title: Expression and purification of a single-chain Type IV restriction enzyme Eco94GmrSD and determination of its substrate preference.
abstract: 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 ECSTEC94C_1402 from E. coli strain STEC_94C 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 (Δip1) 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.
authors.affiliation: 1] New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA [2] State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai, 200030, China.
authors.forename: Xinyi
authors.initials: X
authors.lastname: He
authors.affiliation: New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Victoria
authors.initials: V
authors.lastname: Hull
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Green St, Baltimore, MD 21201-1503, USA.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Thomas
authors.affiliation: New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Fu
authors.affiliation: New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Sonal
authors.initials: S
authors.lastname: Gidwani
authors.affiliation: Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, Box 1677, 1425 Madison Avenue, New York, NY 10029, USA.
authors.forename: Yogesh K
authors.initials: YK
authors.lastname: Gupta
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Green St, Baltimore, MD 21201-1503, USA.
authors.forename: Lindsay W
authors.initials: LW
authors.lastname: Black
authors.affiliation: New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Shuang-yong
authors.initials: SY
authors.lastname: Xu
mesh_terms: D020134:Catalytic Domain; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression; D007477:Ions / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D013379:Substrate Specificity
chemical_list: D007477:Ions; D008670:Metals; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D004262:DNA Restriction Enzymes