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1. Carotenoid cleavage enzymes evolved convergently to generate the visual chromophore

   https://doi.org/10.1038/s41589-024-01554-z  

   Solano, Yasmeen_J; Everett, Michael_P; Dang, Kelly_S; Abueg, Jude; Kiser, Philip_D (February 2024, Nature Chemical Biology)

   Abstract The retinal light response in animals originates from the photoisomerization of an opsin-coupled 11-cis-retinaldehyde chromophore. This visual chromophore is enzymatically produced through the action of carotenoid cleavage dioxygenases. Vertebrates require two carotenoid cleavage dioxygenases, β-carotene oxygenase 1 and retinal pigment epithelium 65 (RPE65), to form 11-cis-retinaldehyde from carotenoid substrates, whereas invertebrates such as insects use a single enzyme known as Neither Inactivation Nor Afterpotential B (NinaB). RPE65 and NinaB coupletrans–cisisomerization with hydrolysis and oxygenation, respectively, but the mechanistic relationship of their isomerase activities remains unknown. Here we report the structure of NinaB, revealing details of its active site architecture and mode of membrane binding. Structure-guided mutagenesis studies identify a residue cluster deep within the NinaB substrate-binding cleft that controls its isomerization activity. Our data demonstrate that isomerization activity is mediated by distinct active site regions in NinaB and RPE65—an evolutionary convergence that deepens our understanding of visual system diversity. 

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2. A Conformational Study of the 10-23 DNAzyme via Programmed DNA Self-Assembly

   https://doi.org/10.1039/D2CC01144A  

   Mao, Dake; Li, Qian; Li, Qian; Wang, Pengfei; Mao, Chengde (January 2022, Chemical Communications)

   This communication measures the inter-helical angle of the 10-23 DNAzyme-substrate complex by atomic force microscopy (AFM). specificity. Herein, we have devised a strategy to assemble the DNAzyme-substrate complex into a periodic DNA 2D array, which allows reliable study of the conformation of the 10-23 DNAzyme by AFM imaging and fast Fourier transform (FFT). Specifically, the angle between the two flanking helical domains of the catalytic core has been determined via the repeating distance of 2D array. We expect that the same strategy can generally be applicable for studying other nucleic acid structures. 

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3. Long Oligos: Direct Chemical Synthesis of Genes with up to 1,728 Nucleotides

   https://doi.org/10.26434/chemrxiv-2024-zb7vk  

   Yin, Yipeng; Arneson, Reed; Yuan, Yinan; Fang, Shiyue (July 2024, RSC and ACS)

   The longest oligos that can be chemically synthesized using known methods are typically considered to be 200-mers. Here, we report direct synthesis of an 800-mer green fluorescent protein (GFP) gene and a 1,728-mer Φ29 DNA polymerase gene on an automated synthesizer. Key innovations that enabled the breakthrough include conducting the synthesis on the smooth surface of glass wool or glass bead rather than within the pores of traditional solid supports, and the use of the powerful catching-by-polymerization (CBP) method for the isolation of the full-length oligos from the crude mixture. Conducting the synthesis on smooth surface not only eliminated the steric hindrance that would otherwise prevent long oligo assembly, but also, surprisingly, drastically reduced the errors that commonly occur in traditional oligo synthesis. The long oligos were characterized by cloning followed by Sanger sequencing. We anticipate that the new method for long oligo synthesis will have a significant impact on projects in areas such as synthetic biology, gene editing, protein engineering, and many others. 

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4. Calcium-Dependent Chemiluminescence Catalyzed by a Truncated c-MYC Promoter G-Triplex DNA

   https://doi.org/10.3390/molecules29184457  

   Das, Malay Kumar; Williams, Elizabeth P; Myhre, Mitchell W; David, Wendi M; Kerwin, Sean M (September 2024, Molecules)

   The dynamic landscape of non-canonical DNA G-quadruplex (G4) folding into G-triplex intermediates has led to the study of G-triplex structures and their ability to serve as peroxidase-mimetic DNAzymes. Here we report the formation, stability, and catalytic activity of a 5′-truncated c-MYC promoter region G-triplex, c-MYC-G3. Through circular dichroism, we demonstrated that c-MYC-G3 adopts a stable, parallel-stranded G-triplex conformation. The chemiluminescent oxidation of luminol by the peroxidase mimicking DNAzyme activity of c-MYC-G3 was increased in the presence of Ca2+ ions. We utilized surface plasmon resonance to characterize both c-MYC-G3 G-triplex formation and its interaction with hemin. The detailed study of c-MYC-G3 and its ability to form a G-triplex structure and its DNAzyme activity identifies issues that can be addressed in future G-triplex DNAzyme designs. 

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5. Two-Metal Ion Mechanism of DNA Cleavage by Activated, Filamentous SgrAI

   https://doi.org/10.1016/j.jbc.2024.107576  

   Shan, Zelin; Rivero-Gamez, Andres; Lyumkis, Dmitry; Horton, Nancy C (July 2024, Journal of Biological Chemistry)

   Toker, Alex (Ed.)

   Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-electron microscopy (cryo-EM). The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes. 

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