Essential cysteine residues in Bacillus subtilis spore photoproduct lyase identified by alanine scanning mutagenesis.
Endospore-forming bacteria (Bacillus and Clostridium spp.) are highly ultraviolet (UV) resistant and repair UV-induced DNA damage in part using the spore-specific DNA repair enzyme spore photoproduct (SP) lyase. SP lyase in all known sporeformers contains four conserved cysteine residues; three absolutely conserved residues are located at the "Radical SAM" consensus (C91xxxC95xxC98), which presumably participates in [4Fe-4S] cluster formation. A fourth conserved cysteine, the function of which is unknown, is located at C141 in SP lyase from all Bacillus spp. sequenced to date. To probe the function of the fourth cysteine, each conserved cysteine in the B. subtilis SP lyase was systematically altered to alanine by site-directed mutagenesis. UV-visible spectroscopy of wild-type and mutant SP lyases indicated that C141 does not participate in [4Fe-4S] formation and redox chemistry; however, in vivo SP lyase activity was abolished in all mutants, indicating an essential role for C141 in SP lyase activity.
Fajardo-Cavazos
Patricia
P
Department of Microbiology and Cell Science, University of Florida, Room 201-B Space Life Sciences Laboratory, Building M6-1025/SLSL, Kennedy Space Center, FL 32899, USA.
Rebeil
Roberto
R
Nicholson
Wayne L
WL
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics* / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis
D011506:Proteins; C088014:spore photoproduct lyase; D003545:Cysteine; D000409:Alanine