期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 423, 期 4, 页码 540-554出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2012.08.014
关键词
pyrimidine nucleotidase; HAD superfamily; substrate complex; induced fit; nonspherocytic hemolytic anemia
资金
- Research Corporation for Science Advancement [19888]
- Georgian Court University Summer Research grant
- U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-06CH11357]
- Michigan Economic Development Corporation
- Michigan Technology Tr-Corridor [085P1000817]
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 1.74 angstrom resolution. The structure captures in an atomic detail the closed conformation that 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 pi-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 stabili7e 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. (C) 2012 Elsevier Ltd. All rights reserved.
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