GGAT2 (glutamate:glyoxylate aminotransferase 2; also AOAT2/GGT2; At1g70580) is a peroxisomal, pyridoxal 5'-phosphate-dependent class-I aminotransferase of the alanine aminotransferase subfamily, 93% identical to its segmentally duplicated paralog GGAT1 (At1g23310). Purified recombinant GGAT2 transaminates glyoxylate to glycine with glutamate as amino donor (Km for glyoxylate 0.14 mM) and also catalyses alanine:glyoxylate, alanine:2-oxoglutarate and glutamate:pyruvate transaminations, but not serine:glyoxylate or aspartate transamination. It ends in a type 1 peroxisomal targeting signal and is detected by isoform-specific peptides in the mature leaf peroxisome proteome. GGAT2 is expressed at much lower levels than GGAT1, in leaves, roots, siliques and light-grown seedlings; its promoter is active in leaves and its mRNA rises at the start of the light period while GGAT1 mRNA falls. It is considered a minor, partially redundant photorespiratory GGAT isoform that contributes to the residual leaf GGAT activity of ggat1 knockouts; no ggat2 mutant has been described.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003729 mRNA binding | IDA PMID:32344669 Discovering the RNA-Binding Proteome of Plant Leaves with an... | MARK AS OVER ANNOTATED | Summary: Glutamate-glyoxylate aminotransferase was captured in a leaf RNA interactome capture (RIC) screen after UV crosslinking. Reason: The authors list glutamate-glyoxylate aminotransferase among metabolic enzymes newly found in the RBPome with no prior link to RNA biology. There is no follow-up showing specific mRNA binding or a function for it, and GGAT2 acts in the peroxisomal matrix, away from mRNA. The screen hit is retained as data but not as a function of the gene. Same action as the corresponding GGAT1 row. Supporting Evidence: PMID:32344669 Additional moonlighting enzymes were identified in the leaf RBPome, such as COBALAMIN-INDEPENDENT METHIONINE SYNTHASE, (S)-2-HYDROXY-ACID OXIDASE, GLYCINE DEHYDROGENASE and GLUTAMATE-GLYOXYLATE AMINOTRANSFERASE, which, to our knowledge, have not previously been linked to RNA biology |
| GO:0003729 mRNA binding | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA machine-learning prediction of mRNA binding. Reason: The ARBA rule most likely reflects the same class of RNA interactome screen hits; there is no evidence that mRNA binding is a function of this peroxisomal aminotransferase. Consistent with the IDA row for the same term. Supporting Evidence: PMID:32344669 Additional moonlighting enzymes were identified in the leaf RBPome, such as COBALAMIN-INDEPENDENT METHIONINE SYNTHASE, (S)-2-HYDROXY-ACID OXIDASE, GLYCINE DEHYDROGENASE and GLUTAMATE-GLYOXYLATE AMINOTRANSFERASE, which, to our knowledge, have not previously been linked to RNA biology |
| GO:0004021 L-alanine:2-oxoglutarate transaminase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT inference of alanine:2-oxoglutarate (AlaAT) activity for the alanine aminotransferase clade; GGAT2 itself is among the experimentally characterized descendants. Reason: Purified recombinant GGAT2 catalyses the reversible Ala:2-oxoglutarate / Glu:pyruvate transamination characteristic of AlaATs, so the activity is real and ancestral to the clade. As for GGAT1, it is secondary to the glutamate:glyoxylate reaction and is kept as non-core. Supporting Evidence: PMID:12529529 Both purified recombinant proteins catalyzed GGT, GPT, and AKT reactions |
| GO:0004021 L-alanine:2-oxoglutarate transaminase activity | IDA PMID:12529529 Alanine aminotransferase homologs catalyze the glutamate:gly... | KEEP AS NON CORE | Summary: Lysates of E. coli expressing GGT2 and the purified recombinant GGAT2 catalyse the Ala:2-oxoglutarate (AKT) reaction, with Km values of 3.56 mM for alanine and 0.51 mM for 2-oxoglutarate. Reason: Directly demonstrated on the GGAT2 protein itself, but a secondary activity relative to the glutamate:glyoxylate reaction. Same action as for GGAT1. Supporting Evidence: PMID:12529529 Recombinant GGT2 was soluble when expressed in E. coli ; lysates of bacteria expressing this construct exhibited significantly increased levels of GGT, GPT, and AKT activity compared with empty vector controls file:ARATH/GGAT2/GGAT2-uniprot.txt KM=3.56 mM for alanine |
| GO:0004021 L-alanine:2-oxoglutarate transaminase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic mapping from EC 2.6.1.2 / RHEA:19453. Reason: Consistent with the experimentally demonstrated AKT/GPT activity of GGAT2; non-core relative to the glutamate:glyoxylate activity. Supporting Evidence: PMID:12529529 Both purified recombinant proteins catalyzed GGT, GPT, and AKT reactions |
| GO:0005737 cytoplasm | ISM GO_REF:0000122 | MODIFY | Summary: AtSubP sequence-based prediction of cytoplasmic localization. Reason: Peroxisomes are part of the cytoplasm, so the term is not wrong, but it is far less specific than the evidence: GGAT2 carries a C-terminal PTS1 and is identified by isoform-specific peptides in the leaf peroxisome proteome. The specific term, peroxisome, is already carried by the gene. Same action as for GGAT1. Proposed replacements: peroxisome Supporting Evidence: PMID:17951448 Of the two isoforms each of Glu-glyoxylate aminotransferase (GGT) and peroxisomal malate dehydrogenase (pMDH), GGT1 (At1g23310; SKM>) and pMDH2 (At5g09660; RIx 5 HL) dominated over the second isoform, both of which were identified as well |
| GO:0005739 mitochondrion | HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati... | MARK AS OVER ANNOTATED | Summary: Detection in a cell-fractionation / size-exclusion profiling study of membrane-associated protein complexes. Reason: GGAT2 has no mitochondrial presequence (the mitochondrial AlaAT paralog At1g72330 carries a long N-terminal extension that GGAT2 lacks) and ends in a PTS1; GGT activity is exclusively peroxisomal in Arabidopsis, with very little in the mitochondrial peak. Detection in crude organelle/membrane fractions is best regarded as contamination. Following projects/ARATH/organelle-audit.md rule 3, this single proteomics hit for a protein localized elsewhere is marked over-annotated rather than removed. Supporting Evidence: PMID:28887381 Here we combined cell fractionation of Arabidopsis leaves with nondenaturing detergent solubilization and LC/MS-based profiling of size exclusion chromatography fractions PMID:12529529 Peak GGT activity cofractionated with the catalase peak, very little GGT activity cofractionated with the mitochondrial and chloroplast peaks PMID:12529529 whereas the putative mitochondrial isozyme contains a long amino-terminal extension |
| GO:0005777 peroxisome | HDA PMID:17951448 Proteome analysis of Arabidopsis leaf peroxisomes reveals no... | ACCEPT | Summary: GGAT2 was identified, by peptides distinguishing it from GGAT1, in the proteome of isolated mature leaf peroxisomes, at lower abundance than GGAT1. Reason: This is the most direct GGAT2-specific localization evidence: GFP targeting was done only with GGAT1, and activity fractionation cannot separate the two isoforms. It agrees with the GGAT2 PTS1 and the exclusively peroxisomal GGT activity. Core location. Supporting Evidence: PMID:17951448 On the basis of the MS data (see Supplemental Table 2 online), closely related isoforms of high sequence similarity could often be distinguished, allowing conclusions on isoform expression and predominance in mature leaves. PMID:17951448 Of the two isoforms each of Glu-glyoxylate aminotransferase (GGT) and peroxisomal malate dehydrogenase (pMDH), GGT1 (At1g23310; SKM>) and pMDH2 (At5g09660; RIx 5 HL) dominated over the second isoform, both of which were identified as well |
| GO:0005777 peroxisome | IDA PMID:12154131 Proteomic analysis of leaf peroxisomal proteins in greening ... | ACCEPT | Summary: Leaf peroxisomes isolated from greening cotyledons were analysed by MALDI-TOF MS; glycolate pathway enzymes were among the identified proteins. Reason: The cached record is abstract-only, so whether the peptide mass fingerprint distinguished GGAT2 from the 93%-identical GGAT1 cannot be checked. The claim itself is independently supported for GGAT2 (isoform-specific peptides in the leaf peroxisome proteome, PTS1, peroxisomal GGT activity), so the curator's annotation is accepted, as for GGAT1. Supporting Evidence: PMID:12154131 Among them, five proteins are related to the glycolate pathway PMID:17951448 Of the two isoforms each of Glu-glyoxylate aminotransferase (GGT) and peroxisomal malate dehydrogenase (pMDH), GGT1 (At1g23310; SKM>) and pMDH2 (At5g09660; RIx 5 HL) dominated over the second isoform, both of which were identified as well |
| GO:0005777 peroxisome | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic mapping of the UniProt subcellular location (Peroxisome). Reason: Consistent with the experimental peroxisomal localization; the UniProt location is itself curated from PMID:12529529. Supporting Evidence: PMID:12529529 These subcellular fractionation data are consistent with a solely peroxisomal localization of GGT activity in Arabidopsis. |
| GO:0005777 peroxisome | ISS PMID:12631323 Identification of photorespiratory glutamate:glyoxylate amin... | ACCEPT | Summary: Similarity-based transfer from AOAT1 (GGAT1), whose GFP fusion is targeted to peroxisomes in a PTS1-dependent way; AOAT2 also carries a PTS1. Reason: GGAT2 shares the C-terminal PTS1 and 93% identity with GGAT1, and GGAT2-specific peroxisome proteomics confirms the inference. Supporting Evidence: PMID:12631323 Arabidopsis has four genes encoding AOAT-like proteins and two of them (namely AOAT1 and AOAT2) contain peroxisomal targeting signal 1 (PTS1). file:ARATH/GGAT2/GGAT2-uniprot.txt Peroxisomal targeting signal |
| GO:0005829 cytosol | HDA PMID:25293756 A proteomic strategy for global analysis of plant protein co... | MARK AS OVER ANNOTATED | Summary: Detection in size-exclusion profiling of a crude cytosolic fraction of leaves. Reason: The study analysed a crude cytosolic fraction, into which matrix enzymes leak from peroxisomes broken during homogenization. GGT activity fractionates exclusively with peroxisomes, and the NOT cytosol annotation from the cytosolic proteome study (accepted below) agrees. The observation does not establish a cytosolic pool. Same action as for GGAT1. Following projects/ARATH/organelle-audit.md rule 3, this single proteomics hit for a protein localized elsewhere is marked over-annotated rather than removed. Supporting Evidence: PMID:25293756 Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography PMID:12529529 GGT activity was localized exclusively to Arabidopsis peroxisomes |
| GO:0005829 cytosol | RCA NOT PMID:21166475 Analysis of the Arabidopsis cytosolic proteome highlights su... | ACCEPT | Summary: NOT annotation: GGAT2 was not part of the experimentally defined cytosolic proteome of Arabidopsis cell cultures. Reason: The negative assertion agrees with the PTS1 and the exclusively peroxisomal GGT activity. Same action as for GGAT1. Supporting Evidence: PMID:21166475 defines an experimental set of plant protein sequences that are not targeted to subcellular locations following translation and folding in the cytosol PMID:12529529 GGT activity was localized exclusively to Arabidopsis peroxisomes |
| GO:0008453 L-alanine:glyoxylate transaminase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT inference for the plant peroxisomal GGAT subclade (GGAT1/GGAT2), seeded by their own experimental data; GGAT2 appears among its own sources. Reason: Alanine:glyoxylate (AGT) activity was detected with recombinant GGAT2, so the inference is sound. Its importance for photorespiratory glycine formation, compared with glutamate as amino donor, is disputed, so it is kept as non-core, as for GGAT1. Supporting Evidence: PMID:12529529 Both recombinant proteins and the purified plant protein displayed AGT activity (data not shown), but this activity was not characterized kinetically because of technical limitations of the AGT assay. PMID:12529529 but the physiological significance of the AGT reaction for photorespiration has been disputed |
| GO:0008453 L-alanine:glyoxylate transaminase activity | IDA PMID:12529529 Alanine aminotransferase homologs catalyze the glutamate:gly... | KEEP AS NON CORE | Summary: AGT activity detected with purified recombinant GGAT2 (data not shown in the paper). Reason: Real activity of the GGAT2 protein, but not kinetically characterized and of disputed physiological weight relative to the glutamate:glyoxylate reaction. Unlike GGAT1, there is no mutant evidence for GGAT2's contribution to leaf AGT activity. Supporting Evidence: PMID:12529529 Both recombinant proteins and the purified plant protein displayed AGT activity (data not shown), but this activity was not characterized kinetically because of technical limitations of the AGT assay. PMID:12529529 Arabidopsis GGT1 and GGT2 both catalyze an AGT reaction in addition to the GGT reaction |
| GO:0008453 L-alanine:glyoxylate transaminase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic mapping from EC 2.6.1.44 / RHEA:24248. Reason: Consistent with the experimental AGT activity; non-core as for the other rows of this term. Supporting Evidence: PMID:12529529 Four aminotransferase activities were specifically associated with GGT1 and GGT2, using the substrate pairs glutamate (Glu):glyoxylate, Ala:glyoxylate, Glu:pyruvate, and Ala:2-oxoglutarate. |
| GO:0008483 transaminase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the ALAT1/2-like family signature. Reason: Correct but general. The specific, experimentally supported core activity is glutamate:glyoxylate transamination (GO:0047958 glycine:2-oxoglutarate transaminase activity), already carried by the gene. Same action as for GGAT1. Proposed replacements: glycine:2-oxoglutarate transaminase activity Supporting Evidence: file:ARATH/GGAT2/GGAT2-uniprot.txt IPR045088; ALAT1/2-like PMID:12529529 Both purified recombinant proteins catalyzed GGT, GPT, and AKT reactions |
| GO:0009507 chloroplast | HDA PMID:18431481 Sorting signals, N-terminal modifications and abundance of t... | MARK AS OVER ANNOTATED | Summary: Identification in a large-scale MS analysis of isolated chloroplast preparations. Reason: GGAT2 has no chloroplast transit peptide and ends in a PTS1. Peroxisomes co-purify with chloroplasts, the study itself considers protein contaminants in chloroplast preparations, and very little GGT activity cofractionates with chloroplasts. The detection does not show a plastid pool. Same action as for GGAT1. Following projects/ARATH/organelle-audit.md rule 3, this single proteomics hit for a protein localized elsewhere is marked over-annotated rather than removed. Supporting Evidence: PMID:18431481 This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. PMID:12529529 Peak GGT activity cofractionated with the catalase peak, very little GGT activity cofractionated with the mitochondrial and chloroplast peaks |
| GO:0009570 chloroplast stroma | HDA PMID:18633119 Quantitative proteomics of a chloroplast SRP54 sorting mutan... | MARK AS OVER ANNOTATED | Summary: Identification in quantitative chloroplast proteomes of the cpSRP54 mutant ffc1-2 and wild type. Reason: The cached record is abstract-only, but the abstract itself reports photorespiratory proteins of peroxisomes and mitochondria in the dataset, showing that the chloroplast preparations carried peroxisomal contaminants. GGAT2 has no transit peptide and ends in a PTS1, and GGT activity does not cofractionate with chloroplasts. This is consistent with the chloroplast row from a second plastid proteome (also marked over-annotated). Following projects/ARATH/organelle-audit.md rule 3, this single proteomics hit for a protein localized elsewhere is marked over-annotated rather than removed. Supporting Evidence: PMID:18633119 with an up-regulation of the Calvin cycle and the photorespiratory pathway in peroxisomes and mitochondria in young leaves PMID:12529529 GGT activity was localized exclusively to Arabidopsis peroxisomes |
| GO:0030170 pyridoxal phosphate binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the class I/II aminotransferase domain. Reason: Class I aminotransferases are PLP-dependent; the PLP-binding lysine (K291) and other catalytic residues are conserved in GGAT2. Retained as the cofactor-binding function supporting the core activity, as for GGAT1. Supporting Evidence: file:ARATH/GGAT2/GGAT2-uniprot.txt IPR004839; Aminotransferase_I/II_large PMID:12529529 residues G224, D249, K291, and R444 of GGT1 and GGT2 correspond to the conserved residues involved in PLP cofactor binding and enzymatic catalysis |
| GO:0042853 L-alanine catabolic process | IEA GO_REF:0000041 | MARK AS OVER ANNOTATED | Summary: UniPathway mapping (L-alanine degradation via transaminase pathway) based on the generic alanine aminotransferase reaction. Reason: GGAT2 can use alanine as an amino donor in vitro, but there is no evidence that it degrades alanine in vivo. Alanine breakdown, for example after hypoxia, is attributed to the mitochondrial AlaAT1 isozyme. Same action as for GGAT1. Supporting Evidence: PMID:17319845 These results indicate that the primary role of AlaAT1 is to break down alanine when it is in excess. PMID:12529529 but the physiological significance of the AGT reaction for photorespiration has been disputed |
| GO:0047958 glycine:2-oxoglutarate transaminase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of glutamate:glyoxylate (GGT) activity for the plant peroxisomal GGAT subclade; GGAT2 appears among its own sources because it carries experimental annotations for this term. Reason: Core molecular function. The GO term is written in the reverse (glycine + 2-oxoglutarate) direction of the same reversible reaction (EC 2.6.1.4) that operates toward glycine in photorespiration. Supporting Evidence: PMID:12529529 Both purified recombinant proteins catalyzed GGT, GPT, and AKT reactions |
| GO:0047958 glycine:2-oxoglutarate transaminase activity | IDA PMID:12529529 Alanine aminotransferase homologs catalyze the glutamate:gly... | ACCEPT | Summary: Purified recombinant GGAT2 catalyses the glutamate:glyoxylate (GGT) reaction with kinetics similar to GGAT1 and to the enzyme purified from leaves (Km 0.14 mM glyoxylate, 3.32 mM glutamate). Reason: Direct biochemical demonstration of the core activity on the GGAT2 protein itself. Supporting Evidence: PMID:12529529 Recombinant GGT2 was soluble when expressed in E. coli ; lysates of bacteria expressing this construct exhibited significantly increased levels of GGT, GPT, and AKT activity compared with empty vector controls PMID:12529529 These purified aminotransferases had very similar K m values for the amino acid substrates Glu and Ala, as well as the 2-oxoacid substrates glyoxylate, pyruvate, and 2-oxoglutarate. file:ARATH/GGAT2/GGAT2-uniprot.txt KM=0.14 mM for glyoxylate |
| GO:0047958 glycine:2-oxoglutarate transaminase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic mapping from EC 2.6.1.4 / RHEA:14089. Reason: Consistent with the experimentally demonstrated core activity. Supporting Evidence: PMID:12529529 Both purified recombinant proteins catalyzed GGT, GPT, and AKT reactions |
| GO:0047958 glycine:2-oxoglutarate transaminase activity | ISS PMID:12631323 Identification of photorespiratory glutamate:glyoxylate amin... | ACCEPT | Summary: Similarity-based transfer of GGAT activity from AOAT1 (GGAT1), identified by Igarashi et al. as the photorespiratory GGAT. Reason: The inference is correct and is confirmed directly by the activity of purified recombinant GGAT2 (IDA row above). Supporting Evidence: PMID:12631323 Here, we report that an alanine:2-oxoglutarate aminotransferase (AOAT)-like protein functions as GGAT in peroxisomes. PMID:12529529 Both purified recombinant proteins catalyzed GGT, GPT, and AKT reactions |
| GO:0009853 photorespiration | IC PMID:16950862 Glutamate:glyoxylate aminotransferase modulates amino acid c... | NEW | Summary: Proposed process annotation: GGAT2 is a leaf-expressed, light-induced peroxisomal enzyme that catalyses the photorespiratory glutamate:glyoxylate transamination itself, as a minor isoform partially redundant with GGAT1. Reason: GOA has photorespiration for GGAT1 but no process annotation for GGAT2 other than L-alanine catabolism. The GGAT2-specific evidence is (1) the purified recombinant protein catalyses the photorespiratory GGT reaction with a glyoxylate Km of 0.14 mM; (2) GGAT2 is identified by isoform-specific peptides in mature leaf peroxisomes, the compartment of this step; (3) its promoter is strongly active in leaves and its mRNA rises at the start of the light period while GGAT1 mRNA falls, yet leaf GGAT activity stays constant through the day. Participation test: GGAT2 does the work of the step (it is the catalyst, not a substrate). Comparator: the term is carried by the other catalysts of the C2 cycle, including GGAT1. Caveat: there is no ggat2 mutant, ggat1 knockouts lose most leaf GGAT activity and still accumulate glyoxylate in air, so GGAT2's contribution in vivo is minor and inferred from expression and biochemistry rather than genetics; hence IC rather than IMP. Supporting Evidence: PMID:16950862 GUS stain was strongly detected in leaves of both GGAT1 - and GGAT2-GUS seedlings ( Fig. 1A ), which suggests that GGAT1 and GGAT2 function as photorespiratory GGAT enzymes. PMID:16950862 However, the enzymatic activity of GGAT remained constant during the day ( Fig. 2A ), suggesting that GGAT1 and GGAT2 may function redundantly in leaf photorespiration. PMID:22303256 Whereas Ggat 2 is induced at the beginning of the photoperiod, Ggat 1 is rather repressed. However, GGAT activity in leaves remains constant during the day ( Igarashi et al., 2006 ) suggesting that both loci contribute to the total leaf GGAT content. PMID:17951448 Of the two isoforms each of Glu-glyoxylate aminotransferase (GGT) and peroxisomal malate dehydrogenase (pMDH), GGT1 (At1g23310; SKM>) and pMDH2 (At5g09660; RIx 5 HL) dominated over the second isoform, both of which were identified as well PMID:12529529 The GGT activity observed in Arabidopsis peroxisomes is likely the sum of the contributions from GGT1 and GGT2 because both catalyze this reaction. |
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Download this section (compressed HTML)Q: How much of the residual leaf GGAT activity in ggat1 knockouts is due to GGAT2, and does GGAT2 make a measurable contribution to photorespiratory glycine formation in wild-type leaves?
Q: Is GGAT2 the main GGAT isoform in roots, as the RT-PCR data of Liepman and Olsen (2003) suggest, or is GGAT1 also strongly expressed there, as GGAT1 promoter-GUS staining indicates?
Experiment: Isolate ggat2 single and ggat1 ggat2 double mutants and compare air-sensitivity, high-CO2 rescue, leaf GGAT/AGT activities and glyoxylate, glycine and serine levels, to quantify GGAT2's in vivo contribution to photorespiration.
Experiment: Express GGAT2-GFP (with and without its C-terminal SRM tripeptide) in planta to confirm PTS1-dependent peroxisomal import of GGAT2 itself.
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