RESUMEN
The Geobacillus stearothermophilus splG gene encodes a thermophilic spore photoproduct lyase (SplG) that belongs to the family of radical S-adenosylmethionine (AdoMet) enzymes. The aerobically purified apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holoform. Formation of the [4Fe-4S] cluster was proven by quantification of the amount of iron and sulfur per homodimer and by UV and EPR spectroscopy. The UV spectrum features a characteristic absorbance at 420 nm typical for [4Fe-4S] clusters, and the EPR data were found to be identical to those of other proteins containing an [4Fe-4S]+ center. Probing of the activity of the holo-SplG with oligonucleotides containing one spore photoproduct lesion at a defined site proved that the enzyme is able to turn over substrate. In addition to repair, we observed cleavage of AdoMet to generate 5'-deoxyadenosine. In the presence of aza-AdoMet the SplG is completely inhibited, which provides direct support for the repair mechanism.
Asunto(s)
Bacillaceae/fisiología , ADN/química , Proteínas/fisiología , Catálisis , Cromatografía Liquida , Reparación del ADN , ADN de Cadena Simple/química , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/química , Modelos Químicos , Oligonucleótidos/química , Estructura Secundaria de Proteína , Proteínas/química , Factores de Tiempo , Rayos UltravioletaRESUMEN
Investigation of the DNA repair process performed by the spore photoproduct (SP) lyase repair enzyme is strongly hampered by the lack of defined substrates needed for detailed enzymatic studies. The problem is particularly severe because the repair enzyme belongs to the class of strongly oxygen-sensitive radical (S)-adenosylmethionine (SAM) enzymes, which are notoriously difficult to handle. We report the synthesis of the spore photoproduct analogues 1 a and 1 b, which have open backbones and are diastereoisomers. In order to solve the problem of stereochemical assignment, two further derivatives 2 a and 2 b with closed backbones were prepared. The key step of the synthesis of 2 a/b is a metathesis-based macrocyclization that strongly increases the conformational rigidity of the synthetic spore photoproduct derivatives. NOESY experiments of the cyclic isomers furnished a clear cross-peak pattern that allowed the unequivocal assignment of the stereochemistry. The results were transferred to the data for isomers 1 a and 1 b, which were subsequently used for enzymatic-repair studies. These studies were performed with the novel spore photoproduct lyase repair enzyme from Geobacillus stearothermophilus. The studies showed an accordance with a recent investigation performed by us with the spore photoproduct lyase from Bacillus subtilis, in that only the S isomer 1 a is recognized and repaired. The ability to prepare a defined functioning substrate now paves the way for detailed enzymatic studies of the SP-lyase lesion recognition and repair process.
Asunto(s)
Proteínas/química , Timidina/análogos & derivados , Timidina/química , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Conformación Molecular , Estereoisomerismo , Rayos UltravioletaRESUMEN
The spore photoproduct lyase is a Fe-S/AdoMet DNA repair enzyme, which directly repairs spore lesions, induced by UV irradiation of spores, using an unknown radical mechanism. The air sensitive radical SAM enzyme was for the first time challenged with synthetically pure substrates. It was found that the enzyme recognizes a synthetic 5S-configured spore lesion without the central phosphodiester bond. The 5R-configured lesion is in contrast to current belief not a substrate.
Asunto(s)
Bacillus subtilis/enzimología , Reparación del ADN , Proteínas/metabolismo , Cromatografía Líquida de Alta Presión , Daño del ADN/efectos de la radiación , Organofosfatos/química , Organofosfatos/metabolismo , Proteínas/química , Estereoisomerismo , Rayos UltravioletaRESUMEN
Thietanes were used in the past as mimics for an unstable oxetane intermediate formed during the repair of mutagenic (6-4) lesions. The thietane derivatives were found to be not repaired, raising the question of how well thietanes are cleaved by single electron donation compared to oxetanes. We have prepared two flavin-containing oxetane and thietane model compounds for the (6-4) photolyase catalyzed repair process and we show that both are efficiently cleaved by a reduced and deprotonated flavin. Thietanes are therefore excellent models. The lack of their repair can be attributed to lack of binding.