title: Structural basis of substrate specificity and selectivity of murine cytosolic 5'-nucleotidase III.
abstract: Cytosolic 5'-nucleotidase III (cN-III) is responsible for selective degradation of pyrimidine 5'-monoribonucleotides during maturation of reticulocytes to erythrocytes. The lack of this enzymatic activity due to genetic aberrations or lead poisoning results in a mild to moderate nonspherocytic hemolytic anemia. In affected individuals, pyrimidine nucleotides as well as their precursor polymers and their off-path metabolites accumulate in erythrocytes, interfering with their proper function in ways that are not yet fully understood. This report describes the first X-ray structure of a catalytically inactivated variant of murine cN-III with a natural substrate, uridine 5'-monophosphate, in the active site at 1.74Å resolution. The structure captures in an atomic detail the closed conformation that cN-III adopts upon substrate binding. Structure and sequence analysis coupled with enzymatic characterization of several mutants confirmed that the aromatic ring of a nitrogenous base of substrate nucleotide is stabilized by parallel π-stacking interactions with conserved aromatic rings of Trp113 and His68. The nitrogenous base is further stabilized by T-shaped stacking with the conserved aromatic ring of Tyr114, as well as by polar contacts with side chains of Thr66 and Ser117. Two water molecules help to stabilize the nucleotide binding by bridging it to protein residues Asp72 and His68 via hydrogen bonds. Finally, fully conserved Glu96 is responsible for recognition of ribose ring via two hydrogen bonds. The presented substrate complex structure elucidates how cN-III achieves specificity for pyrimidine 5'-nucleotides and how it selects against purine 5'-nucleotides.
authors.affiliation: Department of Chemistry, Georgian Court University, Lakewood, NJ 08701, USA.
authors.forename: Christina L
authors.initials: CL
authors.lastname: Grobosky
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Lopez
authors.forename: Sarahbeth
authors.initials: S
authors.lastname: Rennie
authors.forename: Dionysus J
authors.initials: DJ
authors.lastname: Skopelitis
authors.forename: Amanda T
authors.initials: AT
authors.lastname: Wiest
authors.forename: Craig A
authors.initials: CA
authors.lastname: Bingman
authors.forename: Eduard
authors.initials: E
authors.lastname: Bitto
mesh_terms: D015720:5'-Nucleotidase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004912:Erythrocytes / Q000378:metabolism; D004920:Erythropoiesis; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D012156:Reticulocytes / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014542:Uridine Monophosphate / Q000378:metabolism*
chemical_list: D014542:Uridine Monophosphate; D015720:5'-Nucleotidase; C503010:Nt5c3 protein, mouse