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 (N 2,3-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 O 9-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.
Highlights
Non-natural deoxynucleosides and deoxynucleoside analogs are important as therapeutic drugs [1,2,3], as probes for mechanisms of parasite-transmitted disease [4] and mechanisms of DNA repair [5] and for identifying and characterizing DNA damage
The purine deoxyribosyl transferase (PDT) and NDT enzymes show some structural similarity [6], NDT enzymes have been favored as biocatalysts since they are more flexible than PDT enzymes with regard to the type of donor base while retaining absolute stereospecificity for generating the bdeoxyribose anomer [3], expanding the pool of available donors and acceptors for transfer of modified sugars
N3 is hydrogen bonded to Asp75 with a bond distance of 2.5 A ; N1 and O9 participate in two hydrogen bonds with the active site water molecules
Summary
Non-natural deoxynucleosides and deoxynucleoside analogs are important as therapeutic drugs [1,2,3], as probes for mechanisms of parasite-transmitted disease [4] and mechanisms of DNA repair [5] and for identifying and characterizing DNA damage. Enzymic deoxyribosylation of modified nucleobases or glycosylation with non-natural sugars can offer an alternative synthetic pathway with high yields and stereo- and regioselectivity. The synthetic utility of deoxyribosyltransferase (DRT) and purine nucleoside phosphorylase (PNP) enzymes has been explored. Type I DRT (purine deoxyribosyltransferase; PDT) enzymes transfer deoxyribose groups exclusively from purine to purine, while Type II DRT (nucleoside deoxyribosyltransferase; NDT) enzymes can utilize purines and pyrimidines as both donors and acceptors. Escherichia coli (E. coli) PNP in the presence of uridine or thymidine phosphorylases and the appropriate deoxyribose donor has been used for this purpose [1, 2, 7]
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