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1. Cofactor Biogenesis in Cysteamine Dioxygenase: C−F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine

   https://doi.org/10.1002/ange.201803907  

   Wang, Yifan; Griffith, Wendell P.; Li, Jiasong; Koto, Teruaki; Wherritt, Daniel J.; Fritz, Elizabeth; Liu, Aimin (June 2018, Angewandte Chemie)

   Abstract Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein‐derived cofactor. Reported herein is the discovery and elucidation of a Cys‐Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C−S) bond. By genetically incorporating an unnatural amino acid, 3,5‐difluoro‐tyrosine (F₂‐Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon–fluorine bond activation and fluoride release were identified by mass spectrometry and¹⁹F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low‐resolution techniques. Finally, a new sequence motif, C‐X‐Y‐Y(F), is proposed for identifying the Cys‐Tyr crosslink. 

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2. Cleavage of a carbon–fluorine bond by an engineered cysteine dioxygenase

   https://doi.org/10.1038/s41589-018-0085-5  

   Li, Jiasong; Griffith, Wendell P.; Davis, Ian; Shin, Inchul; Wang, Jiangyun; Li, Fahui; Wang, Yifan; Wherritt, Daniel J.; Liu, Aimin (May 2018, Nature Chemical Biology)

   Cysteine dioxygenase (CDO) plays an essential role in sulfur metabolism by regulating homeostatic levels of cysteine. Human CDO contains a post-translationally generated Cys93–Tyr157 cross-linked cofactor. Here, we investigated this Cys–Tyr cross-linking by incorporating unnatural tyrosines in place of Tyr157 via a genetic method. The catalytically active variants were obtained with a thioether bond between Cys93 and the halogen-substituted Tyr157, and we determined the crystal structures of both wild-type and engineered CDO variants in the purely uncross-linked form and with a mature cofactor. Along with mass spectrometry and 19F NMR, these data indicated that the enzyme could catalyze oxidative C–F or C–Cl bond cleavage, resulting in a substantial conformational change of both Cys93 and Tyr157 during cofactor assembly. These findings provide insights into the mechanism of Cys–Tyr cofactor biogenesis and may aid the development of bioinspired aromatic carbon–halogen bond activation. 

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3. Comparison of the pH‐dependent formation of His and Cys heptapeptide complexes of nickel(II), copper(II), and zinc(II) as determined by ion mobility‐mass spectrometry

   https://doi.org/10.1002/jms.4489  

   Yousef, Enas_N; Angel, Laurence_A (February 2020, Journal of Mass Spectrometry)

   Abstract The analog methanobactin (amb) peptide with the sequence ac‐His₁‐Cys₂‐Gly₃‐Pro₄‐Tyr₅‐His₆‐Cys₇(amb_5A) will bind the metal ions of zinc, nickel, and copper. To further understand how amb_5Abinds these metals, we have undertaken a series of studies of structurally related heptapeptides where one or two of the potential His or Cys binding sites have been replaced by Gly, or the C‐terminus has been blocked by amidation. The studies were designed to compare how these metals bind to these sequences in different pH solutions of pH 4.2 to 10 and utilized native electrospray ionization (ESI) with ion mobility‐mass spectrometry (IM‐MS) which allows for the quantitative analysis of the charged species produced during the reactions. The native ESI conditions were chosen to conserve as much of the solution‐phase behavior of the amb peptides as possible and an analysis of how the IM‐MS results compare with the expected solution‐phase behavior is discussed. The oligopeptides studied here have applications for tag‐based protein purification methods, as therapeutics for diseases caused by elevated metal ion levels or as inhibitors for metal‐protein enzymes such as matrix metalloproteinases. 

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4. Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from Gluconacetobacter diazotrophicus

   https://doi.org/10.1007/s10930-023-10158-w  

   Standke, Heidi G; Kim, Lois; Owens, Cedric P (December 2023, The Protein Journal)

   Abstract NifA is a σ⁵⁴activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in NifA is poorly understood. In α- and β-proteobacteria, redox sensing involves two pairs of Cys residues within and immediately following the protein’s central AAA⁺domain. In this work, we examine if an additional Cys pair that is part of a C(X)₅ C motif and located immediately upstream of the DNA binding domain of NifA from the α-proteobacteriumGluconacetobacter diazotrophicus(Gd) is involved in redox sensing. We hypothesize that the Cys residues’ redox state may directly influence the DNA binding domain’s DNA binding affinity and/or alter the protein’s oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. TheK_dof NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. TheK_dof 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 µM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain ofGd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner. 

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5. Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach

   https://doi.org/10.1016/bs.mie.2024.05.018  

   Duan, Ran; Li, Jiasong; Liu, Aimin (September 2024, Elsevier)

   Bridwell-Rabb, Jennifer (Ed.)

   Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes. 

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