Functional Cloning, Heterologous Expression, and Purification of Two Different N-Deoxyribosyltransferases fromLactobacillus helveticus

Kaminski, P.A.

doi:10.1074/jbc.m111995200

Cited by 51 publications

(62 citation statements)

References 36 publications

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“…This substrate specificity is similar to other well-known NDTs [12,18,19] except for the strong preference for Hyp as acceptor, only comparable with that of L. lactis subsp. lactis NDT [26].…”

Section: Discussionsupporting

confidence: 72%

“…In turn, the most and least preferred acceptors are hypoxanthine (Hyp) and uracil (Ura), respectively. Compared to previously described NDTs [18,19,26,27], BpNDT exhibits higher specific activities, except when compared to LrNDT. The synthesis of dIno from Hyp and dAdo proceeds 5.4 times faster in the presence of BpNDT than with LrNDT or PDT from Lactobacillus helveticus (LhPDT), whereas the transfer between Thd and Ura is twice faster with LrNDT or Lactococcus lactis NDT (LlcNDT) than with BpNDT.…”

Section: Substrate Specificitycontrasting

confidence: 59%

“…lactis NDT [26]. This result suggests that high specificity for Hyp is not exclusive for type I NDTs, traditionally associated with the metabolism of dIno for that reason, among others [19].…”

Section: Discussionmentioning

confidence: 84%

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2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium

et al. 2018

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Structure-function relationships of a novel 2 -deoxyribosyltransferase from the psychrotolerant bacterium Bacillus psychrosaccharolyticus (BpNDT) have been exhaustively studied by biochemical and high resolution crystallographic analyses. Despite BpNDT exhibiting some structural features characteristic of cold-adapted enzymes such as localized flexibility in critical loops, its biochemical properties are typical of mesophilic enzymes. BpNDT is a highly symmetrical homohexamer with tightly associated subunits that possesses flexible and short loops bordering the active sites. The catalytic center is essentially identical to that of other mesophilic homologues. Moreover, BpNDT shows that it is a mesophilic-like enzyme since it is not heat-labile and exhibits an apparent unfolding temperature (T m ) of 49 • C, being active during 96 h at 40 and 50 • C. Finally, BpNDT synthesizes natural and modified nucleosides, with preference for purines as acceptors and pyrimidine nucleosides as donors. Remarkably, the synthesis of several therapeutic nucleosides has been efficiently carried out. In this sense, 5-hydroxymethyl-2 -deoxyuridine (5-HMdUrd), 7-deaza-6-hydroxypurine-2 -deoxyriboside (7-DHPdRib) and theophylline-2 -deoxyriboside were synthesized for the first time by an NDT enzyme, showing the biotechnological interest of BpNDT.

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Section: Discussionsupporting

confidence: 72%

Section: Substrate Specificitycontrasting

confidence: 59%

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2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium

et al. 2018

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“…Another possibility involves nucleoside deoxyribosyltransferases in B. burgdorferi (20), which our ERGO database assigns to BB0426 but which we have not found in relapsing fever spirochetes. These enzymes catalyze the transfer of the deoxyribosyl group from a nucleoside to another base (1,28). What we interpreted as hypoxanthine PRT activity may have been the transfer of a deoxyribosyl group to hypoxanthine catalyzed by this enzyme.…”

Section: Discussionmentioning

confidence: 90%

Purine Salvage Pathways among Borrelia Species

Pettersson¹,

Schrumpf²,

Raffel³

et al. 2007

Infect Immun

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Genome sequencing projects on two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed differences in genes involved in purine metabolism and salvage compared to those in the Lyme disease spirochete Borrelia burgdorferi. The relapsing fever spirochetes contained six open reading frames that are absent from the B. burgdorferi genome. These genes included those for hypoxanthine-guanine phosphoribosyltransferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), the ribonucleotide-diphosphate reductase alpha subunit (nrdE), and the ribonucleotide-diphosphate reductase beta subunit (nrdF). Southern blot assays with multiple Borrelia species and isolates confirmed the presence of these genes in the relapsing fever group of spirochetes but not in B. burgdorferi and related species. TaqMan real-time reverse transcription-PCR demonstrated that the chromosomal genes (hpt, purA, and purB) were transcribed in vitro and in mice. Phosphoribosyltransferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did the B. burgdorferi cell lysate, and enzymatic activity was observed with adenine, hypoxanthine, and guanine as substrates. B. burgdorferi showed low but detectable phosphoribosyltransferase activity with hypoxanthine even though the genome lacks a discernible ortholog to the hpt gene in the relapsing fever spirochetes. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA to a much greater extent than did B. burgdorferi. This complete pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to these spirochetes achieving high cell densities in blood.

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“…NDT catalyzes the reversible transfer of the deoxyribosyl moiety from a deoxynucleoside donor to an acceptor nucleobase (11). On the contrary, Rcl is a hydrolase (or a transferase using only water molecule as an acceptor) (9).…”

mentioning

confidence: 99%

Probing the Active Site of the Deoxynucleotide N-Hydrolase Rcl Encoded by the Rat Gene c6orf108

Dupouy

Zhang

Padilla

et al. 2010

Journal of Biological Chemistry

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Rcl is a potential anti-angiogenic therapeutic target that hydrolyzes the N-glycosidic bond of 2-deoxyribonucleoside 5-monophosphate, yielding 2-deoxyribose 5-phosphate and the corresponding base. Its recently elucidated solution structure provided the first insight into the molecular basis for the substrate recognition. To facilitate the development of potent and specific inhibitors of Rcl, the active site was probed by sitedirected mutagenesis and by the use of substrate analogs. The nucleobase shows weak interactions with the protein, and the deoxyribose binding pocket includes the catalytic triad Tyr-13, Asp-69, and Glu-93 and the phosphate binding site Ser-87 and Ser-117. The phosphomimetic mutation of Ser-17 to Glu prevents substrate binding and, thus, abolishes the activity of Rcl. The synthetic ligand-based analysis of the Rcl binding site shows that substitutions at positions 2 and 6 of the nucleobase as well as large heterocycles are well tolerated. The phosphate group at position 5 of the (deoxy)ribose moiety is the critical binding determinant. This study provides the roadmap for the design of small molecules inhibitors with pharmacological properties.Rcl is a c-Myc target (1) that becomes tumorigenic when the metabolic environment of the cell is permissive (2). Its participation in tumorigenesis is supported by several pieces of evidence as follows. rcl is up-regulated in several cancers such as human prostate, breast cancers, and chronic lymphocytic leukemia (3)(4)(5). rcl is repressed in response to histone deacetylase inhibitors, anticancer agents that induce tumor cell death, differentiation, and/or cell cycle arrest (6). rcl is over-expressed in response to chronic administration of the synthetic glucocorticoid methylprednisolone or estrogen diethylstilbestrol. Altogether these data indicate that Rcl has a role in cell growth and/or cell proliferation (7,8). Moreover, a causal role of Rcl in breast tumorigenesis was suggested as the up-regulation of Rcl correlates with the tumor grade (4). Thus, Rcl is a potential therapeutic target. However, its function remains to be determined.We have shown that Rcl is a 2Ј-deoxynucleoside 5Ј-phosphate N-hydrolase that had not been previously described (9). Rcl hydrolyzes 2Ј-deoxyribonucleoside 5Ј-monophosphate (dNMP) 4 to form a free nucleobase moiety (N) and 2-deoxyribose 5-phosphate. 2-Deoxyribose 5-phosphate may be converted to 2-deoxyribose, a downstream mediator of thymidine phosphorylase (also known as the angiogenic factor endothelial cell growth factor 1, ECGF1) that regulates tumor angiogenesis and progression (10). 2-Deoxyribose is also a substrate for glycolysis through its conversion to glyceraldehyde 3-phosphate, which may indirectly increase the development and malignancy of cancer cell overexpressing Rcl.Rcl belongs to the nucleoside 2-deoxyribosyltransferase (NDT) family (EC 2.4.2.6; pfam 05014). NDT catalyzes the reversible transfer of the deoxyribosyl moiety from a deoxynucleoside donor to an acceptor nucleobase (11). On the contrary, Rcl ...

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Functional Cloning, Heterologous Expression, and Purification of Two Different N-Deoxyribosyltransferases fromLactobacillus helveticus

Cited by 51 publications

References 36 publications

2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium

2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium

Purine Salvage Pathways among Borrelia Species

Probing the Active Site of the Deoxynucleotide N-Hydrolase Rcl Encoded by the Rat Gene c6orf108

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