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Overexpression, Purification, and Preliminary Crystallographic Analysis of the Archaeosine Synthase GAT‐QueC
Author(s) -
Susanto Albert,
Honberger Matthew,
Murphy Ryan,
Bayooz Susan,
Yi Stuart,
Swairjo Manal A.
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.s1.06647
Subject(s) - transfer rna , glutamine amidotransferase , sulfolobus acidocaldarius , enzyme , biochemistry , biology , sulfolobus , chemistry , crystallography , stereochemistry , rna , archaea , glutamine , gene , amino acid
Archaeosine (G+) is a highly modified nucleosides found exclusively at position‐15 in the dihydrouridine loops (D‐loop) of archaeal tRNAs where it contributes to tRNA structural stability. In a late step in G+ biosynthesis, the precursor nucleoside 7‐cyano‐7‐deazaguanine (preQ0) is inserted into tRNA by the enzyme tRNA‐guanine transglycosylase (arcTGT) before conversion to G+ by archaeosine synthases that vary among archaeal phyla: ArcS in Euryarchaeota, and QueF‐like or GAT‐QueC in Chrenoarchaeota. GAT‐QueC is a two‐domain enzyme harboring an N‐terminal glutamine amidotransferase (GAT) domain, and a C‐terminal domain homologous to QueC, the enzyme that produces preQ0. Consistent with its 2‐domain structure, GAT‐QueC functions in vivo as both a preQ0 synthase and an amidinotransferase, generating G+‐modified tRNA. However, it remains unknown whether the GAT domain acts on preQ0 before or after its insertion into tRNA by arcTGT. Here we describe overexpression, purification and preliminary biochemical and crystallographic characterization of GAT‐QueC from Sulfolobus tokodaii (StGAT‐QueC). Analytical gel filtration chromatography and electrophoretic mobility shift analysis reveal that StGAT‐QueC is a monomeric protein with molecular weight of 55 kDa and no affinity for tRNA, suggesting that the enzyme acts on free preQ0, not preQ0‐modified tRNA. High throughput crystallization of the apoenzyme (and in the presence of preQ0) using the vapor diffusion method yielded crystals that grew in 21 days from solutions containing methyl pentanediol and sodium acetate, and diffracted to 5.8 Å without cryo protection. The crystals belong to cubic space group F23, exhibit unit cell length=292 Å and contain 4 protein molecules per asymmetric unit. Support or Funding Information NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).