Methods in enzymology最新文献_第9页

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":"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.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Super-resolution microscopy methods to study membrane pores in situ. 原位研究膜孔的超分辨率显微镜方法。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-31 DOI: 10.1016/bs.mie.2024.03.020

Timo Dellmann, Raed Shalaby, Ana J Garcia-Saez

引用次数: 0

Preface. 序言

4区生物学

Methods in enzymology Pub Date : 2024-01-01 DOI: 10.1016/S0076-6879(24)00271-4

Tobias Baumgart, Markus Deserno

引用次数: 0

The spectral phasor approach to resolving membrane order with environmentally sensitive dyes. 用环境敏感染料解析膜阶的光谱相量法。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-05 DOI: 10.1016/bs.mie.2024.01.024

Agustín Mangiarotti, Rumiana Dimova

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High-speed measurements of SNARE-complexin interactions using magnetic tweezers. 利用磁性镊子高速测量 SNARE-复合蛋白的相互作用。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-15 DOI: 10.1016/bs.mie.2024.01.002

Seokyun Hong, Taehyun Yang, Ara Go, Haesoo Kim, Tae-Young Yoon, Min Ju Shon

引用次数: 0

Synthesis of fluorinated amino acids by low-specificity, promiscuous aldolases coupled to in situ fluorodonor generation. 通过低特异性的杂合醛缩酶与原位氟对映体生成相结合合成含氟氨基酸。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-15 DOI: 10.1016/bs.mie.2024.02.016

Alberto De Maria, Manuel Nieto-Domínguez, Pablo I Nikel

{"title":"Synthesis of fluorinated amino acids by low-specificity, promiscuous aldolases coupled to in situ fluorodonor generation.","authors":"Alberto De Maria, Manuel Nieto-Domínguez, Pablo I Nikel","doi":"10.1016/bs.mie.2024.02.016","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.02.016","url":null,"abstract":"Fluorine (F) is an important element in the synthesis of molecules broadly used in medicine, agriculture, and materials. F addition to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to produce fluorometabolites (such as fluorinated amino acids, key building blocks for synthesis) are relatively scarce. This chapter discusses the use of L-threonine aldolase enzymes (LTAs), a class of enzymes that catalyze reversible aldol addition to the α-carbon of glycine. The C-C bond formation ability of LTAs, together with their known substrate promiscuity, make them ideal for in vitro F biocatalysis. Here, we describe protocols to harness the activity of the low-specificity LTAs isolated from Escherichia coli and Pseudomonas putida on 2-fluoroacetaldehyde to efficiently synthesize 4-fluoro-L-threonine in vitro. This chapter also provides a comprehensive account of experimental protocols to implement these activities in vivo. These methods are illustrative and can be adapted to produce other fluorometabolites of interest.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140852186","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

Ancestral terpene cyclases: From fundamental science to applications in biosynthesis. 原始萜烯环化酶：从基础科学到生物合成中的应用。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-05-03 DOI: 10.1016/bs.mie.2024.04.025

Per-Olof Syrén

{"title":"Ancestral terpene cyclases: From fundamental science to applications in biosynthesis.","authors":"Per-Olof Syrén","doi":"10.1016/bs.mie.2024.04.025","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.04.025","url":null,"abstract":"Terpenes constitute one of the largest family of natural products with potent applications as renewable platform chemicals and medicines. The low activity, selectivity and stability displayed by terpene biosynthetic machineries can constitute an obstacle towards achieving expedient biosynthesis of terpenoids in processes that adhere to the 12 principles of green chemistry. Accordingly, engineering of terpene synthase enzymes is a prerequisite for industrial biotechnology applications, but obstructed by their complex catalysis that depend on reactive carbocationic intermediates that are prone to undergo bifurcation mechanisms. Rational redesign of terpene synthases can be tedious and requires high-resolution structural information, which is not always available. Furthermore, it has proven difficult to link sequence space of terpene synthase enzymes to specific product profiles. Herein, the author shows how ancestral sequence reconstruction (ASR) can favorably be used as a protein engineering tool in the redesign of terpene synthases without the need of a structure, and without excessive screening. A detailed workflow of ASR is presented along with associated limitations, with a focus on applying this methodology on terpene synthases. From selected examples of both class I and II enzymes, the author advocates that ancestral terpene cyclases constitute valuable assets to shed light on terpene-synthase catalysis and in enabling accelerated biosynthesis.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469483","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

Mechanistic docking in terpene synthases using EnzyDock. 使用 EnzyDock 对萜烯合成酶进行机理对接。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-05-09 DOI: 10.1016/bs.mie.2024.04.005

Renana Schwartz, Shani Zev, Dan T Major

引用次数: 0

Methods for the preparation and analysis of the diterpene cyclase fusicoccadiene synthase. 二萜环化酶 fusicoccadiene 合酶的制备和分析方法。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2023-12-08 DOI: 10.1016/bs.mie.2023.11.003

Eliott S Wenger, David W Christianson

{"title":"Methods for the preparation and analysis of the diterpene cyclase fusicoccadiene synthase.","authors":"Eliott S Wenger, David W Christianson","doi":"10.1016/bs.mie.2023.11.003","DOIUrl":"10.1016/bs.mie.2023.11.003","url":null,"abstract":"Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11213977/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469495","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

Advancing the study of protein-G4 interactions in DNA repair: Insights from biolayer interferometry. 推进 DNA 修复中蛋白质-G4 相互作用的研究：生物层干涉测量法的启示。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-20 DOI: 10.1016/bs.mie.2023.12.005

Kaitlin Lowran, Vereena Salib, Emma Cismas, Colin G Wu

引用次数: 0