title: Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis.
abstract: The Moco (molybdenum cofactor) sulfurase ABA3 from Arabidopsis thaliana catalyses the sulfuration of the Moco of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes. ABA3 consists of an N-terminal NifS-like domain that exhibits L-cysteine desulfurase activity and a C-terminal domain that binds sulfurated Moco. The strictly conserved Cys430 in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the Moco of aldehyde oxidase and xanthine oxidoreductase. In addition to Cys⁴³⁰, another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among Moco sulfurase proteins and, at the same time, being in close proximity to Cys⁴³⁰. By determination of the number of surface-exposed cysteine residues and the number of persulfide-binding cysteine residues in combination with the sequential substitution of each of the nine cysteine residues, a second persulfide-binding cysteine residue, Cys²⁰⁶, was identified. Furthermore, the active-site Cys⁴³⁰ was found to be located on top of a loop structure, formed by the two flanking residues Cys⁴²⁸ and Cys⁴³⁵, which are likely to form an intramolecular disulfide bridge. These findings are confirmed by a structural model of the NifS-like domain, which indicates that Cys⁴²⁸ and Cys⁴³⁵ are within disulfide bond distance and that a persulfide transfer from Cys⁴³⁰ to Cys²⁰⁶ is indeed possible.
authors: Department of Plant Biology, Braunschweig University of Technology, Humboldtstrasse 1, 38023 Braunschweig, Germany.
authors: Markus
authors: M
authors: Lehrke
authors: Steffen
authors: S
authors: Rump
authors: Torsten
authors: T
authors: Heidenreich
authors: Josef
authors: J
authors: Wissing
authors: Ralf R
authors: RR
authors: Mendel
authors: Florian
authors: F
authors: Bittner
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain / Q000235:genetics; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D006801:Humans; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D000090006:Molybdenum Cofactors; D016297:Mutagenesis, Site-Directed / Q000379:methods; D011485:Protein Binding / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D060066:Protein Interaction Maps; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism; D017385:Sequence Homology; D013329:Structure-Activity Relationship; D013440:Sulfides / Q000378:metabolism*; D013466:Sulfurtransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D003067:Coenzymes; D003545:Cysteine; D004220:Disulfides; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D011621:Pteridines; D013440:Sulfides; D013466:Sulfurtransferases; C080001:nifS protein, Bacteria; C051552:persulfides; C022261:molybdopterin; C440026:ABA3 protein, Arabidopsis