Structure and mechanism of Mycobacterium smegmatis polynucleotide phosphorylase Journal Article

   


Authors: Unciuleac, M. C.; Ghosh, S.; de la Cruz, M. J.; Goldgur, Y.; Shuman, S.
Article Title: Structure and mechanism of Mycobacterium smegmatis polynucleotide phosphorylase
Abstract: Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3′′-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction, PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3′′-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of M. tuberculosis militates for mycobacterial PNPase as a potential drug target. A cryo-EM structure of Mycobacterium smegmatis PNPase (MsmPNPase) reveals a characteristic ring-shaped homotrimer in which each protomer consists of two RNase PH-like domains and an intervening α-helical module on the inferior surface of the ring. The carboxy-terminal KH and S1 domains, which impart RNA specificity to MsmPNPase, are on the opposite face of the core ring and are conformationally mobile. Single particle reconstructions of MsmPNPase in the act of poly(A) synthesis highlight a 3′′-termi-nal (rA)4 oligonucleotide and two magnesium ions in the active site and an adenine nucleobase in the central tunnel. We identify amino acids that engage the 3′′ segment of the RNA chain (Phe68, Arg105, Arg112, Arg430, Arg431) and the two metal ions (Asp526, Asp532, Gln546, Asp548), and we infer those that bind inorganic phosphate (Thr470, Ser471, His435, Lys534). Alanine mutagenesis pinpointed RNA and phosphate contacts as essential (Arg105, Arg431, Lys534, Thr470 + Ser471), important (Arg112, Arg430), or unimportant (Phe68) for PNPase activity. Severe phosphorolysis and polymerase defects accompanying alanine mutations of the enzymic metal ligands suggest a two-metal mechanism of catalysis by MsmPNPase. © 2021 Unciuleac et al.
Keywords: 3 ́ polymerase; metal-dependent catalysis; rna phosphorylase; single particle cryo-em
Journal Title: RNA
Volume: 27
Issue: 8
ISSN: 1355-8382
Publisher: Cold Spring Harbor Laboratory Press  
Date Published: 2021-08-01
Start Page: 959
End Page: 969
Language: English
DOI: 10.1261/rna.078822.121
PUBMED: 34088850
PROVIDER: scopus
PMCID: PMC8284320
DOI/URL:

https://www.scopus.com/inward/record.uri?eid=2-s2.0-85110521770&doi=10.1261%2frna.078822.121&partnerID=40&md5=2126652aff2b25e0e3577e94327e8486
Notes: Article -- Export Date: 2 August 2021 -- Source: Scopus
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MSK Authors
  1. Stewart H Shuman
    546 Shuman
  2. Mihaela Carmen Sandu
    19 Sandu
  3. Yehuda Goldgur
    42 Goldgur
  4. Shreya Ghosh
    12 Ghosh
  5. Michael Jason V De La Cruz
    12 De La Cruz
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