Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
Daniel M. Roberts
Elizabeth Howell, Gladys Alexandre, Beth Mullin, Feng Chen
Glutamine synthetase (GS) is a major ammonia assimilatory enzyme in soybean nodules. The four isoforms of cytosolic glutamine synthetase (GS1[glutamine synthetase 1]β[beta]1, GS1β2, GS1γ[gamma]1 and GS1γ2) present in soybean nodules are 80% identical with respect to amino acid sequence, and share similar kinetic properties. It is shown all major GS1 isoforms interact with nodulin 26, a member of the aquaporin family of membrane channels. Nodulin 26 is the major protein component of the symbiosome membrane (SM), where it serves a function as an ammonia and water channel. The site of interaction of GS on nodulin 26 is the cytosolic C-terminus, where it binds with 1:1 stoichiometry. The binding of GS is proposed to dock the enzyme to the cytosolic surface of the SM. This would promote efficient assimilation of fixed nitrogen, as well as prevent potential ammonia toxicity, by futile cycling of ammonia/ammonium across the SM. Quantitative PCR analysis of the transcripts of all the isoforms from soybean tissues shows that GS1γ are the nodule-specific isoforms, but that the GS1β isoforms are highly expressed and the highest transcripts in nodules is GS1β1. Further investigation of GS1 isoforms showed that they are subjected to differential regulation by thiol based disulfide bond formation. Specifically, GS1γ1 is sensitive to inhibition by reversible oxidation whereas the GS1β1 is not sensitive to oxidizing conditions. Site-directed mutagenesis of the GS1γ1 isoform showed that the oxidation observed is due to reversible disulfide bond formation through intersubunit cys92 and cys159 across the shared active site. Analytical ultracentrifugation studies showed a difference in the native oligomeric molecular weight of the two isoforms, with GS1β1 forming a decamer and GS1γ1 forming a dodecamer. It is hypothesized that these differences in quaternary structure is linked to their different sensitivities to thiol based regulation, possibly due to distinct positioning of the intersubunit cysteine sulfhydryls. The reversible oxidation observed for GS1γ1 is unique to this isoform and may serve as an additional level of regulation in response to oxygen tension in the infected cell, as well as in response to reactive oxygen production during stress responses.
Masalkar, Pintu Daulatrao, "Structure, Function and Regulation of Two Isoforms of Glutamine Synthetase from Soybean Root Nodules. " PhD diss., University of Tennessee, 2014.