Structure of ctAlkD Reveals High Structural Homology to HLR Alkypurine Glycosylases Despite Low Sequence Homology

Methylating compounds are a common cause of DNA damage, especially in soil dwelling bacteria. To counter such environmental effects, bacteria such as Clostridium thermocellum possess a class of DNA-alkylation repair enzymes, which excise methylated purines from the genome, thereby ensuring genomic stability and preventing the corresponding mutations. C. thermocellum is an anaerobic, gram-positive, soil-dwelling bacterium. In its environment it is exposed to toxic exogenous substances, among which are compounds that cause methylation of DNA bases. Under these conditions, similar bacteria have evolved a special class of DNA glycosylases that remove methylated purines. These enzymes were recently shown to excise adenine methylated by yatakemycin (YTM); a natural product that belongs to the duocarmycin family of minor groove alkylating agents. Analysis of the C. thermocellum genome revealed a distant homolog, ctAlkD, which shares only less than 21% sequence identity with known members of the HLR glycosylase family. To investigate the structural and functional­­­­­­ relationships of this distant homolog, the gene was cloned and over-expressed, the protein purified, and subsequently subject to crystallization. After optimization, protein crystals grew within several hours, shaped as elongated hexagonal rods. Diffraction data collected at the ESRF synchrotron facility to 2.5 Å resolution, identified the crystals as belonging to a rhombohedral space group, with two protein monomers in the crystallographic asymmetric unit. Despite low sequence identity, the main structural features of ctAlkD resemble those of known HLR family homologs and the active site residues are conserved. However, the N-terminal domain that in recent studies was shown to interact with the YTM moiety, is not conserved, and positioned in a manner which suggests a different mode of binding and possibly different substrate specificity.