Gap2 (sll1342) is the photosynthetic, phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.59) of the cyanobacterium Synechocystis sp. PCC 6803. It is a cytoplasmic homotetramer of 337-residue subunits that belongs to the type I GAPDH family and is most similar to the chloroplast GAPDH of plants, whereas its paralog Gap1 (slr0884) resembles plant cytosolic GAPDH. Gap2 uses both NADPH and NADH, and in vivo works mainly in the reductive (anabolic) direction, reducing 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate. It catalyses this step in the Calvin-Benson-Bassham (reductive pentose-phosphate) cycle in the light and in gluconeogenesis. gap2 insertion mutants cannot grow photoautotrophically or on organic acids but still grow on glucose, whereas gap1 mutants show the reciprocal phenotype; Gap1 is the NAD-specific glycolytic enzyme. In the dark or under oxidizing conditions Gap2 is held inactive in a CP12-mediated ternary complex with phosphoribulokinase. NADPH and reducing conditions release it from this complex in the light.
EXP PMID:9226260 Functional complementation of an Escherichia coli gap mutant...
ACCEPT
Summary: Purified native and recombinant Gap2 show NAD-dependent as well as NADP-dependent GAPDH activity.
Reason: The dual pyridine nucleotide specificity of Gap2 was shown for the native and the recombinant enzyme, and Koksharova et al. confirmed in cell extracts that Gap2 is the major GAPDH with dual NAD/NADP cosubstrate specificity. NAD+ use is part of the enzyme's core catalytic activity.
Summary: Phylogenetic inference of NAD-dependent phosphorylating GAPDH activity from the type I GAPDH family node.
Reason: Gap2 lies in the type I GAPDH clade and retains the catalytic Cys154 and the NAD-binding residues. Its own experimental annotation (P80505 in the WITH/FROM list) is among the descendant evidence for the node, which is expected. The IBA agrees with the direct biochemical data.
Summary: Cytoplasmic location from the UniProt subcellular-location mapping.
Reason: Gap2 is a soluble Calvin-cycle enzyme. Tagged GapDH2-eYFP is seen inside living cells, where it forms CP12-dependent complexes in the dark. Cytoplasm is the appropriate compartment for a bacterium.
These results show for the first time the CP12-dependent complex formation with GapDH2 or PRK during light/dark transitions inside living Synechocystis cells.
Summary: Family-level inference of a role in glucose metabolism.
Reason: True but general. For Gap2 the glucose-metabolism role is specifically gluconeogenesis (GO:0006094, is_a GO:0006006); gap2 mutants cannot grow on organic acids, and Gap2 works in the anabolic direction. Glycolytic glucose breakdown depends on the paralog Gap1. The Calvin-cycle role is recorded separately as a NEW annotation, because GO:0019253 is not a descendant of glucose metabolic process.
Propagation Review
Root cause:TERM SCOPING PROBLEM
Failure modes:GRANULARITY MISMATCH
Sources checked:
PANTHER:PTN001652504 · PAINT type I GAPDH family node SUPPORTS TRANSFER
Placing glucose metabolism at the type I GAPDH node is sound, and Gap2 is inside the clade. The term is true for Gap2 but too general; its glucose-metabolism role is gluconeogenesis rather than glycolysis.
While Gap2 operates in the photosynthetic Calvin cycle and in non-photosynthetic gluconeogenesis, Gap1 seems to be essential only for glycolytic glucose breakdown
While Gap2 operates in the photosynthetic Calvin cycle and in non-photosynthetic gluconeogenesis, Gap1 seems to be essential only for glycolytic glucose breakdown
Summary: UniPathway mapping placing Gap2 in glycolysis (G3P to pyruvate, step 1/5).
Reason: The UniProt pathway line is a curator inference (ECO:0000305) applied to all GAPDHs. Gap2 is the anabolic GAPDH of Synechocystis. gap2 mutants still grow on glucose, while gap1 mutants cannot, and Gap2 catabolic activity in extracts is very low. Gap2 may contribute some catabolic flux (an amphibolic role was proposed when the gene was cloned), so the term is not clearly wrong, but it misrepresents the paralog's main role.
While Gap2 operates in the photosynthetic Calvin cycle and in non-photosynthetic gluconeogenesis, Gap1 seems to be essential only for glycolytic glucose breakdown
A possible amphibolic role of the cyanobacterial GAPDH2, namely, anabolic for photosynthetic carbon assimilation and catabolic for carbohydrate degradative pathways, is discussed.
GO:0016620 oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor
IEA GO_REF:0000002
MODIFY
Summary: InterPro2GO mapping to the generic aldehyde dehydrogenase (NAD/NADP) class.
Reason: Correct but general. The specific activities are established experimentally and the gene already carries both, the NADP+ and NAD+ phosphorylating GAPDH activities (each is_a GO:0043891, is_a GO:0016620).
Summary: EC2GO mapping from the curated EC 1.2.1.59.
Reason: GO:0043891 is the GO counterpart of EC 1.2.1.59, the NAD(P)-dependent phosphorylating GAPDH. It describes the dual cosubstrate specificity of Gap2 exactly, which is what distinguishes Gap2 from the NAD-specific Gap1. The two cofactor-specific children are also accepted.
EXP PMID:9226260 Functional complementation of an Escherichia coli gap mutant...
ACCEPT
Summary: Purified native and recombinant Gap2 have NADP-dependent GAPDH activity.
Reason: NADP(H)-dependent activity is the defining feature of the photosynthetic GAPDH and is what Gap2 uses in the Calvin-Benson cycle, reducing 1,3-bisphosphoglycerate with photosynthetic NADPH. This is the core activity.
Summary: InterPro2GO mapping of NADP binding from the type I GAPDH signature.
Reason: Correct, because NADP(H) is a cosubstrate of Gap2. Cosubstrate binding is part of the catalytic activity already captured by GO:0047100, so this term is kept as non-core.
Summary: Phylogenetic inference of NAD binding from the type I GAPDH family.
Reason: Correct, because Gap2 uses NAD+ as well as NADP+ and conserves the Rossmann-fold NAD-binding residues. Cosubstrate binding is subsumed by the catalytic activity (GO:0004365), so it is non-core.
IMP PMID:9484473 Genetic and biochemical evidence for distinct key functions ...
NEW
Summary: gap2 insertion mutants cannot survive photoautotrophically, and Gap2 is the major anabolic (1,3-bisphosphoglycerate-reducing) GAPDH in Synechocystis extracts.
Reason: Participation test: Gap2 itself catalyses the reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate with NADPH, one of the steps of the Calvin-Benson-Bassham cycle. Its role is therefore catalytic and not just necessity. The mutant phenotype (no photoautotrophic growth) and the activity measurements in mutant extracts identify Gap2, not Gap1, as the isoenzyme that performs this step. Comparator check: in QuickGO, the plant chloroplast GAPDH subunits GAPA1 and GAPB (P25856, P25857) and the other CBB-cycle enzymes of cyanobacteria (PRK, RbcL/S, FBPase) carry GO:0019253. The term is not an ancestor or descendant of any other term on Gap2.
Therefore, GapDH2 is mainly part of the CBB cycle, in which it reduces G1,3BP using photosynthetic NADPH
Core Functions
NADPH-dependent reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate in the Calvin-Benson-Bassham cycle. Gap2 is the photosynthetic GAPDH of Synechocystis, held inactive in the dark by CP12-mediated complex formation with phosphoribulokinase.
The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme
Phosphorylating GAPDH activity with dual NAD/NADP specificity in the anabolic direction during gluconeogenesis. gap2 mutants cannot grow on organic acids under dim light.
Q: Does Gap2 contribute significant catabolic (glycolytic) flux in vivo under mixotrophic or heterotrophic conditions, or is its oxidative activity fully repressed (by CP12 or by the low-molecular-weight inhibitor reported by Koksharova et al.)?
Q: Is the NAD-dependent activity of Gap2 physiologically relevant in vivo, or does NADPH dominate under all conditions where Gap2 is active?
Q: Should GO represent Gap2's CP12-dependent inactivation, for example as membership of a PRK/CP12/GAPDH complex term, which no current annotation captures?
Suggested Experiments
Experiment: Use 13C-glucose labelling and metabolic flux analysis in wild type, a gap1 mutant and a gap1 mutant expressing a CP12-insensitive Gap2, to measure oxidative GAPDH flux attributable to Gap2.
Hypothesis: Gap2 carries glycolytic flux in a gap1 background under mixotrophic conditions.
Experiment: Express cofactor-specificity variants of Gap2 (NADP-only and NAD-only, by mutating the residues that bind the adenosine 2'-phosphate) in a gap2 background and compare photoautotrophic growth and CBB-cycle intermediate pools.
Hypothesis: The ratio of NADPH to NADH use by Gap2 in vivo changes with light intensity.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
NCBI PGAP names WP_010874115.1 (Gap2, sll1342) with the NCBIfam equivalog NF005641.0 (PRK07403, "type I glyceraldehyde-3-phosphate dehydrogenase"), which supplies the product name and EC 1.2.1.- but no GO terms. PGAP passes on GO only from the naming model, so the protein receives no GO annotation at all. The second matching equivalog, TIGR01534, has the same product name and maps GO:0006006, GO:0016620, GO:0050661 and GO:0051287, but its GO is discarded because it was not the naming model. The single PGAP prediction, EC 1.2.1.-, is correct but less precise than the experimentally supported EC 1.2.1.59 (NAD(P)-dependent phosphorylating GAPDH) curated in UniProt from PMID:9226260. Neither PGAP model distinguishes the NADP-using Calvin-cycle Gap2 from the NAD-specific glycolytic Gap1, although EC 1.2.1.59 could be assigned at the cyanobacterial/plastid Gap2-subfamily level.
LSP — Less precise than existing annotation Review score: 2/2
Prediction method: NCBI PGAP · Version: GCF_000009725.1-RS_2026_01_07 (live); GO from WP_ record · PMID:33270901
Review rationale: EC 1.2.1.- (oxidoreductase acting on aldehydes, NAD+ or NADP+ acceptor) is correct for Gap2 but less precise than its known activity. Purified native and recombinant Gap2 catalyse the phosphorylating oxidation of glyceraldehyde 3-phosphate with either NAD+ or NADP+, which is EC 1.2.1.59, and UniProt curates both reactions with experimental evidence. The incomplete EC comes from the naming model NF005641, which carries EC 1.2.1.- because the type I GAPDH family includes NAD-specific (EC 1.2.1.12), NADP-specific (EC 1.2.1.13) and dual-specificity (EC 1.2.1.59) members. No error type applies, because the prediction is not wrong, only underspecified.