HPR2 (At1g79870; also called HPPR2) is a cytosolic NAD(P)H-dependent hydroxypyruvate reductase of the D-isomer specific 2-hydroxyacid dehydrogenase family (GyaR subfamily). It reduces 3-hydroxypyruvate to D-glycerate, preferring NADPH over NADH, and also reduces glyoxylate to glycolate and 4-hydroxyphenylpyruvate to 4-hydroxyphenyllactate with lower efficiency; its activity is strongly inhibited by oxalate. In leaves HPR2 provides a cytosolic bypass to the peroxisomal NADH-dependent hydroxypyruvate reductase HPR1 in the photorespiratory C2 cycle, converting hydroxypyruvate that leaves the peroxisome into glycerate for return to the chloroplast. hpr2 single mutants are nearly wild type, but hpr1 hpr2 double mutants are air-sensitive and recover at elevated CO2, showing that at least one of the two enzymes is needed for normal growth in air. HPR2 also contributes to formation of the tyrosine-derived metabolite 4-hydroxyphenyllactate, and binds salicylic acid in vitro.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IDA PMID:18952776 A cytosolic pathway for the conversion of hydroxypyruvate to... | MODIFY | Summary: Timm et al. 2008 found that GFP-HPR2 showed no organelle targeting and that the enzyme is predicted to be cytosolic; a later protoplast GFP study placed it in the cytosol. Reason: Correct but less specific than the evidence supports. HPR2 lacks any targeting signal and two GFP studies place it in the cytosol, which the gene already carries (GO:0005829, IDA PMID:30233632). Replace cytoplasm with cytosol. Proposed replacements: cytosol Supporting Evidence: PMID:18952776 Initial analyses with green fluorescent protein–HPR2 fusion proteins in transgenic Arabidopsis also gave no indication for organelle targeting PMID:30233632 HPPR2, HPPR3, and HPPR4 are localized in the cytosol. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | MODIFY | Summary: UniProt SubCell mapping of the curated "Cytoplasm" location derived from Timm et al. 2008. Reason: Correct but general; the experimental evidence supports the more specific cytosol (GO:0005829), which the gene already carries. Proposed replacements: cytosol Supporting Evidence: PMID:30233632 HPPR2, HPPR3, and HPPR4 are localized in the cytosol. |
| GO:0005737 cytoplasm | ISM GO_REF:0000122 | MODIFY | Summary: AtSubP sequence-based prediction of cytoplasmic location. Reason: The prediction agrees with experiment, but the experimentally supported location is the cytosol (GO:0005829), which the gene already carries. Proposed replacements: cytosol Supporting Evidence: PMID:30233632 HPPR2, HPPR3, and HPPR4 are localized in the cytosol. |
| GO:0005739 mitochondrion | HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati... | MARK AS OVER ANNOTATED | Summary: High-throughput size-exclusion profiling of detergent-solubilized membrane fractions from Arabidopsis leaves; HPR2 is not discussed in the text and the mitochondrial call comes from dataset-level assignment. Reason: HPR2 has no mitochondrial targeting sequence and two independent GFP studies show cytosolic localization. The study itself reports that many soluble proteins partition between cytosolic and membrane-associated pools, so detection in an organelle membrane fraction is weak evidence of residence. Not removed because the supplementary data behind the call were not inspected. Supporting Evidence: PMID:28887381 Over 150 proteins had a complicated localization pattern, and were clearly partitioned between cytosolic and membrane-associated pools. PMID:30233632 HPPR2, HPPR3, and HPPR4 are localized in the cytosol. |
| GO:0005829 cytosol | IDA PMID:30233632 Characterization of Arabidopsis thaliana Hydroxyphenylpyruva... | ACCEPT | Summary: N-terminal GFP-HPPR2 fusions expressed in mesophyll protoplasts were cytosolic. Reason: Direct localization evidence, consistent with the absence of targeting signals and with the earlier GFP and biochemical work defining HPR2 as the cytosolic hydroxypyruvate reductase. This is the core location. Supporting Evidence: PMID:30233632 HPPR2, HPPR3, and HPPR4 are localized in the cytosol. |
| GO:0008465 hydroxypyruvate reductase (NADH) activity | IEA GO_REF:0000116 | KEEP AS NON CORE | Summary: Rhea-based mapping of the NADH-dependent hydroxypyruvate reduction reaction recorded by UniProt from Timm et al. 2008. Reason: Recombinant HPR2 does reduce hydroxypyruvate with NADH, but at about one quarter of the NADPH rate; the NADH reaction is a secondary activity. The core activity is captured by GO:0016618 (NAD(P)H) and GO:0120509 (NADPH). Supporting Evidence: PMID:18952776 Tag-purified recombinant At1g79870 was most active in the presence of NADPH and hydroxypyruvate, whereas NADH-dependent rates were about four times lower. |
| GO:0009853 photorespiration | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA-based assignment of photorespiration. Reason: Correct. HPR2 catalyses the reduction of photorespiratory hydroxypyruvate in a cytosolic bypass of peroxisomal HPR1, and hpr1 hpr2 double mutants are air-sensitive and rescued by elevated CO2. The more specific GO:0009854 (part_of photorespiration) is also accepted; this pathway-level term remains informative and is the one the core function uses, so that every C2-cycle step and ammonia refixation share one process term. Supporting Evidence: PMID:18952776 We have identified a second hydroxypyruvate reductase, HPR2, and present genetic and biochemical evidence that the enzyme provides a cytosolic bypass to the photorespiratory core cycle |
| GO:0009854 oxidative photosynthetic carbon pathway | IMP PMID:18952776 A cytosolic pathway for the conversion of hydroxypyruvate to... | ACCEPT | Summary: hpr2 knockouts accumulate hydroxypyruvate and show altered gas exchange; hpr1 hpr2 double knockouts are air-sensitive and recover in 1% CO2, and 13C labelling shows impaired hydroxypyruvate-to-glycerate conversion. Reason: HPR2 catalyses a step of the C2 carbon pathway (hydroxypyruvate to glycerate), and the mutant analysis shows that it carries photorespiratory flux in vivo. This is the most specific appropriate process term (part_of GO:0009853). The core function nonetheless uses the broader GO:0009853 photorespiration, so that every C2-cycle step and ammonia refixation share one process term (see modules/photorespiration_c2_cycle.yaml). Supporting Evidence: PMID:18952776 Growth under the nonphotorespiratory conditions of air enriched with 1% CO 2 restored the phenotype to normal PMID:18952776 From these kinetic data, which support and extend the results from our analysis of steady state metabolite levels, we infer a contribution of HPR2 to photosynthetic–photorespiratory metabolism not only in HPR1-deficient plants but also in wild-type plants. |
| GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the D-isomer 2-hydroxyacid dehydrogenase catalytic domain. Reason: Correct but very general. The specific activities supported by biochemistry are hydroxypyruvate reductase [NAD(P)H] activity (core) and glyoxylate reductase (NADPH) activity, both already carried by the gene. Proposed replacements: hydroxypyruvate reductase [NAD(P)H] activity glyoxylate reductase (NADPH) activity Supporting Evidence: file:ARATH/HPR2/HPR2-uniprot.txt Belongs to the D-isomer specific 2-hydroxyacid dehydrogenase family. GyaR subfamily. PMID:18952776 Tag-purified recombinant At1g79870 was most active in the presence of NADPH and hydroxypyruvate, whereas NADH-dependent rates were about four times lower. |
| GO:0016618 hydroxypyruvate reductase [NAD(P)H] activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference from a GyaR-subfamily ancestral node; HPR2 itself (AT1G79870) is among the experimentally characterized descendants. Reason: Consistent with direct biochemical characterization of HPR2. The NAD(P)H term accurately describes an enzyme that uses both cofactors, and HPR2 lies within the GyaR clade that carries the function. Supporting Evidence: PMID:18952776 Tag-purified recombinant At1g79870 was most active in the presence of NADPH and hydroxypyruvate, whereas NADH-dependent rates were about four times lower. |
| GO:0016618 hydroxypyruvate reductase [NAD(P)H] activity | IDA PMID:18952776 A cytosolic pathway for the conversion of hydroxypyruvate to... | ACCEPT | Summary: HPR2 was purified from hpr1 leaves as the NADPH-dependent hydroxypyruvate reductase activity, and recombinant At1g79870 reduced hydroxypyruvate with NADPH and, more slowly, NADH. Reason: Direct enzymatic evidence; this is the core molecular function of HPR2. Supporting Evidence: PMID:18952776 Tag-purified recombinant At1g79870 was most active in the presence of NADPH and hydroxypyruvate, whereas NADH-dependent rates were about four times lower. PMID:18952776 These combined data from protein identification, interaction with antibodies raised against the spinach enzyme, and catalytic parameters establish At1g70870 as the cytosolic NAD(P)H-dependent hydroxypyruvate reductase, HPR2, in Arabidopsis . |
| GO:0016618 hydroxypyruvate reductase [NAD(P)H] activity | IDA PMID:30233632 Characterization of Arabidopsis thaliana Hydroxyphenylpyruva... | ACCEPT | Summary: Recombinant HPPR2 (HPR2) reduced hydroxypyruvic acid with its highest catalytic efficiency among the substrates tested. Reason: Independent kinetic confirmation of the core activity. Supporting Evidence: PMID:30233632 HPPR2 had the highest affinity for pHPP, although its highest catalytic efficiency was detected with hydroxypyruvic acid. |
| GO:0016618 hydroxypyruvate reductase [NAD(P)H] activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA from EC 1.1.1.81 and an ARBA rule. Reason: Agrees with the experimentally established core activity. Supporting Evidence: PMID:18952776 Tag-purified recombinant At1g79870 was most active in the presence of NADPH and hydroxypyruvate, whereas NADH-dependent rates were about four times lower. |
| GO:0030267 glyoxylate reductase (NADPH) activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT inference of glyoxylate reductase (NADPH) activity for the GyaR-subfamily node, with HPR2 among the experimentally characterized descendants. Reason: HPR2 reduces glyoxylate in vitro, so the activity is real and inherited, but glyoxylate is a poorer substrate than hydroxypyruvate and the physiologically demonstrated role is hydroxypyruvate reduction; glyoxylate is produced and largely consumed in the peroxisome. Non-core. Supporting Evidence: PMID:18952776 The enzyme also accepts glyoxylate as a substrate, but at a considerably lower efficiency in the NADH-driven reaction. |
| GO:0030267 glyoxylate reductase (NADPH) activity | IDA PMID:18952776 A cytosolic pathway for the conversion of hydroxypyruvate to... | KEEP AS NON CORE | Summary: Recombinant HPR2 reduced glyoxylate with NADPH (Table 1 of Timm et al. 2008). Reason: Demonstrated secondary activity of a promiscuous 2-hydroxyacid dehydrogenase; the characterized physiological role is hydroxypyruvate reduction. Supporting Evidence: PMID:18952776 The enzyme also accepts glyoxylate as a substrate, but at a considerably lower efficiency in the NADH-driven reaction. file:ARATH/HPR2/HPR2-uniprot.txt Catalyzes the NADPH-dependent reduction of glyoxylate and hydroxypyruvate (HP) into glycolate and glycerate in the cytoplasm |
| GO:0030267 glyoxylate reductase (NADPH) activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Combined IEA from EC 1.1.1.79 / RHEA:10992. Reason: Consistent with the measured secondary glyoxylate reductase activity; non-core. Supporting Evidence: PMID:18952776 The enzyme also accepts glyoxylate as a substrate, but at a considerably lower efficiency in the NADH-driven reaction. |
| GO:0047995 (2R)-hydroxyphenylpyruvate reductase [NAD(P)H] activity | IDA PMID:30233632 Characterization of Arabidopsis thaliana Hydroxyphenylpyruva... | KEEP AS NON CORE | Summary: Recombinant HPPR2 (HPR2) converted 4-hydroxyphenylpyruvate (pHPP) to 4-hydroxyphenyllactate (pHPL), confirmed by LC-MS/MS; hppr2 mutants contain less pHPL. Reason: A real secondary activity with in planta support for a contribution to pHPL formation from tyrosine, but HPR2's catalytic efficiency is highest with hydroxypyruvate and its main physiological role is photorespiratory. Supporting Evidence: PMID:30233632 While HPR1 and HPPR4 did not exhibit HPPR activity under our assay conditions, HPPR2 and HPPR3 converted pHPP to a product (Figure 3B), which was confirmed to be 4-hydroxyphenyllactic acid (pHPL) PMID:30233632 These findings present strong evidence that TAT1, HPPR2, and HPPR3 contribute to pHPL production in Arabidopsis. |
| GO:0051287 NAD binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the NAD(P)-binding domain of 2-hydroxyacid dehydrogenases. Reason: HPR2 uses NADH as well as (preferentially) NADPH as cosubstrate, so NAD binding is true; cofactor binding is a component of the catalytic activity rather than a separate function. Supporting Evidence: PMID:18952776 Tag-purified recombinant At1g79870 was most active in the presence of NADPH and hydroxypyruvate, whereas NADH-dependent rates were about four times lower. |
| GO:0120509 hydroxypyruvate reductase (NADPH) activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea-based mapping of the NADPH-dependent hydroxypyruvate reduction reaction. Reason: NADPH-dependent hydroxypyruvate reduction is the preferred reaction of HPR2 and distinguishes it from NADH-dependent peroxisomal HPR1. Core, alongside the parent GO:0016618. Supporting Evidence: PMID:18952776 Tag-purified recombinant At1g79870 was most active in the presence of NADPH and hydroxypyruvate, whereas NADH-dependent rates were about four times lower. PMID:21205613 In addition, a NADPH-dependent cytosolic HPR2 provides an efficient extraperoxisomal bypass. |
| GO:1901149 salicylic acid binding | HDA PMID:25628632 Identification of multiple salicylic acid-binding proteins u... | KEEP AS NON CORE | Summary: HPR2 was a candidate SA-binding protein from a protein-microarray screen and was then confirmed with recombinant protein by SA-inhibitable 3AESA binding (SPR) and SA-competable 4AzSA photo-crosslinking. Reason: Binding met the authors' two-assay criterion, so the in vitro observation is supported. No effect of SA on HPR2 activity and no physiological role for the binding were shown, so it is not part of the core function. Supporting Evidence: PMID:25628632 Five of these eight, including glutathione peroxidase 2 (GPX2), GSR2, hydroxypyruvate reductase 2 (HPR2), UDP-glucose 4-epimerase 2 (UGE2), and RBC small subunit 1A (RBCS1A), bound 3AESA and crosslinked to 4AzSA in an SA-inhibitable manner, indicating that they are SABPs |
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Download this section (compressed HTML)Q: Under which natural conditions (light intensity, temperature, peroxisomal NADH supply) does the cytosolic HPR2 route carry a substantial fraction of photorespiratory hydroxypyruvate flux in wild-type plants?
Q: Does salicylic acid binding alter HPR2 catalytic activity at physiological SA concentrations, and is this relevant to SA-regulated photorespiratory or redox processes?
Q: How much of the in vivo 4-hydroxyphenyllactate formation depends on HPR2 relative to HPPR3, and does this have any physiological function in Arabidopsis, which does not accumulate rosmarinic acid?
Experiment: 13C or 14C flux analysis of hydroxypyruvate reduction in wild type, hpr1 and hpr2 under high light and high temperature to quantify the share of photorespiratory flux passing through cytosolic HPR2.
Experiment: Steady-state kinetics of recombinant HPR2 in the presence of salicylic acid to test whether SA binding inhibits or activates hydroxypyruvate reduction.
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