Structural Evidence of a Passive Base-flipping Mechanism for β-Glucosyltransferase

Abstract

β-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. β-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. The base-flipping mechanism has been widely studied and largely discussed in several reviews with the publication of the co-crystal structures of DNA methyltransferases, DNA base excision repair glycosylases, and endonucleases (1Chen X. Roberts R.J. Nucleic Acids Res. 2001; 19: 3784-3795Crossref Scopus (408) Google Scholar, 2Mol C.D. Parikh S.S. Putnam C.D. Lo T.P. Tainer J.A. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 101-128Crossref PubMed Scopus (172) Google Scholar, 3Hosfield D.J. Daniels D.S. Mol C.D. Putnam C.D. Parikh S.S. Tainer J.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 315-347Crossref PubMed Google Scholar, 4Scharer O.D. Jiricny J. BioEssays. 2001; 23: 270-281Crossref PubMed Scopus (235) Google Scholar). Rotating the sugar-phosphate backbone around the flipped-out target base (or abasic site) allows the deoxyribose and the base being carried along to enter into the enzyme's active site before the chemistry step is performed. Whether rotation is initiated by an active process in which the enzyme rotates the sugar-phosphate backbone or a passive one in which the enzyme binds to a spontaneously flipped-out base in a transient conformation remains unclear. An active mechanism such as “push-pull” (5Slupphaug G. Mol C.D. Kavli B. Arvai A.S. Krokan H.E. Tainer J.A. Nature. 1996; 384: 87-92Crossref PubMed Scopus (