Dataset Information

Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases.

ABSTRACT: Terminal RNA uridylyltransferases (TUTases) catalyze template-independent UMP addition to the 3' hydroxyl of RNA. TUTases belong to the DNA polymerase beta superfamily of nucleotidyltransferases that share a conserved catalytic domain bearing three metal-binding carboxylate residues. We have previously determined crystal structures of the UTP-bound and apo forms of the minimal trypanosomal TUTase, TbTUT4, which is composed solely of the N-terminal catalytic and C-terminal base-recognition domains. Here we report crystal structures of TbTUT4 with bound CTP, GTP, and ATP, demonstrating nearly perfect superposition of the triphosphate moieties with that of the UTP substrate. Consequently, at physiological nucleoside 5'-triphosphate concentrations, the protein-uracil base interactions alone are not sufficient to confer UTP selectivity. To resolve this ambiguity, we determined the crystal structure of a prereaction ternary complex composed of UTP, TbTUT4, and UMP, which mimics an RNA substrate, and the postreaction complex of TbTUT4 with UpU dinucleotide. The UMP pyrimidine ring stacks against the uracil base of the bound UTP, which on its other face also stacks with an essential tyrosine. In contrast, the different orientation of the purine bases observed in cocrystals with ATP and GTP prevents this triple stacking, precluding productive binding of the RNA. The 3' hydroxyl of the bound UMP is poised for in-line nucleophilic attack while contributing to the formation of a binding site for a second catalytic metal ion. We propose a dual role for RNA substrates in TUTase-catalyzed reactions: contribution to selective incorporation of the cognate nucleoside and shaping of the catalytic metal binding site.

SUBMITTER: Stagno J

PROVIDER: S-EPMC1976215 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases.

Stagno Jason J Aphasizheva Inna I Aphasizhev Ruslan R Luecke Hartmut H

Proceedings of the National Academy of Sciences of the United States of America 20070904 37

Terminal RNA uridylyltransferases (TUTases) catalyze template-independent UMP addition to the 3' hydroxyl of RNA. TUTases belong to the DNA polymerase beta superfamily of nucleotidyltransferases that share a conserved catalytic domain bearing three metal-binding carboxylate residues. We have previously determined crystal structures of the UTP-bound and apo forms of the minimal trypanosomal TUTase, TbTUT4, which is composed solely of the N-terminal catalytic and C-terminal base-recognition domain ...[more]

PMID: 17785418

Similar Datasets

Project description:Hexachlorobenzene (HCB), as one of the persistent organic pollutants (POPs) and a possible human carcinogen, is especially resistant to biodegradation. In this study, HcbA1A3, a distinct flavin-N5-peroxide-utilizing enzyme and the sole known naturally occurring aerobic HCB dechlorinase, was biochemically characterized. Its apparent preference for HCB in binding affinity revealed that HcbA1 could oxidize only HCB rather than less-chlorinated benzenes such as pentachlorobenzene and tetrachlorobenzenes. In addition, the crystal structure of HcbA1 and its complex with flavin mononucleotide (FMN) were resolved, revealing HcbA1 to be a new member of the bacterial luciferase-like family. A much smaller substrate-binding pocket of HcbA1 than is seen with its close homologues suggests a requirement of limited space for catalysis. In the active center, Tyr362 and Asp315 are necessary in maintaining the normal conformation of HcbA1, while Arg311, Arg314, Phe10, Val59, and Met12 are pivotal for the substrate affinity. They are supposed to place HCB at a productive orientation through multiple interactions. His17, with its close contact with the site of oxidation of HCB, probably fixes the target chlorine atom and stabilizes reaction intermediates. The enzymatic characteristics and crystal structures reported here provide new insights into the substrate specificity and catalytic mechanism of HcbA1, which paves the way for its rational engineering and application in the bioremediation of HCB-polluted environments.IMPORTANCE As an endocrine disrupter and possible carcinogen to human beings, hexachlorobenzene (HCB) is especially resistant to biodegradation, largely due to difficulty in its dechlorination. The lack of knowledge of HCB dechlorinases limits their application in bioremediation. Recently, an HCB monooxygenase, HcbA1A3, representing the only naturally occurring aerobic HCB dechlorinase known so far, was reported. Here, we report its biochemical and structural characterization, providing new insights into its substrate selectivity and catalytic mechanism. This research also increases our understanding of HCB dechlorinases and flavin-N5-peroxide-utilizing enzymes.

Dataset Information

Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases.

Publications

Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets