The c-Myc Target Gene Rcl (C6orf108) Encodes a Novel Enzyme, Deoxynucleoside 5′-monophosphate N-Glycosidase

Yoan Konto Ghiorghi,Karen I Zeller,Chi V Dang,P Alexandre Kaminski

doi:10.1074/jbc.m610648200

Abstract

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.

Highlights

The c-Myc transcription factor plays an important role in the regulation of the cell cycle, cellular transformation, and apoptosis [1] as well as in the genesis of many human cancers [2]
In clustering of orthologous groups (COGs) 3613, only two crystal structures of N-deoxyribosyltransferases in the presence of different ligands were described [16, 19]. These structures allowed the determination of the amino acid residues important in substrate binding and catalysis and explained the substrate specificity
RCL was chosen for several reasons: (i) it was the more distant protein in the COG, (ii) its function was unknown, (iii) it was only present in mammals, and (iv) the Rcl gene is a Myc target gene, which is up-regulated in human cancers and has tumorigenic potential

Summary

EXPERIMENTAL PROCEDURES

Chemicals—Ribonucleosides and deoxyribonucleosides, mono-, di-, and triphosphate derivatives of adenine, cytosine, guanine, hypoxanthine, thymine, and uracil were from SigmaAldrich. 6-Methylthio-guanosine 5Ј-monophosphate, 2Ј-deoxyguanosine 3Ј-monophosphate, and 8-oxo-deoxyguanosine 5Ј-monophosphate were from Jena Bioscience. The reaction medium (1 ml final volume) contained 50 mM Tris acetate, pH 6.0, 0.1 mM NADH, and 1.5 units each of glycerol-3-phosphate dehydrogenase, triose phosphate isomerase (Roche Applied Sciences), deoxyribose-5-phosphate aldolase, and 50 –200 ␮g of purified RCL. The reverse reaction (Gua ϩ dR5P 3 dGMP) was performed at 37 °C in 50 mM Tris acetate, pH 6.0, containing 3 mM guanine, 3 mM deoxyribose 5-phosphate, and 200 ␮g of purified RCL. Deoxyribose 5-phosphate transferase reactions (dNMP ϩ X 3 dXMP ϩ N) were performed at 37 °C in Tris acetate, pH 6.0 (or 50 mM Tris-HCl, pH 7.5, or 50 mM citrate-sodium, pH 6.5), containing 1 mM dCMP or dGMP and 1 mM either adenine, cytosine, guanine, thymine, uracil, or hypoxanthine and 200 ␮g of purified RCL.

RESULTS

Nucleotide kcat

DISCUSSION