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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
Prediction of mitochondrial targeting signals and their cleavage sites. 线粒体靶向信号及其裂解位点的预测。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-11 DOI: 10.1016/bs.mie.2024.07.026
Fukasawa Yoshinori, Kenichiro Imai, Paul Horton
{"title":"Prediction of mitochondrial targeting signals and their cleavage sites.","authors":"Fukasawa Yoshinori, Kenichiro Imai, Paul Horton","doi":"10.1016/bs.mie.2024.07.026","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.026","url":null,"abstract":"<p><p>In this chapter we survey prediction tools and computational methods for the prediction of amino acid sequence elements which target proteins to the mitochondria. We will primarily focus on the prediction of N-terminal mitochondrial targeting signals (MTSs) and their N-terminal cleavage sites by mitochondrial peptidases. We first give practical details useful for using and installing some prediction tools. Then we describe procedures for preparing datasets of MTS containing proteins for statistical analysis or development of new prediction methods. Following that we lightly survey some of the computational techniques used by prediction tools. Finally, after discussing some caveats regarding the reliability of such methods to predict the effects of mutations on MTS function; we close with a discussion of possible future directions of computer prediction methods related to mitochondrial proteins.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"161-192"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504057","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
Isolation of yeast mitochondria by differential centrifugation. 通过差速离心法分离酵母线粒体。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-03 DOI: 10.1016/bs.mie.2024.07.024
Kuo Song, Heike Rampelt
{"title":"Isolation of yeast mitochondria by differential centrifugation.","authors":"Kuo Song, Heike Rampelt","doi":"10.1016/bs.mie.2024.07.024","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.024","url":null,"abstract":"<p><p>The isolation of intact and functional mitochondria is a powerful approach to characterize and study this organelle. The classical biochemical method of differential centrifugation is routinely used to isolate mitochondria. This method has several advantages, such as a high yield and easy adaptability. The isolated mitochondria are physiologically active and can be used for a variety of follow-up experiments, for example protein import and respiration measurements. Here, we describe the procedure to purify mitochondria from the budding yeast Saccharomyces cerevisiae. In addition, two approaches are introduced to assess the quality of isolated mitochondria, by limited proteinase K digestion or measurement of the membrane potential.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"3-18"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504049","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
Analysis of inner membrane lateral sorting at the presequence translocase. 前序转运酶的内膜横向分拣分析。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-21 DOI: 10.1016/bs.mie.2024.07.058
Hyun Kim
{"title":"Analysis of inner membrane lateral sorting at the presequence translocase.","authors":"Hyun Kim","doi":"10.1016/bs.mie.2024.07.058","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.058","url":null,"abstract":"<p><p>The translocase of the mitochondrial inner membrane (TIM23) complex mediates the import and membrane insertion of presequence-carrying mitochondrial proteins. It is experimentally challenging to determine whether the segment of the polypeptide is imported to the matrix or inserted into the inner membrane. Utilizing the unique topogenesis of Mgm1p, a versatile experimental approach to study the TIM23-mediated membrane insertion is developed and described in this chapter. This method combines a simple SDS-gel based assay with the quantification of the relative fractions of membrane inserted and non-inserted products, enabling the quantitative measurement of the membrane insertion efficiencies of a transmembrane segment into the mitochondrial inner membrane.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"23-38"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564284","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
Analysis of quality control pathways for the translocase of the outer mitochondrial membrane. 线粒体外膜转运酶的质量控制途径分析。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-15 DOI: 10.1016/bs.mie.2024.07.050
Lara Calvo Santos, Fabian den Brave
{"title":"Analysis of quality control pathways for the translocase of the outer mitochondrial membrane.","authors":"Lara Calvo Santos, Fabian den Brave","doi":"10.1016/bs.mie.2024.07.050","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.050","url":null,"abstract":"<p><p>The functionality of mitochondria depends on the import of proteins synthesized on cytosolic ribosomes. Impaired import into mitochondria results in mitochondrial dysfunction and proteotoxic accumulation of precursor proteins in the cytosol. All proteins sorted to inner mitochondrial compartments are imported via the translocase of the outer membrane (TOM) complex. Premature protein folding, a reduction of the mitochondrial membrane potential or defects in translocases can result in precursor arrest during translocation, thereby clogging the TOM channel and blocking protein import. In recent years, different pathways have been identified, which employ the cytosolic ubiquitin-proteasome system in the extraction and turnover of precursor proteins from the TOM complex. Central events in this process are the modification of arrested precursor proteins with ubiquitin, their extraction by AAA-ATPases and subsequent degradation by the 26 S proteasome. Analysis of these processes is largely facilitated by the expression of model proteins that function as efficient \"cloggers\" of the import machinery. Here we describe the use of such clogger proteins and how their handling by the protein quality control machinery can be monitored. We provide protocols to study the extent of clogging, the ubiquitin-modification of arrested precursor proteins and their turnover by the 26 S proteasome.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"565-584"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564299","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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