Role of Two Histidines in the (6-4) Photolyase Reaction

Abstract

The reaction mechanism of Xenopus (6-4) photolyase was investigated using several mutant enzymes. In the active site, which is homologous between the cis,syn-cyclobutane pyrimidine dimer and (6-4) photolyases, four amino acid residues that are specific to (6-4) photolyase, Gln(288), His(354), Leu(355), and His(358), and two conserved tryptophans, Trp(291) and Trp(398), were substituted with alanine. Only the L355A mutant had a lower affinity for the substrate, which suggested a hydrophobic interaction with the (6-4) photoproduct. Both the H354A and H358A mutations resulted in an almost complete loss of the repair activity, although the Trp(291) and Trp(398) mutants retained some activity. Taking the pH profile of the (6-4) photolyase reaction into consideration with this observation, we propose a mechanism in which these histidines catalyze the formation of the four-membered ring intermediate in the repair process of this enzyme. When deuterium oxide was used as a solvent, the repair activity was decreased. The proton transfer shown by this isotope effect supports the proposed mechanism. The substrate binding and the reaction mechanism are discussed in detail using a molecular model.

Electron Nuclear Double Resonance Differentiates Complementary Roles for Active Site Histidines in (6-4) Photolyase

“…Two mutant proteins, H354A and H358A, were also examined by pulsed ENDOR spectroscopy at two extreme pH values, pH 6 and 9.5, to identify the origin of the pH dependence of the principal values of the H 8␣ hyperfine tensor. Both mutant enzymes are inactive in photorepair (16), but the photoreduction reaction is still possible. In Fig.…”

“…These histidines were proposed to play a pivotal role in the formation of the oxetane intermediate from the (6-4) photoproduct (Fig. 2), a step that is believed to occur prior to light-induced reductive cleavage (16). A theoretical structure model of (6-4) photolyase, which was built based on the crystallographic structure of E. coli CPD photolyase (45), suggests that these histidines are located close to (ϳ0.4 nm) the 8␣-methyl group of the isoalloxazine moiety.…”

“…The hyperfine interactions arising from protons attached to the isoalloxazine moiety of the FAD cofactor were exploited to probe the immediate environment of the flavin, in particular hydrogen bonding and the micropolarity. The main focus of this study is to obtain information about the protonation states of His 354 and His 358 , which are essential for DNA repair by photolyase (16). This information is crucial for a thorough understanding of the light-independent catalytic steps preceding blue light-initiated enzymatic DNA repair, and the specific structural traits that distinguish (6-4) photolyase from the related CPD photolyase.…”

“…However, until now it has not been established which histidine acts as an acid and which one functions as a base. The proposed reaction mechanism is based on a biochemical study using alanine-substituted mutants (16), and as yet spectroscopy has not been employed to address this stage of the reaction.…”

“…1) (14 -16). In the initial light-independent step, a 6Ј-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases (His 354 and His 358 in Xenopus laevis photolyase) (16) (Fig. 2).…”