Crystal Structure of Type III Glutamine Synthetase: Surprising Reversal of the Inter-Ring Interface

Abstract

Glutamine synthetases are ubiquitous, homo-oligomeric enzymes essential for nitrogen metabolism. Unlike types I and II, which are well described both structurally and functionally, the larger, type IIIs are poorly characterized despite their widespread occurrence. An understanding of the structural basis for this divergence and the implications for design of type-specific inhibitors has, therefore, been impossible. The first crystal structure of a GSIII enzyme, presented here, reveals a conservation of the GS catalytic fold but subtle differences in protein-ligand interactions suggest possible avenues for the design GSIII inhibitors. Despite these similarities, the divergence of the GSIII enzymes can be explained by differences in quaternary structure. Unexpectedly, the two hexameric rings of the GSIII dodecamer associate on the opposite surface relative to types I and II. The diversity of GS quaternary structures revealed here suggests a nonallosteric role for the evolution of the double-ringed architecture seen in all GS enzymes.

Purification, Characterization, and Expression of Multiple Glutamine Synthetases from Prevotella ruminicola 23

“…S2 in the supplemental material). In addition, GSIII-1 and GSIII-2 displayed strong conservation of all five GS conserved regions and four GS type III-specific conserved regions (11,44). Furthermore, key conserved amino acid residues essential for function of the GSIII protein in signature motifs I to IV and motif C in R. albus 8 were all highly conserved within the P. ruminicola 23 GSIII amino acid sequence (2).…”

“…GSIII was first described in Bacteroides fragilis (25) and also assembles as a homododecamer, comprising larger subunits (M r , ca. 75,000) than those of GSI or GSII (2,40,44). GSIII have been identified in Butyrivibrio fibrisolvens, Ruminococcus albus 8, and some cyanobacteria (2,11,22,35).…”

“…Additionally, four typical conserved regions have been identified in GSIII proteins (11). Recently, the first crystal structure of a GSIII enzyme, from B. fragilis, has been presented (44).…”

“…␥-Transferase activity of all three enzymes (GSI, GSIII-1, and GSIII-2) was observed only in the presence of Mn 2ϩ ions and was not detected in the presence of Mg 2ϩ , Cu 2ϩ , Co 2ϩ , or Ca 2ϩ . The optimal (11,44). Amino acid sequences that are conserved are boxed in black, and those that are similar are gray.…”

Structures of regulatory machinery reveal novel molecular mechanisms controlling B. subtilis nitrogen homeostasis

“…Each B. subtilis GS subunit contains 19 b strands and 15 a helices. All GS structures solved to date are dodecamers or decamers that are comprised of two stacked rings (either hexamers or pentamers) (Liaw and Eisenberg 1994;Eisenberg et al 2000;Unno et al 2006;van Rooyen et al 2011). However, because GS active sites are formed at the interfaces between two subunits within a single ring, hexamer, or pentamer, it has been unclear what role the double-ring structure may play in GS function.…”