Metallophosphoesterases: structural fidelity with functional promiscuity

Matange, Nishad; Podobnik, Marjetka; Visweswariah, Sandhya S.

doi:10.1042/bj20150028

Cited by 52 publications

(89 citation statements)

References 112 publications

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“…H β , where underlined/italicized letters correspond to metal ligands, α and β to the binding pockets in which the ligands reside, and X to any amino acid (26). Despite the conservation of metal-binding residues, the repertoire used by various MPEs is diverse and signature sequence-containing enzymes bind Mn(II), Fe(II), Fe(III), Zn(II), Co(II), Cd(II), and Ni(II) (reviewed in ref.…”

Section: Resultsmentioning

confidence: 99%

“…Because previous work suggested yDbr1 is Mn-dependent (23), 1 mM MnCl 2 was added to the EDTA-treated enzyme before crystallization. The resulting wild type and inactive C14S EhDbr1 structures were unexpectedly mononuclear, with Mn(II) in the β-pocket and a water molecule coordinated to the Mn(II) ion occupying the position expected for the putative α-site metal (26). Alignment of the backbones of structures of EhDbr1 in complex with product analog 5′-GMP and with a synthetic RNA possessing a 2′-phosphate monoester at the branchpoint adenosine (AK65), superimposed their 5′-and 2′-phosphate moieties, respectively, suggesting a model of an intact lariat branchpoint with flanking nucleotides bound at the active site (24).…”

Section: Significancementioning

confidence: 99%

“…The data suggested a mononuclear mechanism of hydrolysis in which the invariant α-pocket Cys serves as a catalytic base to activate the metal-bound water nucleophile for attack on the scissile phosphorus atom, with H91 poised to protonate the 2′-O leaving group. However, because essentially all characterized MPEs harbor metal ions in both binding pockets (26) and because Cys residues are excellent ligands for certain metal ions, a binuclear mechanism could not be ruled out (25).…”

Section: Significancementioning

confidence: 99%

“…The structures revealed the first 261 residues adopt the canonical MPE fold with adjacent α and β metal-binding pockets. Features unique to Dbr1 include a 28-residue insertion loop immediately adjacent to the active site (residues 130-158) and an invariant cysteine residue (Cys14) in the position of the canonical Asp in the α pocket (25,26). Because previous work suggested yDbr1 is Mn-dependent (23), 1 mM MnCl 2 was added to the EDTA-treated enzyme before crystallization.…”

Section: Significancementioning

confidence: 99%

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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: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 2 more Smart Citations

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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“…Previous reviews highlight the importance of promiscuity, and also examined the possible mechanisms [1,6,12]. Other more recent reviews have focused on the practical implications of enzyme promiscuity in the development of biocatalysts involved in organic synthesis [13][14][15], or divergence in certain enzyme families [16][17][18][19]. The aim of writing this review is to encapsulate the advances in the field of enzyme promiscuity.…”

Section: Introductionmentioning

confidence: 99%

Recent advances in enzyme promiscuity

Gupta

2016

Sustain Chem Process

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Enzyme promiscuity is defined as the capability of an enzyme to catalyze a reaction other than the reaction for which it has been specialized. Although, enzyme is known for its specificity, many enzymes are reported to be promiscuous in nature. However, the promiscuous function may not be relevant in physiological conditions. The reasons could be either very low level of catalytic activity or unavailability of the substrates in the cell. Hitherto, the enzyme promiscuity is of great importance because they are the starting point for the evolution of new functions in the nature. In addition, the promiscuous activities are utilized for the development of new catalytic functions by applying directed laboratory evolution and protein engineering techniques. The aim of this review is to provide recent developments on the understanding of the mechanism of catalytic promiscuity, evolvability of promiscuous functions and the applications of enzyme promiscuity in the designing of enhanced or new functional biocatalysts.

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Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn²⁺/Mn²⁺ heterobinucleation

et al. 2017

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The RNA lariat debranching enzyme Dbr1 is a metallophosphoesterase that cleaves 2′-5′ phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni2+, Mn2+, Mg2+, Fe2+ and Zn2+. While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn2+ ion), recent structures determined a Zn2+/Fe2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn2+/Mn2+ occupancy. ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently-labeled branched substrates confirm activity.

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Metallophosphoesterases: structural fidelity with functional promiscuity

Cited by 52 publications

References 112 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

Recent advances in enzyme promiscuity

Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn²⁺/Mn²⁺ heterobinucleation

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Metallophosphoesterases: structural fidelity with functional promiscuity

Cited by 52 publications

References 112 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

Recent advances in enzyme promiscuity

Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn2+/Mn2+ heterobinucleation

Contact Info

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Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn²⁺/Mn²⁺ heterobinucleation