Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana: Characterization of the enzyme's in vitro truncation by thiol endopeptidase activity.

Publisher: Academic Press Country of Publication: United States NLM ID: 9101496 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1096-0279 (Electronic) Linking ISSN: 10465928 NLM ISO Abbreviation: Protein Expr Purif Subsets: MEDLINE

Publication: Orlando, FL : Academic Press
Original Publication: San Diego : Academic Press, c1990-

Arabidopsis*/genetics ; Arabidopsis*/enzymology ; Glutamate Decarboxylase*/genetics ; Glutamate Decarboxylase*/metabolism ; Glutamate Decarboxylase*/chemistry ; Glutamate Decarboxylase*/isolation & purification ; Escherichia coli*/genetics ; Escherichia coli*/metabolism ; Arabidopsis Proteins*/genetics ; Arabidopsis Proteins*/isolation & purification ; Arabidopsis Proteins*/metabolism ; Arabidopsis Proteins*/chemistry ; Recombinant Proteins*/genetics ; Recombinant Proteins*/isolation & purification ; Recombinant Proteins*/chemistry ; Recombinant Proteins*/metabolism ; Recombinant Proteins*/biosynthesis; Proteolysis ; Gene Expression

Plant glutamate decarboxylase (GAD) is a Ca ²⁺ -calmodulin (CaM) activated enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. Our prior research established that in vivo phosphorylation of AtGAD1 (AT5G17330) occurs at multiple N-terminal serine residues following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 (rAtGAD1) following its expression in Escherichia coli to facilitate studies of the impact of phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of an approximate 5 kDa polypeptide from the C-terminus of 59 kDa rAtGAD1 subunits occurred during purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing this partial rAtGAD1 proteolysis. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated rAtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalysis, and that rAtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca ²⁺ /CaM facilitated the purification of non-proteolyzed rAtGAD1 to a specific activity of 27 (μmol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca ²⁺ /CaM at pH 7.3. By contrast, the purified partially proteolyzed rAtGAD1 was >40 % less active at both pH values, and only activated 2-fold by Ca ²⁺ /CaM at pH 7.3. These results emphasize the need to diagnose and prevent partial proteolysis before conducting kinetic studies of purified regulatory enzymes.
Competing Interests: Declaration of Competing interest None declared.
(Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Keywords: Arabidopsis thaliana (mouse-ear Cress); Calcium signaling; Calmodulin; Glutamate decarboxylase; Unwanted proteolysis; γ-aminobutyrate (GABA) shunt

EC 4.1.1.15 (Glutamate Decarboxylase)
0 (Arabidopsis Proteins)
0 (Recombinant Proteins)
EC 4.1.1.15 (glutamate decarboxylase 1)

Date Created: 20240929 Date Completed: 20241117 Latest Revision: 20241117

20241118

10.1016/j.pep.2024.106612

39343154