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

Linlin Yang,Lei Li

doi:10.3389/fchem.2017.00014

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-; 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.

Highlights

DNA photoreaction induced by the UV portion of solar light is one of the most universal reactions occurring on our planet
Using deuterated dinucleotide spore photoproduct (SP) TpT as the substrate, we found that the kinetic isotope effects are changed in similar trends for both G168A and G168R mutants comparing with those observed in the wildtype (WT) enzyme, indicating that these mutations alter the protein function under a similar mechanism
The protein expression level in E. coli is suggested to be similar for all three proteins, i.e., WT spore photoproduct lyase (SPL), G168A and G168R, indicating that the mutations at the glycine do not alter the protein folding drastically enough to change the protein solubility

Summary

Introduction

DNA photoreaction induced by the UV portion of solar light is one of the most universal reactions occurring on our planet. DNA Lesion Repair with a neighboring pyrimidine residue. Such a dimerization reaction in typical vegetative cells leads to the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) (Figure 1). All three dimers can be repaired via a direct reversal mechanism, i.e., the dimer is reverted back to two pyrimidine residues by breaking the crosslinking bonds. Direct reversal repair of CPDs and 6-4PPs is performed by specific photolyases (Sancar, 2003, 2008; Li et al, 2010; Liu et al, 2011; Benjdia, 2012; Kneuttinger et al, 2014), which use light and flavin cofactors to generate radical species in the dimers before bond scission occurs resulting in two pyrimidine residues. Protein sequence analysis has identified a homologous region in the carboxyl-terminal portions of the CPD photolyases and SPL, indicating that these enzymes may have descended from a common ancestral protein (Fajardo-Cavazos et al, 1993)

Methods

Results

Conclusion