Ethenoguanines undergo glycosylation by nucleoside 2'-deoxyribosyltransferases at non-natural sites.

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.

Structural characterization of the mammalian deoxynucleotide N-hydrolase Rcl and its stabilizing interactions with two inhibitors.

The gene Rcl encodes a deoxynucleoside 5'-monophosphate N-glycosidase that catalyzes the hydrolysis of the N-glycosidic bond of the nucleotide to give deoxyribose 5-phosphate and a nucleobase, preferentially a purine. This enzyme is over-expressed in several cancers, and its rate of expression is correlated to the degree of aggressiveness of tumors, which makes it a new and attractive therapeutic target. We describe here its structural characterization in the presence of two inhibitory substrate mimics. One of these ligands corresponds to the monophosphorylated form of acyclovir, which is used in the clinic. This study reveals an important ligand-induced stabilization of the dimer structure of the enzyme. The original structural features of Rcl will be helpful for designing new inhibitors.

The c-Myc target gene Rcl (C6orf108) encodes a novel enzyme, deoxynucleoside 5'-monophosphate N-glycosidase.

RCL is a c-Myc target with tumorigenic potential. Genome annotation predicted that RCL belonged to the N-deoxyribosyltransferase family. However, its putative relationship to this class of enzymes did not lead to its precise biochemical function. The purified native or N-terminal His-tagged recombinant rat RCL protein expressed in Escherichia coli exhibits the same enzyme activity, deoxynucleoside 5'-monophosphate N-glycosidase, never before described. dGMP appears to be the best substrate. RCL opens a new route in the nucleotide catabolic pathways by cleaving the N-glycosidic bond of deoxynucleoside 5'-monophosphates to yield two reaction products, deoxyribose 5-phosphate and purine or pyrimidine base. Biochemical studies show marked differences in the terms of the structure and catalytic mechanism between RCL and of its closest enzyme family neighbor, N-deoxyribosyltransferase. The reaction products of this novel enzyme activity have been implicated in purine or pyrimidine salvage, glycolysis, and angiogenesis, and hence are all highly relevant for tumorigenesis.

Replication of hexitol oligonucleotides as a prelude to the propagation of a third type of nucleic acid in vivo.

No backbone motif other than phospho-ribose and phospho-deoxyribose has been found in natural nucleic acids, currently restricting the molecular types of replicable biopolymers to DNA and RNA. With the aim of propagating and expressing a third type of nucleic acid in vivo, we assessed the replicability of polynucleotides with a phospho-hexitol backbone (HNA) in vivo and in vitro. Faithful polymerisation of up to four deoxynucleotides templated by hexitol oligonucleotides was established in vitro using DNA polymerase from Escherichia coli (PolA Klenow exo-fragment) and Thermus aquaticus (Taq polymerase). Condensation of up to three successive hTTPs (hexitol thymidine triphosphate) in responses to a pentameric hexitol template (hA)5 could also be demonstrated in vitro. Such a marginal HNA-dependent HNA polymerase activity of natural polymerases may be evolved in the future to catalyse in vitro amplification of HNA. The transmission of a two-codon-long genetic message carried on a hexameric hexitol template was also established using a selection screen for restoring thymidylate synthase activity in E. coli. These results exemplify the potential that can be explored by converting artificial substrates with natural enzymes in the field of informational polymer synthesis.