title: Isd11p protein activates the mitochondrial cysteine desulfurase Nfs1p protein.
abstract: Cysteine desulfurases perform pyridoxal phosphate (PLP)-dependent desulfuration of cysteine. The key steps of the enzymatic cycle include substrate binding to PLP, formation of a covalent persulfide intermediate at the active site cysteine, and transfer of sulfur to recipients for use in various metabolic pathways. In Saccharomyces cerevisiae, the cysteine desulfurase Nfs1p and an accessory protein, Isd11p, are found primarily in mitochondria, and both are essential for cell viability. Although cysteine desulfurases are conserved from bacteria to humans, Isd11p is found only in eukaryotes and not in prokaryotes. Here we show that Isd11p activates Nfs1p. The enzyme without Isd11p was inactive and did not form the [(35)S]persulfide intermediate from the substrate [(35)S]cysteine. Addition of Isd11p to inactive Nfs1p induced formation of the persulfide. Remarkably, in a two-step assay, [(35)S]cysteine could be bound to the inactive Nfs1p in a PLP-dependent manner, and the enzyme could be subsequently induced to form the persulfide by addition of Isd11p. A mutant form of Isd11p with the (15)LYK(17) motif changed to (15)AAA(17) was able to bind but failed to activate Nfs1p, thus separating these two functions of Isd11p. Finally, compared with Nfs1p with or without the bound Isd11p mutant, the Nfs1p·Isd11p complex was more resistant to inactivation by an alkylating agent. On the basis of these novel findings, we propose that interaction of Isd11p with Nfs1p activates the enzyme by inducing a conformational change, thereby promoting formation of the persulfide intermediate at the active site cysteine. Such a conformational change may protect the active site cysteine from alkylating agents.
authors: Department of Pharmacology and Physiology, University of Medicine & Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07101.
authors: Alok
authors: A
authors: Pandey
authors: Department of Medicine, Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors: Heeyong
authors: H
authors: Yoon
authors: Department of Medicine, Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors: Elise R
authors: ER
authors: Lyver
authors: Department of Medicine, Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors: Andrew
authors: A
authors: Dancis
authors: Department of Pharmacology and Physiology, University of Medicine & Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07101. Electronic address: painde@umdnj.edu.
authors: Debkumar
authors: D
authors: Pain
mesh_terms: D020816:Amino Acid Motifs; D013437:Carbon-Sulfur Lyases / Q000737:chemistry; D020134:Catalytic Domain; D002499:Centrifugation, Density Gradient; D003545:Cysteine / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D013466:Sulfurtransferases / Q000737:chemistry* / Q000378:metabolism
chemical_list: C508374:Isd11 protein, S cerevisiae; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D013466:Sulfurtransferases; C115912:NFS1 protein, S cerevisiae; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; D003545:Cysteine