A radical transfer pathway in spore photoproduct lyase

Abstract

Spore photoproduct lyase (SPL) repairs a covalent UV-induced thymine dimer, spore photoproduct (SP), in germinating endospores and is responsible for the strong UV resistance of endospores. SPL is a radical S-adenosyl-l-methionine (SAM) enzyme, which uses a [4Fe-4S](+) cluster to reduce SAM, generating a catalytic 5'-deoxyadenosyl radical (5'-dA(•)). This in turn abstracts a H atom from SP, generating an SP radical that undergoes β scission to form a repaired 5'-thymine and a 3'-thymine allylic radical. Recent biochemical and structural data suggest that a conserved cysteine donates a H atom to the thymine radical, resulting in a putative thiyl radical. Here we present structural and biochemical data that suggest that two conserved tyrosines are also critical in enzyme catalysis. One [Y99(Bs) in Bacillus subtilis SPL] is downstream of the cysteine, suggesting that SPL uses a novel hydrogen atom transfer (HAT) pathway with a pair of cysteine and tyrosine residues to regenerate SAM. The other tyrosine [Y97(Bs)] has a structural role to facilitate SAM binding; it may also contribute to the SAM regeneration process by interacting with the putative (•)Y99(Bs) and/or 5'-dA(•) intermediates to lower the energy barrier for the second H abstraction step. Our results indicate that SPL is the first member of the radical SAM superfamily (comprising more than 44000 members) to bear a catalytically operating HAT chain.

Figure 1

SP formation and enzymatic repair mediated by SPL. In this study, we also employed a d₄-SP TpT species, where the H_proR and the three hydrogen atoms of the methyl group on the 5′-T are replaced by deuterium atoms.

Figure 2

Active site of Gt SPL in complex with SP (in white), the [4Fe-4S] cluster and SAM. The C140_(Gt), Y96_(Gt) and Y98_(Gt) residues correspond to the C141_(Bs), Y97_(Bs) and Y99_(Bs) residues in Bs SPL respectively. The distances between protein residues, SP and SAM are indicated by dashed lines (PDB code 4FHD) (17).

Figure 3

HPLC chromatograph of the SP TpT repair mediated by the SPL Y99F_(Bs) mutant with 30 μM enzyme, 150 μM SAM and 1 mM dithionite. SP TpT eluted at 5.4 min, 5′-dA at 8.9 min, and TpT at 14.7 min. See SI for HPLC chromatograph of the Y97F_(Bs) mutant reaction.

Figure 4

Mass spectrometry analysis of 5′-dA isolated from Bs SPL reactions conducted with d₄-SP TpT as the substrate. The d₁-5′-dA forms via H atom abstraction from the d₄-SP TpT and the unlabeled 5′-dA forms via uncoupled SAM cleavage reaction. The exact mass for each 5′-dA species is shown in SI.

Figure 5

Secondary structure of substrate-free WT SPL_(Gt) (cyan) and the Y98F_(Gt) mutant (purple, equivalent to Y99F_(Bs) mutant). The superposition of both structures was performed with Coot using the SSM program and all residues (65). The iron-sulfur cluster is depicted in orange (Fe) and yellow (S). SAM is colored by atom type (green C, blue N, red O and yellow S).

Figure 6

Hypothesized reaction mechanism for SPL (The residues are numbered according to the protein sequence in Bs SPL). This mechanism implies that SPL uses a minimum of four H atom transfer processes (labeled in blue numbers) in each catalytic cycle. One of the four processes occurs between a tyrosine and a cysteine, suggesting that SPL uses a novel HAT pathway for SAM regeneration. The role of Y97_(Bs) in SPL catalysis needs further elucidation and is thus not shown here.