T4 phage beta-glucosyltransferase: Substrate binding and proposed catalytic mechanism

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteri ophage T4 which catalyses the transfer of glucose (Glc) from uridine diphos phoglucose (UDP-Glc) to 5-hydroxymethylcytosine (5-HMC) in double-stranded DNA. The glucosylation of T4 phage DNA is part of a phage DNA protection sy stem aimed at host nucleases. We previously reported the first three-dimens ional structure of BGT determined from crystals grown in ammonium sulphate containing UDP-Glc. In this previous structure, we did not observe electron density for the Glc moiety of UDP-Glc nor for two large surface loop regio ns (residues 68-76 and 109-122). Here we report two further BGT co-crystal structures, in the presence of UDP product (form I) and donor substrate YDP -Glc (form II), respectively. Form I crystals are grown in ammonium sulphat e and the structure has been determined to 1.88 Angstrom resolution (R-fact or 19.1%). Form II crystals are grown in polyethyleneglycol 4000 and the st ructure has been solved to 2.3 Angstrom resolution (R-factor 19.8%). The fo rm I structure is isomorphous to our previous BGT UDP-Glc structure. The fo rm II structure, however, has allowed us to model the two missing surface l oop regions and thus provides the first complete structural description of BGT. In this low-salt crystal form, we see no electron density for the Glc moiety from UDP-Glc similar to previous observations. Biochemical data howe ver, shows that BGT can cleave UDP-Glc in the absence of DNA acceptor, whic h probably accounts for the absence of Glc in our UDP-Glc substrate structu res. The complete BGT structure now provides a basis for detailed modelling of a BGT HMC-DNA ternary complex. By using the structural similarity betwe en the catalytic core of glycogen phosphorylase (GP) and BGT, we have model led the position of the Glc moiety in UDP-Glc. From these two models, we pr opose a catalytic mechanism for BGT and identify residues involved in both DNA binding and in stabilizing a "flipped-out" 5-HMC nucleotide. (C) 1999 A cademic Press.