Structural evidence of a passive base-flipping mechanism for beta-glucosyltransferase.
Beta-glucosyltransferase (BGT) is a DNA-modifying enzyme and a glycosyltransferase. This inverting enzyme transfers glucose from UDP-glucose to the 5-hydroxymethyl cytosine bases of T4 phage DNA. From previous structural analyses we showed that Asp-100 and Asn-70 were, respectively, the catalytic base and the key residue for specific DNA recognition (Larivière, L., Gueguen-Chaignon, V., and Moréra, S. (2003) J. Mol. Biol. 330, 1077-1086). Here, we supply biochemical evidence supporting their essential roles in catalysis. We have also shown previously that BGT uses a base-flipping mechanism to access 5-hydroxymethyl cytosine (Larivière, L., and Moréra, S. (2002) J. Mol. Biol. 324, 483-490). Whether it is an active or a passive process remains unclear, as is the case for all DNA cleaving and modifying enzymes. Here, we report two crystal structures: (i) BGT in complex with a 13-mer DNA containing an A:G mismatch and (ii) BGT in a ternary complex with UDP and an oligonucleotide containing a single central G:C base pair. The binary structure reveals a specific complex with the flipped-out, mismatched adenine exposed to the active site. Unexpectedly, the other structure shows the non-productive binding of an intermediate flipped-out base. Our structural analysis provides clear evidence for a passive process.
Larivière
Laurent
L
Laboratoire d'Enzymologie et Biochimie Structurales, Unité Propre de Recherche 9063 CNRS, Bātiment 34, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Moréra
Solange
S
D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000225:Adenine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D014530:Uridine Diphosphate / Q000737:chemistry
D009841:Oligonucleotides; C011865:5-hydroxymethylcytosine; D001224:Aspartic Acid; D014530:Uridine Diphosphate; D044503:5-Methylcytosine; D001216:Asparagine; D003596:Cytosine; D004247:DNA; D005964:Glucosyltransferases; C026585:DNA beta-glucosyltransferase; D000225:Adenine