Methods in enzymology最新文献_第2页

Determining biosynthetic gene cluster boundaries through comparative bioinformatics. 通过比较生物信息学确定生物合成基因簇边界。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-05-16 DOI: 10.1016/bs.mie.2025.04.001

Jerry Cui, Kou-San Ju

{"title":"Determining biosynthetic gene cluster boundaries through comparative bioinformatics.","authors":"Jerry Cui, Kou-San Ju","doi":"10.1016/bs.mie.2025.04.001","DOIUrl":"10.1016/bs.mie.2025.04.001","url":null,"abstract":"Modern advances in sequencing, \"-omics,\" and bioinformatics have given rise to the field of genome mining, loosely defined as the use of genomic data to guide natural product (NP) discovery. This technique applies our understanding of biosynthetic logic to predict the genes encoding for production of novel compounds. The major steps include identification of these biosynthetic gene clusters (BGCs), their classification, and prioritization for subsequent experimentation. Despite decades of effort, determination of cluster boundaries without experimental validation remains one of the greatest challenges in genome mining. Genes encoded within a BGC are the foundation for all downstream analysis. Thus, accurate determination of gene cluster content is critical for effective prioritization of BGCs and prediction of their products. Synteny, or the conservation of homologous genes and their arrangement, provides an effective solution for predicting these borders. Over evolutionary time, transfer and rearrangement of genes results in variability surrounding BGCs, such that natural breaks in conservation underlie these functional units. In this chapter, we provide a comprehensive approach for using synteny to delineate BGC boundaries.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"717 ","pages":"241-265"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12281633/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144619064","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

Characterization of bacterial transporters involved in the uptake of lignin-derived aromatic compounds. 参与木质素衍生芳香族化合物摄取的细菌转运体的表征。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-18 DOI: 10.1016/bs.mie.2025.01.053

Masaya Fujita, Naofumi Kamimura, Eiji Masai

引用次数: 0

Dye-decolorising peroxidase DyP1B from Pseudomonas fluorescens: Expression, reconstitution and reaction with polymeric lignin substrates. 荧光假单胞菌染料脱色过氧化物酶DyP1B：表达、重组和与聚合木质素底物的反应。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-09 DOI: 10.1016/bs.mie.2025.01.049

Rahman Rahmanpour, Timothy D H Bugg

引用次数: 0

Expression, purification, and crystallization of "humanized" Danio rerio histone deacetylase 10 "HDAC10", the eukaryotic polyamine deacetylase. 真核生物多胺脱乙酰酶“人源化”丹尼奥河组蛋白脱乙酰酶10“HDAC10”的表达、纯化和结晶。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-11 DOI: 10.1016/bs.mie.2025.01.074

Juana Goulart Stollmaier, Corey J Herbst-Gervasoni, David W Christianson

引用次数: 0

Genome mining for natural products made by multinuclear iron-dependent oxidation enzymes (MNIOs). 多核铁依赖性氧化酶（MNIOs）天然产物的基因组挖掘。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-16 DOI: 10.1016/bs.mie.2025.01.060

Yue Yu, Wilfred A van der Donk

引用次数: 0

De novo design of four-helix bundle proteins to bind metalloporphyrin cofactors. 结合金属卟啉辅助因子的四螺旋束蛋白的重新设计。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-14 DOI: 10.1016/bs.mie.2025.06.038

Karen R Coronado, Yixuan Zhu, Samuel I Mann

{"title":"De novo design of four-helix bundle proteins to bind metalloporphyrin cofactors.","authors":"Karen R Coronado, Yixuan Zhu, Samuel I Mann","doi":"10.1016/bs.mie.2025.06.038","DOIUrl":"10.1016/bs.mie.2025.06.038","url":null,"abstract":"The versatility of heme proteins in nature stems from the intricate control exerted by their protein scaffolds. De novo protein design offers a powerful means to dissect and recreate these structure-function relationships, enabling construction of novel metalloproteins with tailored functionalities. Here, we describe the computational design and characterization MPP1, a four-helix bundle protein designed to bind an abiological Mn-diphenylporphyrin (MnDPP) cofactor. Using parameterized coiled-coil backbones, flexible backbone sequence design in Rosetta, and structure-guided loop building, MPP1 was designed to accommodate the cofactor with precise positioning of axial ligands and second-shell interactions, as well as purposeful accessibility for oxidants and substrates. The resulting protein was the first crystallographically characterized de novo designed porphyrin-binding protein. MPP1 demonstrated the ability to stabilize a high-valent Mn(V)-oxo species and mediate thioether oxidation. This chapter details the computational strategies, cofactor incorporation, and solution characterization necessary to design and evaluate four-helix bundle proteins capable of binding porphyrin and porphyrin-like cofactors with atomic-level precision. Keywords: de novo design, protein design, bioinorganic chemistry, metalloporphyrins, heme proteins.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"720 ","pages":"1-22"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12497995/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145232889","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

Design and preparation of artificial heme-copper enzymes. 人造血红素-铜酶的设计与制备。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-09-26 DOI: 10.1016/bs.mie.2025.08.004

Hirbod Heidari, Duong Phan, Danielle Lawson, Yi Lu

引用次数: 0

Cobalt-substituted hemoprotein expression. 钴取代血红蛋白表达。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-04 DOI: 10.1016/bs.mie.2025.06.031

Madeline E Rodemeier, Olivia P Holsinger, Andrew R Buller

{"title":"Cobalt-substituted hemoprotein expression.","authors":"Madeline E Rodemeier, Olivia P Holsinger, Andrew R Buller","doi":"10.1016/bs.mie.2025.06.031","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.06.031","url":null,"abstract":"Substituting the native metal of metalloenzymes can significantly alter the enzymes' reactivity and spectroscopic properties. Cobalt is especially attractive as a substitute for the native iron center in hemoproteins, as it generates metal variants with complementary spectroscopic properties and could enable new modes of reactivity. Here, we describe a detailed protocol for the biosynthesis and incorporation of cobalt protoporphyrin IX (CoPPIX) into hemoproteins, replacing the native heme b cofactor during expression in the common laboratory strain Escherichia coli BL21(DE3). This protocol is described using the model hemoprotein Physeter macrocephalus (sperm whale) myoglobin. Because of cobalt's unique electronic and geometric properties, cobalt-substituted hemoproteins offer a valuable handle for spectroscopic characterization and structural studies. We describe analytic methods of assessing cofactor identity and purity, including electronic absorption spectroscopy, liquid-chromatography/mass-spectrometry, inductively coupled plasma-mass spectrometry, and electron paramagnetic resonance spectroscopy. This method for generating artificial metalloenzymes is effective, easy to implement, and can produce useful quantities of Co-substituted hemoproteins.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"720 ","pages":"55-76"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145232845","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

Preface. 前言。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00184-3

引用次数: 0

Reductive amination: Methods for cell-free and whole-cell biocatalysis. 还原胺化：无细胞和全细胞生物催化的方法。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-09 DOI: 10.1016/bs.mie.2025.01.002

Vasilis Tseliou, Matteo Damian, Josemarco Mendoza-Avila, Marco Rabuffetti, Francesco G Mutti

引用次数: 0