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Measuring carbonic anhydrase activity in alpha-carboxysomes using stopped-flow. 利用停流法测量α-羧酶体中碳酸酐酶的活性
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-10-28 DOI: 10.1016/bs.mie.2024.10.012
Nikoleta Vogiatzi, Cecilia Blikstad
{"title":"Measuring carbonic anhydrase activity in alpha-carboxysomes using stopped-flow.","authors":"Nikoleta Vogiatzi, Cecilia Blikstad","doi":"10.1016/bs.mie.2024.10.012","DOIUrl":"10.1016/bs.mie.2024.10.012","url":null,"abstract":"<p><p>Carboxysomes are protein-based organelles that serve as the centerpiece of the bacterial CO<sub>2</sub> concentration mechanism (CCM). They are present in all cyanobacteria and many chemoautotrophic proteobacteria and encapsulate the key enzymes for CO<sub>2</sub> fixation, carbonic anhydrase and the carboxylase Rubisco, within a protein shell. The CCM actively accumulates bicarbonate in the cytosol, which diffuses into the carboxysome where carbonic anhydrase rapidly equilibrates it to CO<sub>2</sub>. This creates a high CO<sub>2</sub> concentration around Rubisco, ensuring efficient carboxylation. In this chapter, we present a general method for purifying α-carboxysomes and measuring carbonic anhydrase activity within these purified compartments. We exemplify this with α-carboxysomes purified from the chemoautotroph Halothiobacillus neapolitanus c2, a model organism for the α-carboxysome based CCM. However, this purification protocol can be adapted for other species, such as carboxysomes from α-cyanobacteria or carboxysomes expressed in heterologous hosts. Further, we describe the Khalifah/pH indicator assay for measuring steady-state kinetics of carbonic anhydrase catalyzed CO<sub>2</sub> hydration. This method allows us to determine the kinetic parameters k<sub>cat</sub>, K<sub>M</sub> and k<sub>cat</sub>/K<sub>M</sub> for the purified α-carboxysomes. It uses a stopped-flow spectrometer for rapid mixing and detection, crucial for capturing the fast equilibrium between CO<sub>2</sub> and bicarbonate. The reaction progress is monitored by absorbance via a pH indicator that changes color due to the proton release. While the method specifically focuses on measuring carbonic anhydrase activity on carboxysomes, it can be used to measure activity on carbonic anhydrases from other contexts as well.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"708 ","pages":"297-322"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142687427","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
The growth of microcrystals for time resolved serial crystallography. 微晶生长的时间分辨序列晶体学。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-10-29 DOI: 10.1016/bs.mie.2024.10.003
Alexander McPherson
{"title":"The growth of microcrystals for time resolved serial crystallography.","authors":"Alexander McPherson","doi":"10.1016/bs.mie.2024.10.003","DOIUrl":"10.1016/bs.mie.2024.10.003","url":null,"abstract":"<p><p>The production of enzyme microcrystals for time resolved serial crystallography employing free electron laser or synchrotron radiation is a relatively new variation on traditional macromolecular crystallization for conventional single crystal X-ray analysis. While the fundamentals of macromolecular crystal growth are the same, some modifications and special considerations are in order if the objective is to produce uniform size, microcrystals in very large numbers for serial data collection. Presented here are the basic principles of protein crystal growth with particular attention to the approaches best employed to achieve the goal of microcrystals and some novel techniques, as well as old, that may be useful. Also discussed are the advantages of particular precipitants and certain methods of growing protein crystals that might be advantageous for serial data recording.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"709 ","pages":"1-27"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142751308","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
The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import. 用于分析线粒体蛋白质导入机制的 MitoLuc 分析法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-22 DOI: 10.1016/bs.mie.2024.07.033
Hope I Needs, Youmian Yan, Natalie M Niemi, Ian Collinson
{"title":"The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.","authors":"Hope I Needs, Youmian Yan, Natalie M Niemi, Ian Collinson","doi":"10.1016/bs.mie.2024.07.033","DOIUrl":"10.1016/bs.mie.2024.07.033","url":null,"abstract":"<p><p>The NanoLuc split luciferase assay has proven to be a powerful tool for the analysis of protein translocation. Its flexibility has enabled in vivo, ex vivo, and in vitro studies-including systems reconstituting protein transport from pure components. The assay has been particularly useful in the characterization of bacterial secretion and mitochondrial protein import. In the latter case, MitoLuc has been developed for the investigation of the TIM23-pathway via import into the matrix of isolated yeast mitochondria. Subsequent analysis identified three distinct phases of import, rather than in a single continuous step. The assay has also been developed to monitor import into the mitochondrial matrix of intact cultured cells. This latter innovation has laid the foundations for further analysis of the import process in humans, including the consequences of interactions with cytosolic factors and neighboring organelles. The versatility of the MitoLuc assay is conducive for its adaptation to also monitor import into the inter-membrane space (MIA-pathway), and into the inner-membrane via the TIM22- and TIM23-complexes. Here, we present detailed protocols for the application of MitoLuc to mitochondria isolated from yeast and to those within cultured human cells.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"407-436"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11756599/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504061","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
Radical-relay C(sp3)-H azidation catalyzed by an engineered nonheme iron enzyme. 工程非血红素铁酶催化的自由基接力 C(sp3)-H 叠氮化反应。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-07-23 DOI: 10.1016/bs.mie.2024.07.003
Qun Zhao, Jinyan Rui, Xiongyi Huang
{"title":"Radical-relay C(sp<sup>3</sup>)-H azidation catalyzed by an engineered nonheme iron enzyme.","authors":"Qun Zhao, Jinyan Rui, Xiongyi Huang","doi":"10.1016/bs.mie.2024.07.003","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.003","url":null,"abstract":"<p><p>Nonheme iron enzymes are versatile biocatalysts for a broad range of unique and powerful transformations, such as hydroxylation, chlorination, and epimerization as well as cyclization/ring-opening of organic molecules. Beyond their native biological functions, these enzymes are robust for engineering due to their structural diversity and high evolvability. Based on enzyme promiscuity and directed evolution as well as inspired by synthetic organic chemistry, nonheme iron enzymes can be repurposed to catalyze reactions previously only accessible with synthetic catalysts. To this end, our group has engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for new-to-nature radical transformations. In particular, we have demonstrated that a nonheme iron enzyme, (4-hydroxyphenyl)pyruvate dioxygenase from streptomyces avermitilis (SavHppD), can be repurposed to enable abiological radical-relay process to access C(sp<sup>3</sup>)-H azidation products. This represents the first known instance of enzymatic radical relay azidation reactions. In this chapter, we describe the detailed experimental protocol to convert promiscuous nonheme iron enzymes into efficient and selective biocatalyst for radical relay azidation reactions. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation mutagenesis, protein expression and whole-cell reactions screening in a 96-well plate. These protocol details might be useful to engineer various nonheme iron enzymes for other applications.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"703 ","pages":"195-213"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291296","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
Biochemical and biophysical approaches to characterization of the aromatic amino acid hydroxylases. 用生物化学和生物物理方法描述芳香族氨基酸羟化酶的特征。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-05 DOI: 10.1016/bs.mie.2024.05.009
Paul F Fitzpatrick, S Colette Daubner
{"title":"Biochemical and biophysical approaches to characterization of the aromatic amino acid hydroxylases.","authors":"Paul F Fitzpatrick, S Colette Daubner","doi":"10.1016/bs.mie.2024.05.009","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.009","url":null,"abstract":"<p><p>The aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase utilize a non-heme iron to catalyze the hydroxylation of the aromatic rings of their amino acid substrates, with a tetrahydropterin serving as the source of the electrons necessary for the monooxygenation reaction. These enzymes have been subjected to a variety of biochemical and biophysical approaches, resulting in a detailed understanding of their structures and mechanism. We summarize here the experimental approaches that have led to this understanding.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"704 ","pages":"345-361"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291214","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
Methods for production and assaying catalysis of isolated recombinant human aspartate/asparagine-β-hydroxylase. 生产和检测分离重组人天冬氨酸/天冬酰胺-β-羟化酶催化作用的方法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-29 DOI: 10.1016/bs.mie.2024.06.003
Lennart Brewitz, Amelia Brasnett, Lara I Schnaubelt, Patrick Rabe, Anthony Tumber, Christopher J Schofield
{"title":"Methods for production and assaying catalysis of isolated recombinant human aspartate/asparagine-β-hydroxylase.","authors":"Lennart Brewitz, Amelia Brasnett, Lara I Schnaubelt, Patrick Rabe, Anthony Tumber, Christopher J Schofield","doi":"10.1016/bs.mie.2024.06.003","DOIUrl":"10.1016/bs.mie.2024.06.003","url":null,"abstract":"<p><p>Aspartate/asparagine-β-hydroxylase (AspH) is a transmembrane 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of aspartate- and asparagine-residues in epidermal growth factor-like domains (EGFDs) of its substrate proteins. Upregulation of ASPH and translocation of AspH from the endoplasmic reticulum membrane to the surface membrane of cancer cells is associated with enhanced cell motility and worsened clinical prognosis. AspH is thus a potential therapeutic and diagnostic target for cancer. This chapter describes methods for the production and purification of soluble constructs of recombinant human AspH suitable for biochemical and crystallographic studies. The chapter also describes efficient methods for performing turnover and inhibition assays which monitor catalysis of isolated recombinant human AspH in vitro using solid phase extraction coupled to mass spectrometry (SPE-MS). The SPE-MS assays employ synthetic disulfide- or thioether-bridged macrocyclic oligopeptides as substrates; a macrocycle is an apparently essential requirement for productive AspH catalysis and mimics an EGFD disulfide isomer that is not typically observed in crystal and NMR structures. SPE-MS assays can be used to monitor catalysis of 2OG oxygenases other than AspH; the methods described herein are representative for 2OG oxygenase SPE-MS assays useful for performing kinetic and/or inhibition studies.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"704 ","pages":"313-344"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291300","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
Spectroscopic definition of ferrous active sites in non-heme iron enzymes. 非血红素铁酶中亚铁活性位点的光谱定义。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-21 DOI: 10.1016/bs.mie.2024.05.019
Edward I Solomon, Robert R Gipson
{"title":"Spectroscopic definition of ferrous active sites in non-heme iron enzymes.","authors":"Edward I Solomon, Robert R Gipson","doi":"10.1016/bs.mie.2024.05.019","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.019","url":null,"abstract":"<p><p>Non-heme iron enzymes play key roles in antibiotic, neurotransmitter, and natural product biosynthesis, DNA repair, hypoxia regulation, and disease states. These enzymes had been refractory to traditional bioinorganic spectroscopic methods. Thus, we developed variable-temperature variable-field magnetic circular dichroism (VTVH MCD) spectroscopy to experimentally define the excited and ground ligand field states of non-heme ferrous enzymes (Solomon et al., 1995). This method provides detailed geometric and electronic structure insight and thus enables a molecular level understanding of catalytic mechanisms. Application of this method across the five classes of non-heme ferrous enzymes has defined that a general mechanistic strategy is utilized where O<sub>2</sub> activation is controlled to occur only in the presence of all cosubstrates.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"703 ","pages":"29-49"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11391101/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291210","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
Preparation of coenzyme F430 biosynthetic enzymes and intermediates. 辅酶 F430 生物合成酶和中间体的制备。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-07-20 DOI: 10.1016/bs.mie.2024.06.008
Prosenjit Ray, Chelsea R Rand-Fleming, Steven O Mansoorabadi
{"title":"Preparation of coenzyme F430 biosynthetic enzymes and intermediates.","authors":"Prosenjit Ray, Chelsea R Rand-Fleming, Steven O Mansoorabadi","doi":"10.1016/bs.mie.2024.06.008","DOIUrl":"10.1016/bs.mie.2024.06.008","url":null,"abstract":"<p><p>Methyl-coenzyme M reductase (MCR) is the key enzyme in pathways for the formation and anaerobic oxidation of methane. As methane is a potent greenhouse gas and biofuel, investigations of MCR catalysis and maturation are of interest for the development of both methanogenesis inhibitors and natural gas conversion strategies. The activity of MCR is dependent on a unique, nickel-containing coenzyme F430, the most highly reduced tetrapyrrole found in nature. Coenzyme F430 is biosynthesized from sirohydrochlorin in four steps catalyzed by the CfbABCDE enzymes. Here, methods for the expression and purification of the coenzyme F430 biosynthesis enzymes are described along with conditions for the synthesis and purification of biosynthetic intermediates on the milligram scale from commercially available porphobilinogen.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"702 ","pages":"147-170"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142000396","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
Seahorse assay for the analysis of mitochondrial respiration using Saccharomyces cerevisiae as a model system. 以酿酒酵母为模型系统分析线粒体呼吸的海马测定法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-24 DOI: 10.1016/bs.mie.2024.07.061
Abhishek Kumar, Tejashree Pradip Waingankar, Patrick D'Silva
{"title":"Seahorse assay for the analysis of mitochondrial respiration using Saccharomyces cerevisiae as a model system.","authors":"Abhishek Kumar, Tejashree Pradip Waingankar, Patrick D'Silva","doi":"10.1016/bs.mie.2024.07.061","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.061","url":null,"abstract":"<p><p>Eukaryotic cells require energy to perform diverse cellular functions critical for survival. Mitochondria are multifunctional organelles that generate energy in the form of Adenosine triphosphate by oxidative phosphorylation, emphasizing their importance to eukaryotic cell viability. The ability of mitochondria to consume oxygen for respiration is a key parameter in assessing mitochondrial health. Therefore, developing new techniques to monitor mitochondrial respiration are crucial for advancing our understanding of organelle functioning. Recently, Seahorse technology has emerged as a valuable tool to analyze various aspects of mitochondrial bioenergetics. Although the Seahorse assay is well established in adherent cell lines and other model organisms, it remains challenging to employ it efficiently in yeast, a powerful genetic system for studying mitochondrial biology. In this chapter, we provide a comprehensive methodology for assessing oxygen consumption rate in baker's yeast using Seahorse.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"673-683"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564661","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
AP endonuclease 1: Biological updates and advances in activity analysis. AP 内切酶 1：生物学更新和活性分析进展。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-05 DOI: 10.1016/bs.mie.2024.07.011
Karen H Almeida, Morgan E Andrews, Robert W Sobol
{"title":"AP endonuclease 1: Biological updates and advances in activity analysis.","authors":"Karen H Almeida, Morgan E Andrews, Robert W Sobol","doi":"10.1016/bs.mie.2024.07.011","DOIUrl":"10.1016/bs.mie.2024.07.011","url":null,"abstract":"<p><p>Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1, APEX1, REF1, HAP1) is an abasic site-specific endonuclease holding critical roles in numerous biological functions including base excision repair, the DNA damage response, redox regulation of transcription factors, RNA processing, and gene regulation. Pathologically, APE1 expression and function is linked with numerous human diseases including cancer, highlighting the importance of sensitive and quantitative assays to measure APE1 activity. Here, we summarize biochemical and biological roles for APE1 and expand on the discovery of APE1 inhibitors. Finally, we highlight the development of assays to monitor APE1 activity, detailing a recently improved and stabilized DNA Repair Molecular Beacon assay to analyze APE1 activity. The assay is amenable to analysis of purified protein, to measure changes in APE1 activity in cell lysates, to monitor human patient samples for defects in APE1 function, or the cellular and biochemical response to APE1 inhibitors.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"705 ","pages":"347-376"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400628","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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