The sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein conjugation and activation of sulfur carrier proteins.
Because of mechanistic parallels in the activation of ubiquitin and the biosynthesis of several sulfur-containing cofactors, we have characterized the human Urm1 and Saccharomyces cerevisiae Uba4 proteins, which are very similar in sequence to MOCS2A and MOCS3, respectively, two proteins essential for the biosynthesis of the molybdenum cofactor (Moco) in humans. Phylogenetic analyses of MOCS3 homologues showed that Uba4 is the MOCS3 homologue in yeast and thus the only remaining protein of the Moco biosynthetic pathway in this organism. Because of the high levels of sequence identity of human MOCS3 and yeast Uba4, we purified Uba4 and characterized the catalytic activity of the protein in detail. We demonstrate that the C-terminal domain of Uba4, like MOCS3, has rhodanese activity and is able to transfer the sulfur from thiosulfate to cyanide in vitro. In addition, we were able to copurify stable heterotetrameric complexes of Uba4 with both human Urm1 and MOCS2A. The N-terminal domain of Uba4 catalyzes the activation of either MOCS2A or Urm1 by formation of an acyl-adenylate bond. After adenylation, persulfurated Uba4 was able to form a thiocarboxylate group at the C-terminal glycine of either Urm1 or MOCS2A. The formation of a thioester intermediate between Uba4 and Urm1 or MOCS2A was not observed. The functional similarities between Uba4 and MOCS3 further demonstrate the evolutionary link between ATP-dependent protein conjugation and ATP-dependent cofactor sulfuration.
Schmitz
Jennifer
J
Institute of Biochemistry and Biology, University of Potsdam, D-14476 Potsdam, Germany.
Chowdhury
Mita Mullick
MM
Hänzelmann
Petra
P
Nimtz
Manfred
M
Lee
Eun-Young
EY
Schindelin
Hermann
H
Leimkühler
Silke
S
D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D003067:Coenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D019143:Evolution, Molecular; D006801:Humans; D008667:Metalloproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000090006:Molybdenum Cofactors; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D011621:Pteridines / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D040681:Structural Homology, Protein; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism*; D013466:Sulfurtransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*; D014452:Ubiquitins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D003067:Coenzymes; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009713:Nucleotidyltransferases; D011621:Pteridines; D029701:Saccharomyces cerevisiae Proteins; D013455:Sulfur; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D025801:Ubiquitin; D014452:Ubiquitins; C406283:UBA4 protein, S cerevisiae; C406282:URM1 protein, S cerevisiae; C529881:Urm1 protein, human; C022261:molybdopterin; C502790:MOCS3 protein, human; C085585:molybdopterin synthase