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Structural Basis of 7‐Deazaguanine Modification of DNA in Bacteria and Phage
Author(s) -
Hellie Eric,
Alkuzweny Sana,
Bayooz Susan,
Phan Phuc,
Savage Joshua,
Luthra Amit,
Iwata-Reuyl Dirk,
Swairjo Manal
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02684
Subject(s) - guanine , dna , transfer rna , restriction enzyme , biology , chemistry , stereochemistry , nucleotide , rna , biochemistry , gene
Two important modifications of tRNA, the 7‐deazaguanine nucleosides queuosine (Q) and archaeosine (G + ), are biosynthesized from GTP in bacteria and archaea, respectively, in a well characterized multi‐enzyme pathway leading to the shared advanced intermediate, 7‐cyano‐7‐deazaguanine (preQ 0 ). In bacteria, preQ 0 is converted to an aminomethyl derivative that is then inserted in tRNA by the bacterial tRNA‐guanine transglycosylase (TGT) enzyme. In archaea, preQ 0 is inserted directly in tRNA by the archaeal TGT before conversion to G + . Recently, in 230 bacterial and phage genomes, a genomic island that contains a paralog of TGT was identified, which led to the discovery of Q, G + and preQ 0 deoxy derivatives in the DNA of some of these organisms. This gene cluster was renamed DpdA‐K, for “7‐deazapurine in DNA.” The G + ‐modified DNA of E. coli bacteriophage 9g has been shown to resist restriction by >140 Type II restriction endonucleases (REases), consistent with a role of the modification as a defense mechanism. Recent experiments have shown that DpdA is a DNA‐guanine transglycosylase that catalyzes the insertion of preQ 0 at a specific palindromic sequence in DNA. Here we report overexpression, purification, and crystallographic analysis of S. Montevideo DpdA ( Sm DpdA, ~47 kDa). The crystal structure, determined at 2.25‐Å resolution by multi‐wavelength anomalous diffraction methods, reveals a TIM‐barrel enzyme similar to the bacterial TGT in overall fold, active site and structural Zn 2+ site. However, a large DpdA‐specific insertion in the TIM barrel provides an extended positively charged 26‐Å wide surface groove, consistent with a DNA binding surface. Free docking of a DNA duplex using the HADDOCK server places DNA to this surface in a fashion reminiscent of DNA binding to the ubiquitous Zn 2+ ‐dependent DNA repair enzyme apurinic/apyrimidinic endonuclease IV. The structure and docking model suggest that DpdA bends its substrate DNA by 60° and flips the modification‐site guanine base out of the helix for transglycosylation. The results also inform the design of a substrate DNA duplex for future crystallization of the nucleoprotein complex. Support or Funding Information NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).