Structural Insight into the Contributions of the N-Terminus and Key Active-Site Residues to the Catalytic Efficiency of Glutamine Synthetase 2.

Wen-Ting Chen,Szu-Chin Ma,Chih-Yu Lin,Yi-Zong Lee,Wei-Cheng Chen,Hsin-Yu Yang,Hsien-Sheng Yin

doi:10.3390/biom10121671

Abstract

Glutamine synthetase (GS) catalyzes the condensation of ammonia and glutamate, along with ATP, to form glutamine. Despite extensive studies on GSs from eukaryotes and prokaryotes, the roles of the N-terminus and other structural features in catalysis remain unclear. Here we report the decameric structure of Drosophila melanogaster GS 2 (DmGS2). The N-terminal short helices, α1 and α2, constitute a meander region, and form hydrogen bonds with residues 3–5 in the N-terminal loop, which are not present in the GSs of other species. Deletion of α1 or α1-α2 inactivates DmGS2. Notably, the Arg4 in each monomer of one pentamer forms hydrogen bonds with Glu7, and Asp8 in the adjacent monomer of the other pentamer. Replacement of Arg4 with Asp (R4D) abolishes activity. Analytical ultracentrifugation revealed that Arg4 is crucial for oligomerization. Circular dichroism spectra revealed that R4D may alter the secondary structure. We mutated key residues to identify the substrate-binding site. As Glu140 binds glutamate and Glu311 binds ammonia, mutants E140A and E311A have little activity. Conversely, mutant P214A (P contributes to ATP binding) has higher activity than wild-type DmGS2. These findings expand the understanding of the structural and functional features of the N-terminal meander region of DmGS2 and the residues important for catalytic efficiency.

Highlights

Glutamine synthetase (GS) plays a crucial role in nitrogen metabolism by catalyzing the ATP-dependent condensation of glutamate with ammonia to produce glutamine, ADP, and inorganic phosphate [1]
A structure was obtained for the Drosophila melanogaster GS 2 (DmGS2) decamer in complex with ADP (Figure 1)
The hydrolysis of ATP may be promoted by Mg2+, which accelerates the reaction in aqueous solution [32,33]

Summary

Introduction

Glutamine synthetase (GS) plays a crucial role in nitrogen metabolism by catalyzing the ATP-dependent condensation of glutamate with ammonia to produce glutamine, ADP, and inorganic phosphate [1]. GS modulates the cellular level of harmful ammonia, and converts neurotoxic glutamate into nontoxic glutamine [2]. In the relatively late embryonic stage, DmGS2 mRNA is expressed in the nervous system and accumulates in axon bundles, correlating with synapse formation [7]. Recent studies have indicated that GS2 is expressed in the glial cells of the young Drosophila mushroom body [10]. Glial cells take up glutamate released from the synapses and converts it to glutamine via GS. The mushroom body of the Drosophila brain plays vital roles in olfactory memory and learning [11]. Drosophila GS2 may be critical for learning and memory

Methods

Results

Conclusion