title: Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1.
abstract: 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-1Similar 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-1is 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.
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229; clarkn@uthscsa.edu fitzpatrickp@uthscsa.edu masad.damha@mcgill.ca pjh@biochem.uthscsa.edu.
authors.forename: Nathaniel E
authors.initials: NE
authors.lastname: Clark
authors.affiliation: Department of Chemistry, McGill University, Montreal, QC H3A 0B8, Canada.
authors.forename: Adam
authors.initials: A
authors.lastname: Katolik
authors.affiliation: Department of Chemistry & Physics, University of South Carolina Aiken, Aiken, SC 29811.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Roberts
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229.|X-ray Crystallography Core Laboratory, University of Texas Health Science Center, San Antonio, TX 78229.
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Taylor
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Holloway
authors.affiliation: Northeastern Collaborative Access Team, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Schuermann
authors.affiliation: Departments of Biochemistry and Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706.
authors.forename: Eric J
authors.identifier: 0000-0001-7883-7706
authors.initials: EJ
authors.lastname: Montemayor
authors.affiliation: Department of Molecular Biosciences, The University of Texas, Austin, TX 78712.|Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Stevens
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229; clarkn@uthscsa.edu fitzpatrickp@uthscsa.edu masad.damha@mcgill.ca pjh@biochem.uthscsa.edu.
authors.forename: Paul F
authors.initials: PF
authors.lastname: Fitzpatrick
authors.affiliation: Department of Chemistry, McGill University, Montreal, QC H3A 0B8, Canada; clarkn@uthscsa.edu fitzpatrickp@uthscsa.edu masad.damha@mcgill.ca pjh@biochem.uthscsa.edu.
authors.forename: Masad J
authors.initials: MJ
authors.lastname: Damha
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229; clarkn@uthscsa.edu fitzpatrickp@uthscsa.edu masad.damha@mcgill.ca pjh@biochem.uthscsa.edu.|X-ray Crystallography Core Laboratory, University of Texas Health Science Center, San Antonio, TX 78229.|Department of Veterans Affairs, South Texas Veterans Health Care System, San Antonio, TX 78229.
authors.forename: P John
authors.initials: PJ
authors.lastname: Hart
mesh_terms: D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D004748:Entamoeba histolytica / Q000201:enzymology*; D006868:Hydrolysis; D007438:Introns; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D015800:Protozoan Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D012316:RNA Nucleotidyltransferases / Q000737:chemistry*; D012322:RNA Precursors / Q000737:chemistry; D012326:RNA Splicing; D000079962:RNA, Circular; D014965:X-Rays; D015032:Zinc / Q000737:chemistry
chemical_list: D010455:Peptides; D015800:Protozoan Proteins; D012322:RNA Precursors; D000079962:RNA, Circular; D012313:RNA; D007501:Iron; D012316:RNA Nucleotidyltransferases; C048135:lariat debranching enzyme; D015032:Zinc
keywords: Dbr1; RNA debranching; X-ray crystallography; enzyme kinetics; intron lariat