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Glucosyltransferase in the Presence and Absence of the Substrate Uridine Diphosphoglucose

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Glucosyltransferase in the Presence and Absence of the Substrate Uridine Diphosphoglucose The EMBO Journal vol.13 no.15 pp.3413-3422, 1994 Crystal structure of the DNA modifying enzyme :-glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose Alice Vrielinkl, Wolfgang Ruger2, Rabussay, 1982). Moreover, the host DNA is degraded by Huub P.C.Driessen3 and Paul S.Freemont4 phage encoded enzymes (Warner et al., 1970; Mathews et al., 1983). To protect it's own genome against phage Protein Structure Laboratory, Imperial Cancer Research Fund, encoded nucleases and host restriction endonuclease Lincoln's Inn Fields, London WC2A 3PX, UK, 2Arbeitsgruppe Molekulare Genetik, Fakultat fUr Biologie, Ruhr Universitat, Bochum, systems, the phage has evolved a specific DNA modifica- Germany and 3ICRF Unit of Structural Molecular Biology, Birkbeck tion system. In T-even phage this specific DNA modi- College, Malet Street, London WCIE 7HX, UK fication process involves two steps. First, cytosine is 'Present address: Department of Biochemistry, McGill University, replaced by 5-hydroxymethylcytosine which is incorpor- 3655 Drummond Street, Montreal, Canada ated into DNA synthesis forming hydroxymethylated DNA 4Corresponding author (HMC-DNA) (Wyatt and Cohen, 1952; Lamm et al., 1988). As a second step, in a post-replicative mechanism, Communicated by M.Crumpton the hydroxymethylated cytosines are glucosylated forming glucose-HMC-DNA (Revel, 1983): Bacteriophage T4 P-glucosyltransferase (EC 2.4.1.27) UDP-glucose + HMC-DNA - catalyses the transfer of glucose from uridine diphos- glucosyl-HMC-DNA + UDP. phoglucose to hydroxymethyl groups of modified cyto- The enzymes catalysing DNA glucosylation in T4 phage sine bases in T4 duplex DNA forming f-glycosidic are a-glucosyltransferase (AGT) and 0-glucosyltransferase linkages. The enzyme forms part of a phage DNA (BGT) (Kornberg et al., 1961; Josse and Kornberg, 1962; protection system. We have solved and refined the Zimmerman et al., 1962). In T2 and T6 phage ,-glucosyl- crystal structure of recombinant P-glucosyltransferase transferase is replaced by ,B-glucosyl-HMC-a-glucosyl- to 2.2 A resolution in the presence and absence of transferase (Lehman and Pratt, 1960). While AGT and the substrate, uridine diphosphoglucose. The structure BGT form a- and P-glycosidic linkages directly to the comprises two domains of similar topology, each remin- hydroxymethylcytosine bases respectively, 3-glucosyl- iscent of a nucleotide binding fold. The two domains HMC-a-glucosyltransferase links a second glucose are separated by a central cleft which generates a molecule in a 1,6, linkage to bases which have already concave surface along one side of the molecule. The been a-glucosylated (Lehman and Pratt, 1960). The gluco- substrate-bound complex reveals only clear electron sylation pattern occurs in a species-specific fashion for density for the uridine diphosphate portion of the each of the three phage strains (Lehman and Pratt, 1960). substrate. The UDPG is bound in a pocket at the The glucosylation reaction catalysed by these enzymes bottom of the cleft between the two domains and makes involves the transfer of glucose from host-synthesized extensive hydrogen bonding contacts with residues of uridine diphosphoglucose (UDPG) to the hydroxymethyl the C-terminal domain only. The domains undergo a group of cytosine bases in double-stranded DNA. rigid body conformational change causing the structure The genes for these three enzymes have been sequenced to adopt a more closed conformation upon ligand and the proteins overexpressed and purified (Gram and binding. The movement of the domains is facilitated Ruger 1985; Tomaschewski et al., 1985; Winkler and by a hinge region between residues 166 and 172. Ruger 1993). Sequence comparisons among the three Electrostatic surface potential calculations reveal a glucosyltransferase enzymes show only limited homology large positive potential along the concave surface of (Tomaschewski et al., 1985; Winkler and Roger 1993), the structure, suggesting a possible site for duplex suggesting that these enzymes may have different three- DNA interaction. dimensional structures, although convergent structural Key words: DNA modification/enzyme/glucosylation/ evolution cannot be excluded. T-phage/X-ray crystal structure Apart from the protective function, glucosylation of phage DNA has also been implicated as having a con- trol function on phage-specific gene expression. Studies Introduction have shown that non-glucosylated T4 DNA is significantly more active in stimulating transcription and protein T-even bacteriophage, and in particular T4, have been the synthesis (Cox and Conway, 1973; Roger 1978) and subject of extensive biochemical and genetic analyses that this control occurs during late gene expression leading to a detailed molecular understanding of T-phage (Dharmalingam and Goldberg, 1979). This control function infection, replication and assembly (Mosig and Eiserling, may involve the specificity of the phage-induced modifica- 1988). The extreme virulence of these phages is reflected tion of Escherichia coli RNA polymerase (Wu and in a complete cessation of host macromolecular synthesis Geiduschek, 1975) or the structure of the glucosylated immediately after phage infection (for a review see DNA template which may result in an altered susceptibility © Oxford University Press 3413 A.Vrielink et al. of glucosylated phage DNA to nucleases and other This is of particular importance since VSG expression is enzymes. Experimental studies have shown that glucosyl- the primary mechanism for parasitic surface coat replace- ated DNA is unable to undergo the transition from B ment, a mechanism which protects the parasite from to A conformation, whereas non-glucosylated DNA can immune recognition and neutralization (Bemards et al., (Mokulskaya et al., 1966). The glucose group of the 1984). The identification of a glucosylated form of DNA modified B-DNA would lie in the major groove and in organisms other than phage suggests that this form of would sterically prevent major groove narrowing, an event DNA modification may be more widespread than has characteristic of B to A transition. Non-glucosylated DNA previously been thought and leads to further speculation therefore would have greater structural flexibility than on mechanisms of DNA protection and control of gene glucosylated DNA, allowing it potentially to adopt a larger expression. number of conformations. In order to understand, at the molecular level, this For many years DNA modification by cytosine hydroxy- unusual DNA modification process we have solved the methylation and glucosylation have been found only in T- crystal structure of T4-phage [-glucosyltransferase. The even phage. More recently, however, a similar modification BGT structure represents the first example of an enzyme has been observed in the African trypanosome, Trypano- which glucosylates double-stranded DNA and provides soma brucei, where the modified base has been identified as the basis for studies into the mechanisms of non-specific 3-D-glucosyl-hydroxymethyluracil (Gommers-Ampt et al., DNA recognition combined with specific base modifica- 1993). The role of such a modification system in Tbrucei tion. Furthermore, the BGT structure may provide clues is unclear, although it has been suggested to be directly as to the mechanism of the trypanosome-specific DNA involved in the regulation of variant surface glycoprotein modification system which may have important therapeutic (VSG) gene expression (Gommers-Ampt et al., 1993). consequences. A B Fig. 1. Stereo diagram showing the regions of the final 2Fobs-FcaIc electron density maps for I-glucosyltransferase calculated using all reflections between 10 and 2.2 A and phases from the final model. The contour level used is 1.3 times the standard deviation of each map. (A) A view of the electron density for Ile94 and Tyr95 in the substrate-free structure. (B) A view of the density for uridine diphosphate in the substrate-bound structure. 3414 I--glucosyltransferase Results and discussion ferase (Cheng et al., 1993a), guanine DNA methyl- transferase (Moore et al., 1994) and adenine-specific DNA Electron density map and quality of the model methyltransferase (Labahn et al., 1994) shows only limited The final electron density maps for both the substrate free similarity. Both the HhaI DNA methyltransferase and and substrate bound models were calculated using the adenine-specific DNA methyltransferase are monomers Fourier coefficients (2Fobs-Fcalc), (Xcaic. The maps show comprising two domains, one of which contains a nucleo- clear electron density for residues 1-67, 75-107 and tide binding fold that binds the substrate, S-adenosyl-L- 123-351. The substrate-free structure includes 184 water methionine. molecules and the UDPG bound model includes positions for the uridine diphosphate portion of the UDPG substrate UDPG binding and 221 water molecules. Two loops within the structure, In order to obtain a substrate-bound complex, data were 68-74 and 108-122 have no visible electron density and collected using crystals in which UDPG had not been thus could not be modelled. Figure 1 shows examples of removed. Difference electron density maps using data the electron density map where a clear interpretation of collected from these crystals, and the substrate-free struc- the structure was possible. Ramachandran plots of the two ture after rigid body and positional refinement, showed models (not illustrated) show that all non-glycine residues clear density only for the uridine diphosphate portion
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