HVE). In this chapter we describe these delivery methods, with the focus on GDVNs. Instrumentation, sample requirements, and preparation steps for SFX beamtimes are discussed. Other sample delivery methods available at the EuXFEL are briefly introduced at the end of this chapter.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"709 ","pages":"105-129"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142751302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Brewing coral terpenes-A yeast based approach to soft coral terpene cyclases. 酿造珊瑚萜烯--基于酵母的软珊瑚萜烯环化酶研究方法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-04 DOI: 10.1016/bs.mie.2024.03.023
Paul D Scesa, Eric W Schmidt
{"title":"Brewing coral terpenes-A yeast based approach to soft coral terpene cyclases.","authors":"Paul D Scesa, Eric W Schmidt","doi":"10.1016/bs.mie.2024.03.023","DOIUrl":"10.1016/bs.mie.2024.03.023","url":null,"abstract":"<p><p>Coral terpenes are important molecules with numerous applications. Here, we describe a robust and simple method to produce coral terpene scaffolds at scale. As an example of the approach, here we discover, express, and characterize further klysimplexin R synthases, expanding the known enzymology of soft coral terpene cyclases. We hope that the underlying method described will enable widespread basic research into the functions of coral terpenes and their biosynthetic genes, as well as the commercial development of biomedically and technologically important molecules.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"373-394"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Experimental considerations for precise RNA-mediated insertion of transgenes. RNA 介导的转基因精确插入的实验考虑因素。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-10 DOI: 10.1016/bs.mie.2024.07.007
Sarah M Palm, Briana Van Treeck, Kathleen Collins
{"title":"Experimental considerations for precise RNA-mediated insertion of transgenes.","authors":"Sarah M Palm, Briana Van Treeck, Kathleen Collins","doi":"10.1016/bs.mie.2024.07.007","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.007","url":null,"abstract":"<p><p>Precise RNA-mediated insertion of transgenes (PRINT) is a pioneering method for site-specific, safe-harbor transgene supplementation of the human genome that harnesses a eukaryotic retroelement protein and relies solely on the delivery of RNA. Here we outline important considerations in the design of the two required RNAs, details for the production and transfection of these RNAs to cells, and read-outs for successful transgene addition. Throughout, tips and key concepts are laid out to enable general use of this method.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"705 ","pages":"1-24"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400653","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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