Dataset Information

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase.

ABSTRACT: Glutamate decarboxylase (GAD; EC 4.1.1.15) is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce ?-aminobutyric acid (GABA), which exhibits several well-known physiological functions. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, a parallel strategy comprising sequential analysis and free energy calculation was applied to identify critical amino acid sites affecting thermostability of GAD and select proper mutation contributing to improve structure rigidity of the enzyme. Two mutant enzymes, D203E and S325A, with higher thermostability were obtained, and their semi-inactivation temperature (T5015) values were 2.3 °C and 1.4 °C higher than the corresponding value of the wild-type enzyme (WT), respectively. Moreover, the mutant, S325A, exhibited enhanced activity compared to the wild type, with a 1.67-fold increase. The parallel strategy presented in this work proved to be an efficient tool for the reinforcement of protein thermostability.

SUBMITTER: Zhang QF

PROVIDER: S-EPMC7037157 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase.

Zhang Qing-Fei QF Hu Sheng S Zhao Wei-Rui WR Huang Jun J Mei Jia-Qi JQ Mei Le-He LH

Molecules (Basel, Switzerland) 20200206 3

Glutamate decarboxylase (GAD; EC 4.1.1.15) is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA), which exhibits several well-known physiological functions. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, a parallel strategy comprising sequential analysis and free energy calculat ...[more]

PMID: 32041144

Similar Datasets

Project description:BACKGROUND:Gamma-aminobutyric acid (GABA) is a non-protein amino acid present in all living things. GABA is mainly synthesized from glutamate by glutamate decarboxylase (GAD). In plants the enzymatic activity of GAD is activated by Ca2+/calmodulin binding (CaMBD) at the C-terminus in response to various stresses, allowing rapid GABA accumulation in cells. GABA plays a central role in not only stress responses but also many aspects of plant growth and development as a signaling molecules. Furthermore, it is known to be a health-promoting functional substance that exerts improvements in life-style related diseases such as hypertension, diabetes, hyperlipidemia, and so on. Previous reports indicated that CaMBD found plant GADs possess an autoinhibitory function because truncation of GAD resulted in extreme GABA accumulation in plant cells. Therefore, we attempted a genetic modification of rice GAD via genome editing technology to increase GABA levels in the edible part of rice. RESULTS:In this study, we focused on GAD3, one of five GAD genes present in the rice genome, because GAD3 is the predominantly expressed in seeds, as reported previously. We confirmed that GAD3 has an authentic Ca2+/CaMBD that functions as an autoinhibitory domain. CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD off from the OsGAD3 gene, then introducing this transgene into rice scutellum-derived calli using an all-in-one vector harboring guide RNAs and CRISPR/Cas9 via Agrobacterium to regenerate rice plants. Out of 24 transformed rice (T1), a genome-edited rice line (#8_8) derived from two independent cleavages and ligations in the N-terminal position encoding OsGAD3-CaMBD and 40 bp downstream of the termination codon, respectively, displayed a AKNQDAAD peptide in the C-terminal region of the putative OsGAD3 in place of its intact CaMBD (bold indicates the trace of the N-terminal dipeptides of the authentic CaMBD). A very similar rice line (#8_1) carrying AKNRSSRRSGR in OsGAD3 was obtained from one base pair deletion in the N-terminal coding region of the CaMBD. Free amino acid analysis of the seeds (T2) indicated that the former line contained seven-fold higher levels of GABA than wild-type, whereas the latter line had similar levels to the wild-type, although in vitro enzyme activities of recombinant GAD proteins based on the GAD3 amino acid sequence elucidated from these two lines in the absence of Ca2+/bovine CaM were both higher than wild-type counterpart. In addition to high level of GABA in #8_8, the average seed weight per grain and protein content were superior to wild-type and #8_1. CONCLUSIONS:We have successfully established GABA-fortified rice by using CRISPR/Cas9 genome editing technology. Modified rice contained seven-fold higher GABA content and furthermore displayed significantly higher grain weight and protein content than wild-type brown rice. This is the first report of the production of GABA-enriched rice via a genome editing.

Dataset Information

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase.

Publications

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets