TAILIEUCHUNG - Báo cáo khoa học: Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3

Sequence alignments of human molybdopterin synthase sulfurase, MOCS3, showed that the N-terminal domain is homologous to Escherichia coli MoeB, whereas the C-terminal domain is homologous to rhodanese-like proteins. Previous studies showed that the activity of the separately purified rhodanese-like domain of MOCS3 displayed 1000-fold lower activity in comparison to bovine rhodanese with thiosulfate as sulfur source. | ễFEBS Journal Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3 Major differences in substrate specificity between eukaryotic and bacterial homologs Karsten Krepinsky and Silke Leimkuhler University of Potsdam Institute of Biochemistry and Biology Potsdam Germany Keywords evolution Moco MOCS3 MoeZ rhodanese Correspondence S. Leimkuhler University of Potsdam Institute of Biochemistry and Biology D-14476 Potsdam Germany Fax 49 331 977 5419 Tel 49 331 977 5603 E-mail sleim@ Received 21 February 2007 revised 20 March 2007 accepted 27 March 2007 doi Sequence alignments of human molybdopterin synthase sulfurase MOCS3 showed that the N-terminal domain is homologous to Escherichia coli MoeB whereas the C-terminal domain is homologous to rhodanese-like proteins. Previous studies showed that the activity of the separately purified rhodanese-like domain of MOCS3 displayed 1000-fold lower activity in comparison to bovine rhodanese with thiosulfate as sulfur source. When the six amino acid active site loop of MOCS3 rhodanese-like domain was exchanged with the loop found in bovine rhodanese thiosulfate cyanide sulfurtransferase activity was increased 165-fold. Site-directed mutagenesis of each individual residue of the active site loop of the MOCS3 rhodanese-like domain showed that the charge of the last amino acid determines thiosulfate sulfurtransferase activity. Replacing Asp417 by threonine resulted in 90-fold increased activity whereas replacing it by arginine increased the activity 470-fold. Using a fully defined in vitro system containing precursor Z MOCS2A E. coli MoaE E. coli MoeB Mg-ATP MOCS3 rho-danese-like domain and thiosulfate it was shown that sulfur transfer to MOCS2A was also affected by the alterations but not as drastically. Our studies revealed that in humans and most eukaryotes thiosulfate is not the physiologic sulfur donor for .

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