title: Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P.
abstract: Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5' end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN (Nedd4-BP1, YacP nuclease) metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (nH = 2) under kcat/Km conditions, suggesting that PRORP1 catalysis is optimal with at least two active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified two aspartates important for enhancing metal ion affinity. The single turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pKa ∼ 8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis.
authors.affiliation: From the Departments of Biological Chemistry and.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Howard
authors.affiliation: From the Departments of Biological Chemistry and.
authors.forename: Bradley P
authors.initials: BP
authors.lastname: Klemm
authors.affiliation: From the Departments of Biological Chemistry and Chemistry, University of Michigan, Ann Arbor, Michigan 48109 Fierke@umich.edu.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D016880:Anisotropy; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D008670:Metals; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; C581753:PRORP1 protein, Arabidopsis; D043262:Ribonuclease P
keywords: NYN; PRORP; endoribonuclease; magnesium; metal dependence; metallonuclease; precursor tRNA (pre-tRNA); ribonuclease P (RNase P); ribozyme
references.citation: Nucleic Acids Res. 2014 Nov 10;42(20):e159
references.pmid: 25249623
references.citation: Cell. 2011 Apr 15;145(2):212-23
references.pmid: 21496642
references.citation: Biochemistry. 2004 Aug 17;43(32):10547-59
references.pmid: 15301552
references.citation: Cell. 2005 Jul 1;121(7):1005-16
references.pmid: 15989951
references.citation: Annu Rev Biochem. 1986;55:599-629
references.pmid: 2427016
references.citation: Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8924-8
references.pmid: 8799129
references.citation: RNA Biol. 2013 Jun;10(6):909-14
references.pmid: 23595059
references.citation: Trends Biochem Sci. 1994 Jan;19(1):15-8
references.pmid: 8140615
references.citation: Biochemistry. 2014 Mar 11;53(9):1456-65
references.pmid: 24520994
references.citation: RNA Biol. 2006 Jan-Mar;3(1):18-27
references.pmid: 17114934
references.citation: Biochemistry. 1997 Mar 4;36(9):2425-38
references.pmid: 9054547
references.citation: J Am Chem Soc. 1994 Jun;116(12):5467-8
references.pmid: 21391696
references.citation: Biochemistry. 1995 Jan 17;34(2):683-96
references.pmid: 7819264
references.citation: J Mol Biol. 2002 Mar 15;317(1):21-40
references.pmid: 11916377
references.citation: EMBO J. 2003 Mar 3;22(5):995-1004
references.pmid: 12606565
references.citation: Metallomics. 2010 Sep;2(9):609-20
references.pmid: 21072352
references.citation: RNA. 2000 Apr;6(4):545-53
references.pmid: 10786845
references.citation: Biochemistry. 1991 Oct 1;30(39):9464-9
references.pmid: 1716459
references.citation: Cell. 2000 Apr 28;101(3):295-305
references.pmid: 10847684
references.citation: J Mol Biol. 2007 Apr 27;368(2):421-33
references.pmid: 17355881
references.citation: Cell. 2008 Oct 31;135(3):462-74
references.pmid: 18984158
references.citation: Mol Cell. 2006 Apr 7;22(1):5-13
references.pmid: 16600865
references.citation: RNA. 2009 Feb;15(2):224-34
references.pmid: 19095620
references.citation: Biochemistry. 1994 Aug 30;33(34):10294-304
references.pmid: 7520753
references.citation: Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6498-502
references.pmid: 8341661
references.citation: Biochemistry. 2003 Oct 28;42(42):12418-29
references.pmid: 14567703
references.citation: Biochim Biophys Acta. 2012 Sep-Oct;1819(9-10):1017-26
references.pmid: 22137969
references.citation: Methods Enzymol. 1989;180:51-62
references.pmid: 2482430
references.citation: Crit Rev Biochem Mol Biol. 2006 Mar-Apr;41(2):77-102
references.pmid: 16595295
references.citation: Genes Dev. 2012 May 15;26(10):1022-7
references.pmid: 22549728
references.citation: Proc Natl Acad Sci U S A. 2012 Oct 2;109(40):16149-54
references.pmid: 22991464
references.citation: Biochemistry. 2011 Oct 4;50(39):8264-9
references.pmid: 21888353
references.citation: Wiley Interdiscip Rev RNA. 2012 Jan-Feb;3(1):37-61
references.pmid: 21957054
references.citation: Science. 1999 Oct 1;286(5437):123-6
references.pmid: 10506560
references.citation: J Biol Chem. 2008 Oct 17;283(42):28741-6
references.pmid: 18697748
references.citation: Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14355-60
references.pmid: 9405616
references.citation: Biochemistry. 2005 Dec 13;44(49):16130-9
references.pmid: 16331973
references.citation: Biochemistry. 2002 Jul 30;41(30):9545-58
references.pmid: 12135377
references.citation: Chembiochem. 2012 Oct 15;13(15):2270-6
references.pmid: 22976545
references.citation: Biochemistry. 1993 Dec 7;32(48):13040-5
references.pmid: 8241158
references.citation: Biochemistry. 2001 May 1;40(17):5161-71
references.pmid: 11318638
references.citation: J Mol Biol. 1999 Apr 23;288(1):105-16
references.pmid: 10329129
references.citation: RNA. 2009 Aug;15(8):1565-77
references.pmid: 19549719
references.citation: Nat Struct Mol Biol. 2010 Jun;17(6):740-4
references.pmid: 20473316
references.citation: Nature. 2013 Nov 14;503(7475):229-34
references.pmid: 24196718
references.citation: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W686-9
references.pmid: 15980563
references.citation: Biochemistry. 1998 Feb 24;37(8):2393-400
references.pmid: 9485387