Insights into the Activity Change of Spore Photoproduct Lyase Induced by Mutations at a Peripheral Glycine Residue

Abstract

UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e., the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. This mechanism is consistent with the recently solved SPL structure that shows all three residues are located in proximity and thus able to participate in the radical transfer process during the enzyme catalysis. In contrast, an earlier in vivo mutational study identified a glycine to arginine mutation at the position 168 on the B. subtilis SPL that is >15 Å away from the enzyme active site. This mutation appears to abolish the enzyme activity because endospores carrying this mutant were sensitive to UV light. To understand the molecular basis for this rendered enzyme activity, we constructed two SPL mutations G168A and G168R, examined their repair of dinucleotide SP TpT, and found that both mutants exhibit reduced enzyme activity. Comparing with the wildtype (WT) SPL enzyme, the G168A mutant slows down the SP TpT repair by 3~4-fold while the G168R mutant by ~ 80-fold. Both mutants exhibit a smaller apparent (DV) kinetic isotope effect (KIE) but a bigger competitive (DV/K) KIE than that by the WT SPL. Moreover, the G168R mutant also produces a large portion of the abortive repair product TpT-SO2−; the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate. All these data imply that the mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process.

Very high sensitivity of African rice to artificial ultraviolet-B radiation caused by genotype and quantity of cyclobutane pyrimidine dimer photolyase

“…However, contrary to our expectations, the 3D structure predicted using PyMol showed that this replacement caused structural changes through the loss of polar contacts between rice CPD photolyase and the FAD chromophore. Yang and Li demonstrated that the replacement of a G residue with an A residue at position 168 of a UV-induced spore photoproduct (5-tyminyl-5,6-diydrothymine) repair enzyme, spore photoproduct lyase (SPL), reduces the SPL activity by 3~4-fold relative to that of the WT enzyme 29 . Thus, the replacement of G with A at position 283 of rice CPD photolyase might cause subtle conformational changes that can affect the enzyme activity.…”

Assessment of the Effect of Light, HgCl<sub>2</sub> and Organic Compound on <i>Enterococcus faecalis</i> and <i>Escherichia coli</i> Cells Survive in Aquatic Microcosm

“…Thus, due to the particular conditions inside the spore (high dehydration, presence of proteins modifying the conformation of the DNA, high concentration of dipicolinic acid, among others), the absorption of a UV photon by DNA does not lead to the appearance of 12 different photoproducts as in all other cells but to the formation of only one type of photoproduct. Spore germination thus requires only a single repair enzyme, spore photoproduct lyase, present in large quantities in the spore, to repair its DNA [9].…”