Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase.
Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)], a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an isotropic signal centered at g = 1.99, characteristic of a [3Fe-4S](+) cluster accounting for 3-4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a [4Fe-4S](+) cluster is observed that accounts for 34-45% of total iron. Addition of S-adenosylmethionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-L: -methionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 +/- 0.6 nmol min(-1) mg(-1), whereas no repair was observed for the 5S diastereomer.
Silver
Sunshine C
SC
Department of Chemistry and Biochemistry, The Astrobiology Biogeocatalysis Research Center, Montana State University, 103 CBB, Bozeman, MT 59717, USA.
Chandra
Tilak
T
Zilinskas
Egidijus
E
Ghose
Shourjo
S
Broderick
William E
WE
Broderick
Joan B
JB
D000693:Anaerobiosis; D046969:Clostridium acetobutylicum / Q000201:enzymology / Q000528:radiation effects; D004260:DNA Repair; D015226:Dinucleoside Phosphates / Q000737:chemistry* / Q000378:metabolism*; D055668:Photochemical Processes; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013057:Spectrum Analysis; D013171:Spores, Bacterial / Q000201:enzymology / Q000528:radiation effects; D013237:Stereoisomerism; D013379:Substrate Specificity
D015226:Dinucleoside Phosphates; D011506:Proteins; C017758:thymidylyl-(3'-5')-thymidine; C088014:spore photoproduct lyase
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