Recombinant Solanum lycopersicum Gamma aminobutyrate transaminase 2 (GABA-TP2)

What is the subcellular localization of GABA-TP2 and how does it differ from other tomato GABA-T isoforms?

GABA-TP2 is localized to the cytosol in tomato cells. This distinguishes it from the other two GABA-T isoforms in tomato: GABA-TP1 (SlGABA-T1), which is targeted to mitochondria, and GABA-TP3 (SlGABA-T3), which is localized to plastids. The specific targeting of the mitochondrion and plastid-localized isoforms is mediated by their N-terminal presequences, while GABA-TP2 lacks such targeting sequences .

To determine these localizations, researchers used transient expression of individual full-length GABA-T isoforms fused to green fluorescent protein in tobacco suspension-cultured cells. The distinct subcellular distribution of these enzymes suggests they may have specialized roles in different cellular compartments .

What enzymatic activities does recombinant GABA-TP2 possess?

The enzyme catalyzes the transamination of GABA using pyruvate or glyoxylate as amino group acceptors, producing alanine or glycine, respectively, along with succinic semialdehyde (SSA). This represents a key step in GABA catabolism in plants .

How can recombinant GABA-TP2 be efficiently expressed and purified?

The methodological approach involves:

• Cloning the full-length GABA-TP2 coding sequence into an appropriate expression vector

• Co-transformation with plasmids expressing the GroES/EL chaperones

• Induction of protein expression under optimized conditions

• Purification using affinity chromatography methods

This approach results in good recovery of soluble, functional recombinant GABA-TP2 enzyme suitable for biochemical characterization .

What is the expression pattern of GABA-TP2 in different tomato tissues and developmental stages?

The expression of GABA-TP2 shows tissue-specific patterns that differ from the other GABA-T isoforms. Notably, differential expression patterns are observed particularly in reproductive tissues, but not in vegetative tissues, suggesting unique roles for each enzyme in developmental processes .

Unlike SlGABA-T1, which shows strong correlation with GABA catabolism during fruit ripening, GABA-TP2 appears to have a less prominent role in fruit GABA metabolism. This is evidenced by studies showing that suppression of SlGABA-T1 significantly increases GABA accumulation in fruits (6.8–9.2 times higher in red fruits), while suppression of SlGABA-T2 shows almost no correlation with GABA content in fruits .

What is the relationship between GABA-TP2 and other enzymes in the GABA shunt pathway?

GABA-TP2 functions within the larger GABA shunt pathway, which includes:

• Glutamate decarboxylase (GAD) - converts glutamate to GABA

• GABA transaminases (GABA-T) - convert GABA to succinic semialdehyde

• Succinic semialdehyde dehydrogenase (SSADH) - converts succinic semialdehyde to succinate

GABA-TP2 specifically catalyzes the second step in this pathway. Unlike the α-ketoglutarate-dependent GABA transaminase (GABA-TK) that predominates in animals, plant GABA-TPs like GABA-TP2 primarily use pyruvate or glyoxylate as amino group acceptors .

The compartmentalization of the GABA shunt enzymes across different subcellular locations (cytosol, mitochondria, and plastids) necessitates the transport of metabolites between these compartments, suggesting complex regulation of GABA metabolism .

How do CRISPR/Cas9-mediated mutations in GABA-TP2 affect plant phenotype and GABA accumulation?

CRISPR/Cas9 genome editing has been used to target GABA-TP2 along with other genes involved in GABA metabolism. While specific data on GABA-TP2 single mutants is limited in the search results, multiplex CRISPR/Cas9 systems targeting multiple genes including GABA-Ts have shown significant impacts on GABA accumulation .

The most dramatic effects on GABA accumulation and plant phenotype were observed with SlGABA-T1 mutations or in multiple gene knockouts. Many of these plants exhibited abnormalities in growth, development, and fertility. Notably, single SlGABA-T1 and double SlGABA-T1/SlGABA-T3 mutants were among the few combinations that could still set fruits, suggesting that GABA-TP2 may have distinct roles that impact reproductive development differently .

What are the kinetic properties and substrate specificities of GABA-TP2?

Recombinant GABA-TP2 exhibits pyruvate- and glyoxylate-dependent GABA transaminase activities. While the search results don't provide specific kinetic parameters (Km, Vmax) for GABA-TP2, they do indicate that plant pyruvate-dependent GABA-TPs generally have Km values for GABA, pyruvate, and glyoxylate of 0.18−0.34 mM, 0.14 mM, and 0.11 mM, respectively .

The cytosolic localization of GABA-TP2 compared to the mitochondrial GABA-TP1 suggests it may have access to different pools of substrates and could be regulated by different cellular conditions. It's notable that the mitochondrial GABA-TP1 has significantly higher specific activity than both the cytosolic GABA-TP2 and plastidic GABA-TP3 .

How can researchers differentiate between GABA-TP and GABA-TK activities in plant extracts?

Differentiating between pyruvate-dependent (GABA-TP) and α-ketoglutarate-dependent (GABA-TK) GABA transaminase activities in plant extracts presents methodological challenges. The search results highlight a scientific debate on this topic, with some researchers cautioning that detection of GABA-TK activity in crude extracts should be treated with skepticism .

Methodological approaches to differentiate these activities include:

• Using specific substrates: pyruvate for GABA-TP and α-ketoglutarate for GABA-TK

• Employing recombinant enzymes with known activities as controls

• Using specific inhibitors if available

• Performing enzyme assays under conditions that favor one activity over the other

• Conducting genetic studies with knockout or suppressed enzyme expression

Researchers should be aware that there are contradictory findings regarding GABA-TK activity in tomato. While Akihiro et al. (2008) reported significant GABA-TK activity correlating with GABA catabolism in ripening fruits, Clark et al. (2009) found no GABA-TK activity in the same cultivar and suggested that pyruvate/glyoxylate-dependent GABA-T activity accounts for GABA catabolism in tomato fruits .

What are the challenges in studying GABA-TP2 function specifically among other GABA-T isoforms?

Researching GABA-TP2 function specifically presents several technical challenges:

• Functional redundancy: The presence of three GABA-T isoforms with similar catalytic activities makes it difficult to isolate the specific contribution of GABA-TP2.

• Subcellular compartmentalization: As a cytosolic enzyme, GABA-TP2 functions in a different cellular compartment than the other isoforms, requiring methods to study compartment-specific metabolism.

• Expression levels: GABA-TP2 may have lower expression or activity compared to the predominant mitochondrial GABA-TP1, making its specific contribution harder to detect.

• Specific inhibitors: There is a lack of isoform-specific inhibitors that would allow selective inhibition of GABA-TP2 activity.

• Complex phenotypes: As seen in gene-editing studies, mutations in GABA metabolism genes often result in complex phenotypes that can be difficult to attribute to specific enzymes .

How does GABA-TP2 contribute to GABA homeostasis during tomato fruit development?

The contribution of GABA-TP2 to GABA homeostasis during tomato fruit development appears to be less significant than that of GABA-TP1. Studies have shown that GABA content in tomato fruits changes dramatically during development, accumulating to high levels at the mature green stage (constituting up to 50% of free amino acids) and then rapidly decreasing during ripening .

GABA-TP1 suppression via RNAi resulted in significantly increased GABA levels in fruits (6.8–9.2 times higher in red fruits), while suppression of GABA-TP2 showed almost no correlation with GABA content in fruits . This suggests that GABA-TP1, not GABA-TP2, is the essential isoform for GABA reduction during fruit ripening.

Table 1: Comparison of Tomato GABA Transaminase Isoforms

Table 2: GABA Metabolic Pathway Enzymes and Their Properties