Unique Utilization of a Phosphoprotein Phosphatase Fold by a Mammalian Phosphodiesterase Associated with WAGR Syndrome

Dermol, Urška; Janardan, Vishnu; Tyagi, Richa; Visweswariah, Sandhya S.; Podobnik, Marjetka

doi:10.1016/j.jmb.2011.07.060

Cited by 13 publications

(15 citation statements)

References 31 publications

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“…33). Still, Mn(II) is commonly found in MPEs and was reported to support the highest phosphoesterase activity among divalent cations tested in Mre11 (34,35), 5′-nucleotidase (36, 37), MPPED2 (38,39), and yeast Dbr1 (23). Superposition of the Mre11 and Dbr1 binuclear centers reveals they are indeed similar, including the position of the bridging nucleophile, except for the substitution of the first aspartic acid in the signature sequence with Cys in Dbr1 (Fig.…”

Section: Resultsmentioning

confidence: 93%

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Clark

Katolik

Roberts

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2′,5′-and 3′,5′-phosphodiester linkages. The 2′,5′-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s −1. Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ∼4.0 s −1 is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the β-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease.RNA debranching | intron lariat | enzyme kinetics | X-ray crystallography | Dbr1 T he enzymatic processing of diverse RNA molecules requires selective recognition of their unique physicochemical properties. The sequential trans-esterification reactions catalyzed by the spliceosome yield mature messenger RNA (mRNA) and excised intron lariats (1, 2), the latter of which contain internal branchpoint adenosine nucleotides harboring vicinal 2′,5′-and 3′,5′-phosphodiester linkages (3). Mature mRNA transcripts are exported to the cytosol for protein synthesis, but lariat introns must be linearized before they can be turned over or processed into the subset of small nucleolar RNAs and microRNAs that are derived from intronic RNA (4, 5). The lariat forms when the 2′-hydroxyl group of an adenosine nucleotide near the 3′-end of the intron acts as the nucleophile to attack the 5′-splice site, producing 5′-exon-3′-OH and intron lariat/ 3′-exon intermediates. The 3′-hydroxyl group of the 5′-exon-3′-OH intermediate subsequently acts as the nucleophile to attack the 3′-splice site, resulting in intron excision and exon ligation (6, 7) (Fig. 1A). The resulting vicinal 2′,5′-and 3′,5′-phosphodiester linkages confer unique topological and chemical features to the branchpoint and flanking nucleotides, and these lariats persist in yeast cells lacking active Dbr1 (RNA lariat debranching enzyme) ...

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Section: Resultsmentioning

confidence: 93%

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Clark

Katolik

Roberts

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…7). Although this helix is not present in most other members of the PhoD family, analogous “cap domains” are a feature of certain other enzymes in the metallophosphatase superfamily (33–36). In most cases the role of these cap domains is unclear.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of the Bacillus subtilis Phosphodiesterase PhoD Reveals an Iron and Calcium-containing Active Site

Rodriguez

Lillington

Johnson

et al. 2014

Journal of Biological Chemistry

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“…In the Mpped2 #4 clone there was a significant increase in caspase activity with respect to both the activity. 30,41 As a control, we assayed the Mpped2 H67R mutant for its cAMP-PDE activity. 48 As recently described in reference 41, the structure of the Mpped2 H67R mutant differs from that of the wild-type protein in terms of the position of its W182.…”

Section: Mpped2 Overexpression Impairs Proliferation and Anchorageindmentioning

confidence: 99%

“…The Mpped2 H67R mutation abrogates Mpped2 PDE activity in vitro. 30,34,41 To determine whether this mutation can also abrogate the in vivo function of the wild-type Mpped2 protein observed previously in vitro, we performed a flank tumorigenesis assay in athymic nude mice. Four mice (M1, M2, M3, M4) were inoculated subcutaneously in their paired right and left flanks with the human NB SH-SY5Y cells, as the Mpped2 #4 clone and the Mpped2-H67R #8 clone.…”

Section: Mpped2 Overexpression Impairs Proliferation and Anchorageindmentioning

confidence: 99%

The metallophosphodiesterase Mpped2 impairs tumorigenesis in neuroblastoma

et al. 2012

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Unique Utilization of a Phosphoprotein Phosphatase Fold by a Mammalian Phosphodiesterase Associated with WAGR Syndrome

Cited by 13 publications

References 31 publications

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Crystal Structure of the Bacillus subtilis Phosphodiesterase PhoD Reveals an Iron and Calcium-containing Active Site

The metallophosphodiesterase Mpped2 impairs tumorigenesis in neuroblastoma

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