Open AccessCrystallization and preliminary X‐ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase
Author(s) -
Itoh Yuzuru,
Sekine Shunichi,
Yokoyama Shigeyuki
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112033519
Subject(s) - aquifex aeolicus , selenocysteine , crystallography , transfer rna , chemistry , resolution (logic) , crystallization , serine , crystal structure , stereochemistry , biochemistry , rna , enzyme , escherichia coli , organic chemistry , gene , cysteine , artificial intelligence , computer science
Selenocysteine (Sec), the 21st amino acid, is synthesized on its specific tRNA (tRNA Sec ) via a multi‐step process. In bacteria, tRNA Sec is ligated first with serine by seryl‐tRNA synthetase, which is followed by Ser‐to‐Sec conversion by Sec synthase (SelA). To elucidate its structure and catalytic mechanism, Aquifex aeolicus SelA was crystallized. Although wild‐type SelA crystals diffracted X‐rays poorly (to up to 8 Å resolution), the resolution was improved by introducing a quadruple point mutation targeting the loop regions and by methylating the lysine residues, which yielded 3.9 Å resolution diffraction data from a full‐length SelA crystal. Truncation of the N‐terminal region (ΔN) also improved the resolution. A 3.3 Å resolution data set for phase determination was obtained from a crystal of selenomethionine‐substituted Lys‐methylated SelA‐ΔN.