title: Ethenoguanines undergo glycosylation by nucleoside 2'-deoxyribosyltransferases at non-natural sites.
abstract: Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT) with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N2,3-ethenoguanine) at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2'-deoxyribosyltransferase enzymes (DRT Type II) from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O9-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted in N7 and N1 ribosylation. Absent from the enzymatic and chemical glycosylations is the natural pattern of N3 ribosylation, verified by comparison of spectroscopic and chromatographic properties with an authentic standard synthesized by an unambiguous route.
authors.affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, The University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Ye
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, United States of America.
authors.forename: Debamita
authors.initials: D
authors.lastname: Paul
authors.affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, The University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Lina
authors.initials: L
authors.lastname: Gao
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, United States of America.
authors.forename: Jolita
authors.initials: J
authors.lastname: Seckute
authors.affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, The University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Ramiah
authors.initials: R
authors.lastname: Sangaiah
authors.affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, The University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Karupiah
authors.initials: K
authors.lastname: Jayaraj
authors.affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, The University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Zhenfa
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Institut Pasteur, Unité de Chimie et Biocatalyse, UMR CNRS, Paris, France.
authors.forename: P Alexandre
authors.initials: PA
authors.lastname: Kaminski
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, United States of America.
authors.forename: Steven E
authors.initials: SE
authors.lastname: Ealick
authors.affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, The University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Avram
authors.initials: A
authors.lastname: Gold
authors.affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, The University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Louise M
authors.initials: LM
authors.lastname: Ball
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007778:Lactobacillus / Q000201:enzymology; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D006147:Guanine; D010430:Pentosyltransferases; C021888:nucleoside deoxyribosyltransferase