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Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase

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Abstract

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.

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Abbreviations

SP lyase:

Spore photoproduct lyase

SP:

Spore photoproduct

AdoMet:

S-Adenosyl-l-methionine

dAdo:

5′-Deoxyadenosine

SAH:

S-Adenosyl-l-homocysteine

TpT:

Thymidylyl-(3′–5′)-thymidine

(5R)-SPTpT:

5R stereoisomer of the SPTpT substrate

(5S)-SPTpT:

5S stereoisomer of the SPTpT substrate

EPR:

Electron paramagnetic resonance

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Acknowledgments

We would like to thank David Schwab and Eric Shepard for assistance in running EPR samples, and David Singel for use of his EPR instrument. This research was supported by the National Institutes of Health (GM67804 to J.B.B.). The Astrobiology Biogeocatalysis Research Center is supported by the NASA Astrobiology Institute (NAI05-19). The authors acknowledge support for the Mass Spectrometry Laboratory at the University of Illinois at Urbana-Champaign. The 70-VSE mass spectrometer was purchased in part with a grant from the Division of Research Resources, National Institutes of Health (RR 04648). The Ultima quadrupole TOF mass spectrometer was purchased in part with a grant from the National Science Foundation, Division of Biological Infrastructure (DBI-0100085).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, The Astrobiology Biogeocatalysis Research Center, Montana State University, 103 CBB, Bozeman, MT, 59717, USA

    Sunshine C. Silver, Tilak Chandra, Egidijus Zilinskas, Shourjo Ghose, William E. Broderick & Joan B. Broderick

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  1. Sunshine C. Silver
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  2. Tilak Chandra
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  3. Egidijus Zilinskas
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Correspondence to Joan B. Broderick.

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Silver, S.C., Chandra, T., Zilinskas, E. et al. Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase. J Biol Inorg Chem 15, 943–955 (2010). https://doi.org/10.1007/s00775-010-0656-8

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