Expression and purification of a single-chain Type IV restriction enzyme Eco94GmrSD and determination of its substrate preference

The first reported Type IV restriction endonuclease (REase) GmrSD consists of GmrS and GmrD subunits. In most bacteria, however, the gmrS and gmrD genes are fused together to encode a single-chain protein. The fused coding sequence for ECSTEC94C1402 from E. coli strain STEC94C was expressed in T7 Express. The protein designated as Eco94GmrSD displays modification-dependent ATP-stimulated REase activity on T4 DNA with glucosyl-5-hydroxymethyl-cytosines (glc-5hmC) and T4gt DNA with 5-hydroxymethyl-cytosines (5hmC). A C-terminal 6xHis-tagged protein was purified by two-column chromatography. The enzyme is active in Mg 2+ and Mn 2+ buffer. It prefers to cleave large glc-5hmC-or 5hmC-modified DNA. In phage restriction assays, Eco94GmrSD weakly restricted T4 and T4gt, whereas T4 IPI∗-deficient phage ( "ip1) were restricted more than 10 6-fold, consistent with IPI∗ protection of E. coli DH10B from lethal expression of the closely homologous E. coli CT596 GmrSD. Eco94GmrSD is proposed to belong to the His-Asn-His (HNH)-nuclease family by the identification of a putative C-terminal REase catalytic site D507-H508-N522. Supporting this, GmrSD variants D507A, H508A, and N522A displayed no endonuclease activity. The presence of a large number of fused GmrSD homologs suggests that GmrSD is an effective phage exclusion protein that provides a mechanism to thwart T-even phage infection.