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• W1966248180 abstract "A large number of nucleoside analogues and 2′-deoxynucleoside triphosphates (dNTP) have been synthesized to interfere with DNA metabolism. However, in vivo the concentration and phosphorylation of these analogues are key limiting factors. In this context, we designed enzymes to switch nucleobases attached to a deoxyribose monophosphate. Active chimeras were made from two distantly related enzymes: a nucleoside deoxyribosyltransferase from lactobacilli and a 5′-monophosphate-2′-deoxyribonucleoside hydrolase from rat. Then their unprecedented activity was further extended to deoxyribose triphosphate, and in vitro biosyntheses could be successfully performed with several base analogues. These new enzymes provide new tools to synthesize dNTP analogues and to deliver them into cells.Background: The nucleoside deoxyribosyltransferase family contains hydrolases and transferases with different substrate specificities.Results: Chimeras exchange deoxyribose 5-(mono, di, and tri)-phosphate between natural bases and analogues.Conclusion: Comparison of the structures and catalytic mechanisms of members of the nucleoside deoxyribosyltransferase family allows the design of unprecedented enzymes.Significance: Phosphodeoxyribosyltransferases open the road to new deoxyribonucleotides synthetic pathways. A large number of nucleoside analogues and 2′-deoxynucleoside triphosphates (dNTP) have been synthesized to interfere with DNA metabolism. However, in vivo the concentration and phosphorylation of these analogues are key limiting factors. In this context, we designed enzymes to switch nucleobases attached to a deoxyribose monophosphate. Active chimeras were made from two distantly related enzymes: a nucleoside deoxyribosyltransferase from lactobacilli and a 5′-monophosphate-2′-deoxyribonucleoside hydrolase from rat. Then their unprecedented activity was further extended to deoxyribose triphosphate, and in vitro biosyntheses could be successfully performed with several base analogues. These new enzymes provide new tools to synthesize dNTP analogues and to deliver them into cells. Background: The nucleoside deoxyribosyltransferase family contains hydrolases and transferases with different substrate specificities. Results: Chimeras exchange deoxyribose 5-(mono, di, and tri)-phosphate between natural bases and analogues. Conclusion: Comparison of the structures and catalytic mechanisms of members of the nucleoside deoxyribosyltransferase family allows the design of unprecedented enzymes. Significance: Phosphodeoxyribosyltransferases open the road to new deoxyribonucleotides synthetic pathways. DNA in all known living organisms is synthesized from deoxynucleoside triphosphates (dNTPs), the precursor substrates that are condensed by DNA polymerase enzymes, releasing pyrophosphate as co-product. No exception to this biosynthetic scheme was ever encountered in nature, whether nucleoside triphosphates are condensed onto a DNA template acting as co-catalyst, an RNA template, or no template at all. The pyrimidine and purine base moieties attached to the common triphosphodeoxyribosyl moiety of dNTPs are not universally conserved in nature. In addition to the four canonical dNTPs, four noncanonical dNTPs bearing an exotic pyrimidine and one bearing an exotic purine are condensed by bacterial viruses (1Kirnos M.D. Khudyakov I.Y. Alexandrushkina N.I. Vanyushin B.F. 2-Aminoadenine is an adenine substituting for a base in S-2L cyanophage DNA.Nature. 1977; 270: 369-370Crossref PubMed Scopus (113) Google Scholar, 2Warren R.A. Modified bases in bacteriophage DNAs.Annu Rev. Microbiol. 1980; 34: 137-158Crossref PubMed Scopus (169) Google Scholar). This shows that DNA can host modified nucleobases that could serve, for example, to extend the genetic code or to induce mutagenesis. The demand for purified deoxyribonucleotides is high, for example, for DNA synthesis (PCR or DNA microarrays) and reverse transcription in vitro in academic research and medical diagnosis. It extends to numerous nucleoside/nucleotide analogues that are used for DNA mutagenesis or labeling, as well as mechanistic probes to study nucleotide metabolic enzymes. In parallel, these modified precursors are also used as antibiotics, antiviral, and anticancer agents. The diversification of the “bio-compatible nucleotides” is also a field in expansion with the development of synthetic biology as illustrated by the development of new functional base pairs (3Benner S.A. Understanding nucleic acids using synthetic chemistry.Acc. Chem. Res. 2004; 37: 784-797Crossref PubMed Scopus (308) Google Scholar, 4Khakshoor O. Kool E.T. Chemistry of nucleic acids. Impacts in multiple fields.Chem. Commun. (Camb.). 2011; 47: 7018-7024Crossref PubMed Scopus (49) Google Scholar) or the chemical synthesis and assembly of genomes (5Gibson D.G. Glass J.I. Lartigue C. Noskov V.N. Chuang R.Y. Algire M.A. Benders G.A. Montague M.G. Ma L. Moodie M.M. Merryman C. Vashee S. Krishnakumar R. Assad-Garcia N. Andrews-Pfannkoch C. Denisova E.A. Young L. Qi Z.Q. Segall-Shapiro T.H. Calvey C.H. Parmar P.P. Hutchison 3rd, C.A. Smith H.O. Venter J.C. Creation of a bacterial cell controlled by a chemically synthesized genome.Science. 2010; 329: 52-56Crossref PubMed Scopus (1723) Google Scholar, 6Gibson D.G. Smith H.O. Hutchison 3rd, C.A. Venter J.C. Merryman C. Chemical synthesis of the mouse mitochondrial genome.Nat. Methods. 2010; 7: 901-903Crossref PubMed Scopus (243) Google Scholar). Methods for chemical synthesis of nucleotides are evolving, but nucleotides are still difficult to make, isolate, and characterize (7Burgess K. Cook D. Syntheses of nucleoside triphosphates.Chem. Rev. 2000; 100: 2047-2060Crossref PubMed Scopus (188) Google Scholar). Enzyme-mediated syntheses of natural nucleotides and of some analogues (7Burgess K. Cook D. Syntheses of nucleoside triphosphates.Chem. Rev. 2000; 100: 2047-2060Crossref PubMed Scopus (188) Google Scholar, 8Mikhailopulo I.A. Miroshnikov A.I. Biologically important nucloesides. Modern trends in biotechnology and application.Mendeleev Commun. 2011; 21: 57-68Crossref Scopus (98) Google Scholar) have been developed using whole cells or coupled enzymes, but the available repertoire of enzymes is still limited. The enzymatic synthesis of pure 2′-deoxyribonucleotides and the metabolic engineering for producing such compounds is hindered by the intricacy of biosynthesis and salvage pathways, upstream and downstream of the DNA polymerization step. Each nucleobase A, C, G, and T is indeed processed separately by highly discriminating enzymes that phosphorylate nucleoside monophosphates into diphosphates. In addition, nucleotide reductases are required to convert the ribose moiety of RNA precursors into the deoxyribose moiety of DNA building blocks (rNDP into dNDP or rNTP into dNTP, depending on nucleotide reductase families) through a cumbersome and fragile free radical mechanism (9Nordlund P. Reichard P. Ribonucleotide reductases.Annu. Rev. Biochem. 2006; 75: 681-706Crossref PubMed Scopus (829) Google Scholar). The natural design of this complex metabolic machinery is to prevent the generation of unwanted rNTPs and dNTPs and their incorporation into RNA and DNA. We sought to bypass this intricacy and expand DNA chemistry by elaborating an enzyme that would transfer 5′-phosphorylated deoxyribose between any two nucleobases (N and N′), canonical or synthetic, e.g., dN − (P)x + N′ ⇌ N + dN′ − (P)x, where x stands for 0, 1, 2, or 3. We proceeded using a structure-based and stepwise approach to build a chimera from two distantly related enzymes: a nucleoside deoxyribosyltransferase (EC 2.4.2.6) (NDT)2 from lactobacilli and a 5′-monophosphate-2′-deoxyribonucleoside hydrolase (EC 3.2.2.-) from rat (Rcl) (Fig. 1). Although their sequences are globally dissimilar (∼18% of sequence identity), these two proteins, whose oligomerization states differ (NDT is an hexamer, and Rcl is a dimer in solution), adopt the same Rossmann fold (Fig. 2A), and they share a common catalytic triad. Both enzymes hydrolyze their substrates via the formation of a deoxyribose (5-phosphate)-enzyme covalent intermediate (10Doddapaneni K. Zahurancik W. Haushalter A. Yuan C. Jackman J. Wu Z. RCL hydrolyzes 2′-deoxyribonucleoside 5′-monophosphate via formation of a reaction intermediate.Biochemistry. 2011; 50: 4712-4719Crossref PubMed Scopus (9) Google Scholar, 11Porter D.J. Merrill B.M. Short S.A. Identification of the active site nucleophile in nucleoside 2-deoxyribosyltransferase as glutamic acid 98.J. Biol. Chem. 1995; 270: 15551-15556Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) using a conserved glutamate residue (11Porter D.J. Merrill B.M. Short S.A. Identification of the active site nucleophile in nucleoside 2-deoxyribosyltransferase as glutamic acid 98.J. Biol. Chem. 1995; 270: 15551-15556Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). However, these enzymes differ functionally: NDT can act as a transferase on nucleosides (x = 0), whereas Rcl hydrolyzes monophosphate deoxynucleosides (x = 1) (12Ghiorghi Y.K. Zeller K.I. Dang C.V. Kaminski P.A. The c-Myc target gene Rcl (C6orf108) encodes a novel enzyme, deoxynucleoside 5′-monophosphate N-glycosidase.J. Biol. Chem. 2007; 282: 8150-8156Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) (Fig. 1). These structural and functional features are in agreement with Rcl and NDT diverging from a common enzyme ancestor with broader substrate specificity.FIGURE 2A, overlay of the dimeric structures of Rcl (Protein Data Bank code 2KLH; green) and NDT (Protein Data Bank code 1F8Y; blue). B, zoom in the complexes of Rcl (green) with GMP (5GP) and of NDT (blue) with 5-methyl 2′-deoxy pseudouridine (5MD), respectively. Backbones are represented as ribbons, and ligands are represented as sticks. Important side chains were drawn as sticks. The figures were generated by PyMOL.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Here, we report the structure-based design of Rcl-NDT chimera with mono-, di-, and triphosphodeoxyribosyltransferase activities. These enzymes can transfer deoxyribose 5-phosphate between canonical bases and also between analogues opening the road to new synthetic pathways of deoxyribonucleotides. All of the chemicals were obtained from Sigma-Aldrich except dCTPαS from Biolog. The oligonucleotides used were: T7prom, 5′-CGCGAAATTAATACGACTCACTATAGGGG-3′; T7term, 5′-GGGGTTATGCTAGTTATTGCTCAGCGG-3′; f13R, 5′-CTTCGGTGCCGGCTGGCGCACTGACCGCCAAAAC-3′; f13Q, 5′-CTTCGGTGCCGGCTGGCAGACTGACCGCCAAAACA-3′; f13Q-, 5′-TGTTTTGGCGGTCAGTCTGCCAGCCGGCACCGAAG-3′; D92S, 5′-ATCCCTGACGAAGAAAGCGTCGGCCTGGGCATG-3′; N123S, 5′-CTACGGCAAGCCGATCAGCCTCATGAGCTGGGGCG-3′; N123S-, 5′-CGCCCCAGCTCATGAGGCTGATCGGCTTGCCGTAG-3′; N123T, 5′-CTACGGCAAGCCGATCACCCTCATGAGCTGGGGCG-3′; N123T-, 5′-CGCCCCAGCTCATGAGGGTGATCGGCTTGCCGTAG-3′; E91Q, 5′-TGTCTACATCCCTGACGAACAGGGCACCGGCC-3′; E91Q-, 5′-GGCCGGTGCCCTGTTCGTCAGGGATGTAGACA-3′; Y157K, 5′-GCAAGCTTTTACTTTACGGCACCTTCGTAGAAGTCGAAGC-3′; G92T93, 5′-GTCTACATCCCTGACGAAGAAGGCACCGGCCTGGGCA-3′; and G92T93-, 5′-TGCCCAGGCCGGTGCCTTCTTCGTCAGGGATGTAGAC-3′. Variants S92 and R13 S92 were obtained by using plasmid pET24a NDT as DNA template and oligonucleotides D92S and D92S and f13R, respectively, using the QuikChange® Multi site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. For variants S92 S123, R13 S92 S123, R13 S92 T123, R13 G92 T93 S123, R13 Q91 G92 T93 S123, Q13 S92 S123, and Q13 Q91 S92 S123, oligonucleotide N123S-, N123T-, G92 T93-, E91Q-, f13Q-, E91Q-, and T7 prom and oligonucleotide N123S, N123T, G92 T93, E91Q, f13Q, E91Q, and T7 term were used in two separate PCRs using plasmids pET24a NDT S92, pET24a NDT R13 S92, pET24a NDT R13 S92 S123, pET24a NDT R13 G92 T93 S123, pET24a NDT S92 S123, and pET24a NDT Q13 S92 S123 as DNA templates, respectively. The parameters used 1 cycle of 5 min at 95 °C; 25 cycles of 30 s at 95 °C, 30 s at 53 °C, and 30 s at 72 °C; and 1 cycle of 10 min at 72 °C. The annealing temperature was dependent on the pairs of oligonucleotides used. Oligonucleotides T7prom and T7term were used in a second PCR using aliquots of the first one using the same parameters as above with the exception of an annealing temperature of 61 °C. For variant R13 G92 T93 S123 K157, oligonucleotides T7 prom and Y157K were used in a standard PCR using plasmid pET24a NDT R13 G92 T93 S123 as DNA template. The amplified DNA fragments were purified by using the QIAquick PCR purification kit (Qiagen) and then digested with NdeI and BamHI enzymes over 2 h at 37 °C and repurified. Each PCR product was ligated with plasmid pET24a that had been digested with the same restriction enzymes. The ligation mixtures were used to transform strain DH5α. Plasmids with the correct sequence were used to transform strain Bli5. Plasmids containing an insert of the correct size were sequenced at the Plateforme Génomique Pf1 at the Institut Pasteur. Those with the correct sequences were used to transform strain BLi5. 650 ml of 2× YT medium inoculated with an overnight culture of BLi5 containing any of the pET24a+PDT* was grown under agitation at 37 °C until A600 = 0.6. Isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm, and the cultures were incubated for 2.5 h. Bacteria were centrifuged, and the pellets were frozen at −20 °C. The cells were resuspended in 20 ml of 50 mm MES, pH 6.0, and broken by one passage through a French press at 14000 p.s.i. Cell debris were pelleted by centrifugation at 12,000 rpm for 20 min at 4 °C. The total proteins contained in the supernatant were separated by an anion exchange chromatography on a Hi Trap Q HP with a linear gradient from 0 to 300 mm NaCl gradient in 50 mm MES, pH 6.0, at 2 ml min−1 for 100 min. Fractions containing the enzyme were pooled and precipitated at 4 °C by adding solid ammonium sulfate (0.6 mg/ml). The precipitate was pelleted by centrifugation at 12,000 rpm for 20 min at 4 °C. The pellet was resuspended into 50 mm MES, 100 mm NaCl, pH 6.0, and purified on a Hi Load Superdex S200 column at 1 ml min−1. The purified proteins ran as a single band at ∼18 kDa, consistent with the predicted molecular weight and was >98% pure as judged by SDS-PAGE with Coomassie Blue staining. Protein concentration was determined spectrophotometrically by UV absorption at 280 nm using an ϵ280 = 34,380. The standard assay for the different NDTs consisted of 5–15 μg of NDT in 50 mm MES buffer, pH 6.0, 3 mm dNxP (dNMP, dNDP, or dNTP), and 1 mm base in a total volume of 50 μl. The assays were incubated for 60 min at 37 °C. The products of the reactions were analyzed every 7 min by rapid resolution high performance liquid chromatography using a reverse phase column (ZORBAX Eclipse XDB-C18 2.1*50 mm 1.8 μm) with a flow rate of 0.25 ml/min and a linear gradient of 1–12% CH3CN (2–25% CH3CN or 1–90% CH3CN) in 20 mm triethylammonium acetate, pH 7.5, buffer for 7 min. The low resolution mass spectra of the newly synthesized deoxyribonucleotides were obtained on an Agilent 1200 series LC/MS 6120 quadrupole system using an atmospheric electrospray ionization system. Kinetic parameters kcat and Kmwere obtained by fitting the initial velocity at various substrate concentrations to a Michaelis-Menten equation using the software KaleidaGraph. Crystal growth conditions were screened using cations, anions, and AmSO4 kits from Qiagen. Crystallization was then optimized in medium concentration of NH4SO4 (∼1.2 m) in 0.1 m HEPES at pH 7.7, in the presence of glycerol and/or small PEGs (e.g., 2% PEG400). Co-crystallization with ATP or CMP was also attempted. X-ray diffraction data sets were collected from frozen single crystals at the European Synchrotron Radiation Facility (Grenoble, France, Beamline ID29) and processed with the programs MOSFLM (13Leslie A.G. The integration of macromolecular diffraction data.Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 48-57Crossref PubMed Scopus (967) Google Scholar), SCALA, and TRUNCATE from the CCP4 program suite (14Bailey S. Collaborative Computational Project, Number 4 The CCP4 Suite. Programs for protein crystallography.Acta. Crystallogr. D. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar) (see Table 1).TABLE 1Data collection and refinement statistics for PDTT2PDTT2BeamlineID29Data collectionSpace groupI213Cell dimension, a = b = c (Å)218.5No. of molecules (asymmetric unit)8Wavelength (Å)0.9793Resolution (Å)a2.68Rmerge (%)a, b9.0I/σIa10.9Completeness (%)a99.8Redundancya5.1B-Wilson64.1RefinementResolution (Å)2.69No. of reflections48494Rwork/Rfree (%)c, d19.8/24.9No. of protein atoms10091No. of water molecules213Ligand type14 SO4 + 8 PEGB-factors (Å2)Protein48.1Ligand (SO4/PEG)66.6/75.6Water44.8Root mean square deviationseBond lengths (Å)0.10Bond angles (°)1.179a The values in parentheses refer to the outermost resolution shell.b Rmerge = ΣhklΣi|Ihkl,i − Iaverage,hkl|/|ΣhklΣi|Ihkl,i| × 100.c Rwork = Σhkl|Fobs| − |Fcalc|/Σhkl|Fobs| × 100.d Rfree is calculated in the same way on a subset of reflections that are not used in the refinement (5%).e Deviation from ideal values. Open table in a new tab a The values in parentheses refer to the outermost resolution shell. b Rmerge = ΣhklΣi|Ihkl,i − Iaverage,hkl|/|ΣhklΣi|Ihkl,i| × 100. c Rwork = Σhkl|Fobs| − |Fcalc|/Σhkl|Fobs| × 100. d Rfree is calculated in the same way on a subset of reflections that are not used in the refinement (5%). e Deviation from ideal values. The structure was solved by molecular replacement using the program MolRep (15Vagin A. Teplyakov A. Molecular replacement with MOLREP.Acta Crystallogr. D. 2010; 66: 22-25Crossref PubMed Scopus (2701) Google Scholar) and the crystal structure of the wild-type NDT (wtNDT) from Lactobacillus leichmannii (Protein Data Bank code 1F8Y) (16Armstrong S.R. Cook W.J. Short S.A. Ealick S.E. Crystal structures of nucleoside 2-deoxyribosyltransferase in native and ligand-bound forms reveal architecture of the active site.Structure. 1996; 4: 97-107Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) as a search model. Two crystal forms were obtained both in the I213 trigonal space group, but only the one with the larger cell (a = b = c = 218.5 Å versus 151.3 Å) diffracted well. The structure of PDTT2 was solved at 2.7 Å and refined using the program COOT (17Emsley P. Cowtan K. COOT. Model-building tools for molecular graphics.Acta Crystallogr. D. 2004; 60: 2126-2132Crossref PubMed Scopus (23224) Google Scholar) and the program REFMAC5 (18Murshudov G.N. Vagin A.A. Dodson E.J. Refinement of macromolecular structures by the maximum-likelihood method.Acta Crystallogr. D. 1997; 53: 240-255Crossref PubMed Scopus (13853) Google Scholar), using a translation/liberation/screw model (19Winn M.D. Isupov M.N. Murshudov G.N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement.Acta Crystallogr. D. 2001; 57: 122-133Crossref PubMed Scopus (1651) Google Scholar) (see Table 1). The refined model and structure factors have been deposited in the Research Collaboratory for Structural Biology under the following accession number: Protein Data Bank code 4HX9. Once a crystal structure of a mutated NDT was made available, we used it to model the other variants to analyze the docking of nucleotides. Three-dimensional models were built using @TOME-2 (20Pons J.L. Labesse G. @TOME-2. A new pipeline for comparative modeling of protein-ligand complexes.Nucleic Acids Res. 2009; 37: W485-W491Crossref PubMed Scopus (103) Google Scholar). Three-dimensional conformation of the desired deoxynucleosides/deoxynucleotides (adenosine and cytosine series) were extracted from Pubchem (21Wang Y. Xiao J. Suzek T.O. Zhang J. Wang J. Bryant S.H. PubChem. A public information system for analyzing bioactivities of small molecules.Nucleic Acids Res. 2009; 37: W623-W633Crossref PubMed Scopus (960) Google Scholar) in SDF format and rewritten MOL2 format to dock them using the software PLANTS (22Korb O. StÜtzle T. Exner T.E. Empirical scoring functions for advanced protein-ligand docking with PLANTS.J. Chem. Inf. Model. 2009; 49: 84-96Crossref PubMed Scopus (876) Google Scholar). The results will be described elsewhere.3 To have a deoxyribosyltransferase with the broadest substrate specificity, we attempt to reconstruct the hypothetical common ancestor to Rcl and NDT by phylogenetic means using the software PAML 4.3 (PMID:17483113). The resurrected proteins were poorly soluble and inactive (not shown), so an alternative approach was to compare the sequences and structures of NDT and Rcl to create a chimera with the desired activities. NDT was chosen as a starting template because its transferase activity was more interesting from the synthetic point of view because it exchanges deoxyribose between pyrimidines and purines and, vice versa, between two pyrimidines and to a lesser extent between two purines. Furthermore, it tolerates substitutions on the base. The idea was to keep intact the catalytic core while reshaping the substrate specificity. First, two amino acids, Asp92 and Asn123 of NDT, were substituted to neutral and smaller serines. In wtNDT, these aspartate and asparagine interact with the 5′-OH group of 5-methyl-2′-deoxypseudouridine (Fig. 2B) by forming hydrogen bonds and prevent entrance of a larger and negatively charged phosphate group. At equivalent positions in the active site of Rcl, two serines (Ser87 and Ser117, respectively) were found (Fig. 2B). These changes confer to the NDT single mutant D92S (hereafter PDTM1, for phosphodeoxyribosyltransferase monophosphate) and the double mutant D92S,N123S (PDTM2) the ability to transfer deoxyribose 5-phosphate between cytosine and adenine, although with a low efficiency (Table 1). Overlay of the active sites of Rcl and NDT also predicts a steric clash between f13 and an incoming phosphate group (Fig. 2B). Upon substitution of Phe13 to the positively charged arginine (to give PDTM3 from PDTM1 or PDTM4 from PDTM2), the deoxyribose 5-phosphate transferase activity between C and A is enhanced by a factor of 50. Finally, we also substituted the Phe13 by a polar but neutral glutamine (PDTM5) leading to significant improvements in activity (Table 1). The phosphodeoxyribosyltransferase activity is not restricted to cytosine as donor and to adenine as acceptor (Table 2). PDTM3 is also able to transfer deoxyribose 5-phosphate between A and C and between C and T but with 2- and 4-fold lower activity than between C and A. All mutants transfer also deoxyribose 5-phosphate between A and G, whereas the reaction is almost undetectable when hypoxanthine is the acceptor base in agreement with previous observations showing that the deoxyribose transfer activity of NDT between a purine and hypoxanthine is low (23Kaminski P.A. Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus.J. Biol. Chem. 2002; 277: 14400-14407Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). PDTM4 is twice more active than PDTM3, whatever the couple of bases tested. In contrast, PDTM5 transfers deoxyribose 5-phosphate efficiently between C and A and between A and C but is much less active for the transfer from C to T. Both PDTM4 and PDTM5 are much more active with the couple A and G than PDTM3. This improvement is mainly due to a better affinity for dCMP, the Km for dCMP varying from 22 mm (PDTM3) to 11 or 12 mm (PDTM4 and PDTM5, respectively) (Table 3).TABLE 2Specific activities of alignment-based PDT variants in the presence of different purines and pyrimidines as donors and acceptors of deoxyribose 5-phosphateReactionsaEnzymesbwtNDTPDTM1PDTM2PDTM3PDTM4PDTM5PDTT0PDTT1PDTT2dCMP + A D dAMP + C0.11.81.779.717029646.378286dAMP + C D dCMP + A34.777.620623.7531dCMP + T D dTMP + C18.2521.7<0.1<0.11.1dGMP + A D dAMP + G8.55781493250dAMP + Hx D dIMP + A2<0.1<0.1<0.1<0.1<0.1dCDP + A D dADP + C<0.13166dADP + C D dCDP + A128dCDP + U D dUDP + Cc68dGDP + A D dADP + G1533dADP + Hx D dIDP + A<0.1<0.1dCTP + A D dATP + C<0.11051dATP + C D dCTP + A115dCTP + T D dTTP + C11.4dGTP + A D dATP + G2536dATP + Hx D dITP + A<0.1<0.1a The donor concentration was 3 mm, and the acceptor concentration was 1 mm.b The NDT and PDT sequences are: NDT, Phe13-Glu91-Asp92-Val93-Asn123-Tyr157; PDTM1, Ser92; PDTM2, Ser92-Ser123; PDTM3, Arg13-Ser92; PDTM4, Arg13-Ser92-Ser123; PDTM5, Gln13-Ser92-Ser123; PDTT0, Arg13-Gly92-Thr93-Ser123; PDTT1, Arg13-Gly92-Thr93-Ser123-Lys157; and PDTT2, Arg13-Gln91-Gly92-Thr93-Ser123.c U was used as acceptor base instead of T. The activities are expressed in nanomoles of deoxynucleotides synthesized per min/mg of protein. Open table in a new tab TABLE 3Kinetic parameters of PDT variants with an enlarged phosphate binding site in the presence of adenine as acceptor and deoxycytidine 5′-mono-, -di-, and -triphosphate as donors of deoxyribose 5-phosphateEnzymesaReactionbKmckcatkcat/Kmmms−1s−1 m−1PDTM3dCMP + A D dAMP + C220.313.6PDTM4dCMP + A D dAMP + C112.1192PDTM5dCMP + A D dAMP + C121.99166PDTT0dCMP + A D dAMP + C9.20.4548.9PDTT1dCMP + A D dAMP + C4.10.46112PDTT2dCMP + A D dAMP + C60.9153PDTT1dCDP + A D dADP + C8.90.088.9PDTT2dCDP + A D dADP + C8.70.111.5PDTT1dCTP + A D dATP + C5.50.5103.7PDTT2dCTP + A D dATP + C21.3668.3a The PDT sequences are: PDTM3, Arg13-Ser92; PDTM4, Arg13-Ser92-Ser123; PDTM5, Gln13-Ser92-Ser123; PDTT0, Arg13-Gly92-Thr93-Ser123; PDTT1, Arg13-Gly92-Thr93-Ser123-Lys157; and PDTT2, Arg13-Gln91-Gly92-Thr93-Ser123.b Same as above.c Vmax and Km were obtained from double reciprocal plots of initial velocity measurements. At least five different concentrations of dCMP, dCDP, and dCTP were used. The kcat (s−1) was calculated assuming a molecular mass of 108 kDa for each mutant. Open table in a new tab a The donor concentration was 3 mm, and the acceptor concentration was 1 mm. b The NDT and PDT sequences are: NDT, Phe13-Glu91-Asp92-Val93-Asn123-Tyr157; PDTM1, Ser92; PDTM2, Ser92-Ser123; PDTM3, Arg13-Ser92; PDTM4, Arg13-Ser92-Ser123; PDTM5, Gln13-Ser92-Ser123; PDTT0, Arg13-Gly92-Thr93-Ser123; PDTT1, Arg13-Gly92-Thr93-Ser123-Lys157; and PDTT2, Arg13-Gln91-Gly92-Thr93-Ser123. c U was used as acceptor base instead of T. The activities are expressed in nanomoles of deoxynucleotides synthesized per min/mg of protein. a The PDT sequences are: PDTM3, Arg13-Ser92; PDTM4, Arg13-Ser92-Ser123; PDTM5, Gln13-Ser92-Ser123; PDTT0, Arg13-Gly92-Thr93-Ser123; PDTT1, Arg13-Gly92-Thr93-Ser123-Lys157; and PDTT2, Arg13-Gln91-Gly92-Thr93-Ser123. b Same as above. c Vmax and Km were obtained from double reciprocal plots of initial velocity measurements. At least five different concentrations of dCMP, dCDP, and dCTP were used. The kcat (s−1) was calculated assuming a molecular mass of 108 kDa for each mutant. The better affinity of PDTM4 and PDMT5 over PDTM3 confirms the predicted contact involving the 5′ position of the ligand and the residue occupying the position 123 and the requirement for additional space to accommodate the phosphate group. Surprisingly, the mutant PDTM4 and PDTM5, differing by the nature of the residue at position 13 (arginine versus glutamine), show distinct specificity in the type of nucleobases to transfer. The arginine side chain was expected to mainly stabilize the negatively charged phosphate group, whereas the glutamine was predicted to point into the active site and stabilize the entering ligand through hydrogen bonding. Although the arginine is supposed to have little contact with the incoming nucleobase, the glutamine may contact it. In such a case the hydrophobic nature of the 5-position (methyl in thymine) may disfavor the transfer from C to T. In agreement, 5-halogenated uracils are better incorporated (see below; Table 4).TABLE 4Specific activities of PTM4, PDTM5, and PDTT2 variants in the presence of different donors and nucleoside analogues as acceptors of deoxyribose 5-phosphate or deoxyribose 5-triphosphate At this stage we reasoned that we should try to build a larger phosphate binding site and take advantage of the macrodipole of the central helix bearing the common catalytic glutamate (Glu98 in NDT) as observed in the Rcl-GMP complex (24Yang Y. Padilla A. Zhang C. Labesse G. Kaminski P.A. Structural characterization of the mammalian deoxynucleotide N-hydrolase Rcl and its stabilizing interactions with two inhibitors.J. Mol. Biol. 2009; 394: 435-447Crossref PubMed Scopus (14) Google Scholar). A more drastic reshaping of the active site was attempted by replacing the two amino acids, Asp and Val, with Gly and Thr at positions 92 to 93 (PDTT0) to remove a negatively charged residue and simultaneously make the backbone amide groups more accessible to better accommodate a phosphate group. Subsequently, in the context of the variant PDTT0 (PDTT for phosphodeoxyribosyltransferase triphosphate), we either added another positively charged residue (mutation Y157K; named PDTT1) or removed a negative charge (E91Q; PDTT2) to favor further the entrance of nucleotides. To track possible structural rearrangements induced by the mutations, several variants (PDTM1, PDTM4, and PDTT2) were tested for crystallization. Crystals of the apo form of PDTM1 and PDTT2 were obtained, and the structures were solved by molecular replacement. The crystals of PDTM1 diffracted at best to 3.5 Å, and the resulting structure appeared identical to the parent one and was not further refined. Conversely, the structure of PDTT2 could be refined to 2.7 Å. This crystal possesses a large unit cell that is composed of on" @default.
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• W1966248180 title "Phosphodeoxyribosyltransferases, Designed Enzymes for Deoxyribonucleotides Synthesis" @default.
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