Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3′ terminal RNA ribose 2′O-methyltransferase

Jain, Ruchi; Shuman, Stewart

doi:10.1261/rna.2500711

Cited by 18 publications

(22 citation statements)

References 42 publications

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“…Although changes in the substrate preferences of nucleic acid enzymes in response to varying the divalent cation are often dismissed as in vitro quirks, our experience with enzymes that mediate bacterial NHEJ and bacterial RNA repair has taught us that the ligase, polymerase, 3′-phosphoesterase, and methyltransferase components of these repair pathways require manganese for catalysis and are either inactive or poorly active with magnesium. 26-34 Studies of the radio-resistant bacterium Deinococcus have revealed that manganese exerts profound effects on its sensitivity to radiation and oxidative stress in vivo . 35,36 The observed ablation of both the Mn 2+ and Mg 2+ dependent activities of mycobacterial PNPase by the D526A mutation argues that they bind to the same site on the enzyme, corresponding to the site defined by the crystal structure of an E. coli PNPase·Mn 2+ complex.…”

Section: Discussionmentioning

confidence: 99%

Distinctive Effects of Domain Deletions on the Manganese-Dependent DNA Polymerase and DNA Phosphorylase Activities of Mycobacterium smegmatis Polynucleotide Phosphorylase

Unciuleac

Shuman

2013

Biochemistry

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Polynucleotide phosphorylase (PNPase) plays synthetic and degradative roles in bacterial RNA metabolism; it is also suggested to participate in bacterial DNA transactions. Here we characterize and compare the RNA and DNA modifying activities of Mycobacterium smegmatis PNPase. The full-length (763-aa) M. smegmatis PNPase is a homotrimeric enzyme with Mg2+·PO4-dependent RNA 3′-phosphorylase and Mg2+·ADP-dependent RNA polymerase activities. We find that the enzyme is also a Mn2+·dADP-dependent DNA polymerase and a Mn2+·PO4-dependent DNA 3′-phosphorylase. The Mn2+·DNA and Mg2+·RNA end modifying activities of mycobacterial PNPase are coordinately ablated by mutating the putative manganese ligand Asp526, signifying that both metals likely bind to the same site on PNPase. Deletions of the C-terminal S1 and KH domains of mycobacterial PNPase exert opposite effects on the RNA and DNA modifying activities. Subtracting the S1 domain diminishes RNA phosphorylase and polymerase activity; simultaneous deletion of the S1 and KH domains further cripples the enzyme with respect to RNA substrates. By contrast, the S1 and KH domain deletions enhance the DNA polymerase and phosphorylase activity of mycobacterial PNPase. We observe two distinct modes of nucleic acid binding by mycobacterial PNPase: (i) metal-independent RNA-specific binding via the S1 domain, and (ii) metal-dependent binding to RNA or DNA that is optimal when the S1 domain is deleted. These findings add a new dimension to our understanding of PNPase specificity, whereby the C-terminal modules serve a dual purpose: (i) to help capture an RNA polynucleotide substrate for processive 3′ end additions or resections, and (ii) to provide a specificity filter that selects against a DNA polynucleotide substrate

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Section: Discussionmentioning

confidence: 99%

Distinctive Effects of Domain Deletions on the Manganese-Dependent DNA Polymerase and DNA Phosphorylase Activities of Mycobacterium smegmatis Polynucleotide Phosphorylase

Unciuleac

Shuman

2013

Biochemistry

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“…In methyl transferases with tRNA as the acceptor, while metal ions are required for activity (Hurwitz, et al, 1964), this requirement is believed to stabilize the tertiary structure of the nucleic acid (Gong, et al, 2002). Recently, a metal-dependent O-methyl transferase involved in tRNA repair was identified (Jain and Shuman, 2011), although the precise role of the metal ion is unknown. Thus, most of the AdoMet-dependent methyl transferases require no metal ions for methyl transfer; for those that do, metal ions are used to modulate substrate binding and selectivity but not catalysis.…”

Section: Introductionmentioning

confidence: 99%

A Divalent Metal Ion-Dependent N 1 -Methyl Transfer to G37-tRNA

Sakaguchi¹,

Lahoud²,

Christian³

et al. 2014

Chemistry & Biology

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The catalytic mechanism of the majority of S-adenosyl methionine (AdoMet)-dependent methyl transferases requires no divalent metal ions. Here we report that methyl transfer from AdoMet to N1 of G37-tRNA, catalyzed by the bacterial TrmD enzyme, is strongly dependent on divalent metal ions and that Mg2+ is the most physiologically relevant. Kinetic isotope analysis, metal rescue, and spectroscopic measurements indicate that Mg2+ is not involved in substrate binding, but in promoting methyl transfer. Based on the pH-activity profile indicating one proton transfer during the TrmD reaction, we propose a catalytic mechanism in which the role of Mg2+ is to help to increase the nucleophilicity of N1 of G37 and stabilize the negative developing charge on O6 during attack on the methyl sulfonium of AdoMet. This work demonstrates how Mg2+ contributes to the catalysis of AdoMet-dependent methyl transfer in one of the most crucial post-transcriptional modifications to tRNA.

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“…3,4,10,11 The C-terminal half of Hen1 is an autonomous Mn 2+ -dependent 3′-terminal ribose 2′-O-methyltransferase that places a 2′-OCH 3 mark at the RNA repair junction prior to ligation and thereby protects the junction from recurrent damage. 4,5,12,13 …”

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Structural and Biochemical Analysis of the Phosphate Donor Specificity of the Polynucleotide Kinase Component of the Bacterial Pnkp•Hen1 RNA Repair System

et al. 2013

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Clostridium thermocellum Pnkp is the end-healing and end-sealing subunit of a bacterial RNA repair system. CthPnkp is composed of three catalytic modules: an N-terminal 5′-OH polynucleotide kinase; a central 2′,3′ phosphatase; and a C-terminal ligase. The crystal structure of the kinase domain bound to ATP•Mg2+ revealed a rich network of ionic and hydrogen-bonding contacts to the α, β, and γ phosphates. By contrast, there are no enzymic contacts to the ribose and none with the adenine base other than a π-cation interaction with Arg116. Here we report that the enzyme uses ATP, GTP, CTP, UTP or dATP as a phosphate donor for the 5′-OH kinase reaction. The enzyme also catalyzes the reverse reaction, in which a polynucleotide 5′-PO4 group is transferred to ADP, GDP, CDP, UDP or dADP to form the corresponding NTP. We report new crystal structures of the kinase in complexes with GTP, CTP, UTP and dATP in which the respective nucleobases are stacked on Arg116 but make no other enzymic contacts. Mutating Arg116 to alanine elicits a 10-fold increase in Km for ATP, but has little effect on kcat. These findings illuminate the basis for non-specific donor nucleotide utilization by a P-loop phosphotransferase.

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Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3′ terminal RNA ribose 2′O-methyltransferase

Cited by 18 publications

References 42 publications

Distinctive Effects of Domain Deletions on the Manganese-Dependent DNA Polymerase and DNA Phosphorylase Activities of Mycobacterium smegmatis Polynucleotide Phosphorylase

Distinctive Effects of Domain Deletions on the Manganese-Dependent DNA Polymerase and DNA Phosphorylase Activities of Mycobacterium smegmatis Polynucleotide Phosphorylase

A Divalent Metal Ion-Dependent N 1 -Methyl Transfer to G37-tRNA

Structural and Biochemical Analysis of the Phosphate Donor Specificity of the Polynucleotide Kinase Component of the Bacterial Pnkp•Hen1 RNA Repair System

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