5-thyminyl-5,6-dihydrothymine (also called spore photoproduct or SP) is the exclusive DNA photo-damage product in bacterial endospores. It is repaired by a radical SAM (S-adenosylmethionine) enzyme, the spore photoproduct lyase (SPL), at the bacterial early germination phase. Our previous studies proved that SPL utilizes the 5′-dA• generated by SAM cleavage reaction to abstract the H6proR atom to initiate the SP repair process. The resulting thymine allylic radical was suggested to take an H atom from an unknown protein source, most likely the cysteine 141. Here we show that C141 can be readily alkylated in the native SPL by iodoacetamide treatment, suggesting that it is accessible to the TpT radical. SP repair by the SPL C141A mutant yields TpTSO2− and TpT simultaneously from the very beginning of the reaction; no lag phase is observed for the TpTSO2− formation. Should any other protein residue serve as the H donor, its presence would result in TpT as the major product at least for the first enzyme turnover. These observations provide strong evidence to support C141 as the direct H atom donor. Moreover, due to the lack of this intrinsic H donor, the C141A mutant produces TpT via an unprecedented thymine cation radical reduction (proton coupled electron transfer) process, contrasting to the H atom transfer mechanism in the WT SPL reaction. The C141A mutant repairs SP at a rate which is ~3-fold slower than the WT enzyme. Formation of TpTSO2− and TpT exhibit a Vmax deuterium kinetic isotope effect (KIE) of 1.7 ± 0.2 respectively, which is smaller than the DVmax KIE of 2.8 ± 0.3 determined in the WT SPL reaction. These findings suggest that removing the intrinsic H atom donor disturbs the rate-limiting process in the enzyme catalysis. As expected, the pre-reduced C141A mutant only supports ~ 0.4 turnover, which is in sharp contrast to the > 5 turnovers exhibited by the WT SPL reaction, suggesting that the enzyme catalytic cycle (SAM regeneration) is disrupted by this single mutation.

Spore photoproduct lyase (SPL) repairs a covalent UV-induced thymine dimer, spore photoproduct (SP), in germinating endospores and is responsible for endospores’ strong UV resistance. SPL is a radical SAM enzyme, which uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine (SAM), generating a catalytic 5′-deoxyadenosyl radical (5′-dA•). This in turn abstracts an 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 an H atom to the thymine radical, resulting in a putative thiyl radical. Here we present structural and biochemical data which 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-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 44,000 members) to bear a catalytically operating HAT chain.

We describe here the design, construction and validation of ALTHEA Gold Libraries™. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/J H (H3J) fragments. One IGHV gene provided a universal V H scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal V H scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries™ with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated K D values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C-80°C, demonstrating that ALTHEA Gold Libraries™ are a valuable source of specific, high affinity and highly stable antibodies. ARTICLE HISTORY

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