Solution Structure of RCL, a Novel 2′-Deoxyribonucleoside 5′-Monophosphate N-glycosidase

Doddapaneni, Kiran Kumar; Mahler, Bryon; Pavlovicz, Ryan E.; Haushalter, Adam; Yuan, Chunhua; Wu, Zhengrong

doi:10.1016/j.jmb.2009.08.054

Cited by 15 publications

(37 citation statements)

References 33 publications

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“…The K d value of Rcl with GMP was 10.6 Ϯ 0.4 M (Fig. 3), similar to that described by Doddapaneni et al (14). The Y13F mutant shows a K d value comparable with Rcl wild type, whereas their K m values for dGMP differ by a factor of 80.…”

Section: Resultssupporting

confidence: 87%

“…We hypothesize that understanding Rcl substrate specificity and catalytic mechanism should help in elucidating its cellular role. Its solution structure was recently solved and has provided insights into the molecular basis for substrate recognition (13,14). Tyr-13, Glu-93, and His-45 are located in the vicinity of the 3Ј-OH of the ribose.…”

Section: Discussionmentioning

confidence: 99%

“…Both NDT and Rcl are specific for 2Ј-deoxyribose-containing substrates, whereas the corresponding riboside analogs are inhibitors. Their amino acid sequences display an overall low level of identity but share the catalytic triad composed of a tyrosine, an aspartate, and a glutamate, suggesting a similar mode of action (9, 12).The structure of Rcl in solution was recently described (13,14). Rcl is a symmetric homodimer; each monomer is composed of five buried ␤-strands alternating with five ␣-helices.…”

mentioning

confidence: 99%

“…The structure of Rcl in solution was recently described (13,14). Rcl is a symmetric homodimer; each monomer is composed of five buried ␤-strands alternating with five ␣-helices.…”

mentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

“…The structure of Rcl in solution was recently described (13,14). Rcl is a symmetric homodimer; each monomer is composed of five buried ␤-strands alternating with five ␣-helices.…”

mentioning

confidence: 99%

See 2 more Smart Citations

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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“…8 Rcl, another member of the NDT superfamily, catalyzes the hydrolysis of purine 2′-deoxyribosylnucleotides and is inhibited by ribonucleotides 9 . Its solution structure 10, 11 , and recent crystal structures with bound inhibitors 12 , provide highly detailed information about its substrate-enzyme interactions. Rcl is an unusual because it is the only NDT family member found in mammals.…”

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confidence: 99%

Reversal of the Substrate Specificity of CMP N-Glycosidase to dCMP

et al. 2013

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MilB is a CMP hydrolase involved in the early steps of biosynthesis of the antifungal compound mildiomycin. An enzyme from the bacimethrin biosynthetic pathway, BcmB, is closely related in both sequence and function to MilB. These two enzymes belong to the nucleoside 2′-deoxyribosyltransferase (NDT) superfamily. NDTs catalyze N-glycosidic bond cleavage of 2′-deoxynucleosides via a covalent 2-deoxyribosyl-enzyme intermediate. Conservation of key active site residues suggests that members of the NDT superfamily share a common mechanism; however, the enzymes differ in their substrate preferences. Substrates vary in the type of nucleobase, the presence or absence of a 2′-hydroxyl group, and the presence or absence of a 5′-phosphate group. We have determined the structures of MilB and BcmB and compared them to previously determined structures of NDT superfamily members. The comparisons reveal how these enzymes differentiate between ribosyl and deoxyribosyl nucleotides or nucleosides, and among different nucleobases. The 1.6 Å structure of the MilB-CMP complex reveals an active site feature that is not obvious from sequence comparisons alone. MilB and BcmB that prefer substrates containing 2′-ribosyl groups have a phenylalanine positioned in the active site whereas NDT family members with preference for 2′-deoxyribosyl groups have a tyrosine residue. Further studies show that the phenylalanine is critical for MilB and BcmB specificity towards CMP, and mutation of this phenylalanine residue to tyrosine results in a 1000-fold reversal of substrate specificity from CMP to dCMP.

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Solution structure of the nucleotide hydrolase BlsM: Implication of its substrate specificity

et al. 2020

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Biosynthesis of the peptidyl nucleoside antifungal agent blasticidin S in Streptomyces griseochromogenes requires the hydrolytic function of a nucleotide hydrolase, BlsM, to excise the free cytosine from the 5′‐monophosphate cytosine nucleotide. In addition to its hydrolytic activity, interestingly, BlsM has also been shown to possess a novel cytidine deaminase activity, converting cytidine, and deoxycytidine to uridine and deoxyuridine. To gain insight into the substrate specificity of BlsM and the mechanism by which it performs these dual function, the solution structure of BlsM was determined by multi‐dimensional nuclear magnetic resonance approaches. BlsM displays a nucleoside deoxyribosyltransferase‐like dimeric topology, with each monomer consisting of a five‐stranded β‐sheet that is sandwiched by five α‐helixes. Compared with the purine nucleotide hydrolase RCL, each monomer of BlsM has a smaller active site pocket, enclosed by a group of conserved hydrophobic residues from both monomers. The smaller size of active site is consistent with its substrate specificity for a pyrimidine, whereas a much more open active site, as in RCL might be required to accommodate a larger purine ring. In addition, BlsM confers its substrate specificity for a ribosyl‐nucleotide through a key residue, Phe19. When mutated to a tyrosine, F19Y reverses its substrate preference. While significantly impaired in its hydrolytic capability, F19Y exhibited a pronounced deaminase activity on CMP, presumably due to an altered substrate orientation as a result of a steric clash between the 2′‐hydroxyl of CMP and the ζ‐OH group of F19Y. Finally, Glu105 appears to be critical for the dual function of BlsM.

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Solution Structure of RCL, a Novel 2′-Deoxyribonucleoside 5′-Monophosphate N-glycosidase

Cited by 15 publications

References 33 publications

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

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

Reversal of the Substrate Specificity of CMP N-Glycosidase to dCMP

Solution structure of the nucleotide hydrolase BlsM: Implication of its substrate specificity

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