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Hydroxypyruvate (HP), the key intermediate of photorespiration, is converted to glycerate via the catalysis of hydroxypyruvate reductases (HPRs) with NADH/NADPH as cofactors. The non-lethal phenotype resulting from HPR defects allows for the use of mutants to investigate interactions between photorespiration and other cellular processes, facilitating the establishment of plant chassis with compromised photorespiration. Considering that establishing a plant chassis of the HPR- series would provide great potential in promoting plant synthetic biology to tackle future challenges, the genome-wide identification and bioinformatics analysis of HPR gene family in N. benthamiana are imperative but remains to be solved. In this study, 12 N. benthamiana hydroxypyruvate reductases (NbHPRs) were identified from a genome-wide study. These genes could be classified into three subclasses by phylogenetic analysis, and conserved gene structures or motif compositions were identified in each subclass. A variety of signal-sensing elements were identified in the HPRs promoter regions indicating their regulation by multiple potential transcription factors such as C2H2 proteins. Quantitative real-time PCR (qRTPCR) results further demonstrated the higher expression levels of NbHPRs in functional and young leaves compared to other organs. Subsequently, we confirmed the subcellular localization of NbHPRs with transient expression analysis, which suggests their different functions. Moreover, the relative expression level of the gene under nitrogen (N) treatment was assessed through qRTPCR analysis. These works will offer valuable insights into elucidating the function and mechanism of HPRs in N. benthamiana, thus illuminating the strategies for introducing artificial carbon fixation pathways to tackle future challenges with the modification on photorespiration.

It has been shown repeatedly that exposure to elevated atmospheric CO2 causes an increased C/N ratio of plant biomass that could result from either increased carbon or – in relation to C acquisition - reduced nitrogen assimilation. Possible reasons for diminished nitrogen assimilation are controversial, but an impact of reduced photorespiration at elevated CO2 has frequently been implied. Using a mutant defective in peroxisomal hydroxy-pyruvate reductase (hpr1-1) that is hampered in photorespiratory turnover, we show that indeed, photorespiration stimulates the glutamine-synthetase 2 (GS) / glutamine-oxoglutarate-aminotransferase (GOGAT) cycle, which channels ammonia into amino acid synthesis. However, mathematical flux simulations demonstrated that nitrate assimilation was not reduced at elevated CO2, pointing to a dilution of nitrogen containing compounds by assimilated carbon at elevated CO2. The massive growth reduction in the hpr1-1 mutant does not appear to result from nitrogen starvation. Model simulations yield evidence for a loss of cellular energy that is consumed in supporting high flux through the GS/GOGAT cycle that results from inefficient removal of photorespiratory intermediates. This causes a futile cycling of glycolate and hydroxy-pyruvate. In addition to that, accumulation of serine and glycine as well as carboxylates in the mutant creates a metabolic imbalance that could contribute to growth reduction. [ABSTRACT FROM AUTHOR]

It has been shown repeatedly that exposure to elevated atmospheric CO2 causes an increased C/N ratio of plant biomass that could result from either increased carbon or – in relation to C acquisition - reduced nitrogen assimilation. Possible reasons for diminished nitrogen assimilation are controversial, but an impact of reduced photorespiration at elevated CO2 has frequently been implied. Using a mutant defective in peroxisomal hydroxy-pyruvate reductase (hpr1-1) that is hampered in photorespiratory turnover, we show that indeed, photorespiration stimulates the glutamine-synthetase 2 (GS) / glutamine-oxoglutarate-aminotransferase (GOGAT) cycle, which channels ammonia into amino acid synthesis. However, mathematical flux simulations demonstrated that nitrate assimilation was not reduced at elevated CO2, pointing to a dilution of nitrogen containing compounds by assimilated carbon at elevated CO2. The massive growth reduction in the hpr1-1 mutant does not appear to result from nitrogen starvation. Model simulations yield evidence for a loss of cellular energy that is consumed in supporting high flux through the GS/GOGAT cycle that results from inefficient removal of photorespiratory intermediates. This causes a futile cycling of glycolate and hydroxy-pyruvate. In addition to that, accumulation of serine and glycine as well as carboxylates in the mutant creates a metabolic imbalance that could contribute to growth reduction.

Photorespiration plays an important role in maintaining normal physiological metabolism in higher plants and other oxygenic organisms, such as algae. The unicellular eukaryotic organism Chlamydomonas is reported to have a photorespiration system different from that in higher plants, and only two out of nine genes encoding photorespiratory enzymes have been experimentally characterized. Hydroxypyruvate reductase (HPR), which is responsible for the conversion of hydroxypyruvate into glycerate, is poorly understood and not yet explored in Chlamydomonas. To identify the candidate genes encoding hydroxypyruvate reductases in Chlamydomonas (CrHPR) and uncover their elusive functions, we performed sequence comparison, enzyme activity measurement, subcellular localization, and analysis of knockout/knockdown strains. Together, we identify five proteins to be good candidates for CrHPRs, all of which are detected with the activity of hydroxypyruvate reductase. CrHPR1, a nicotinamide adenine dinucleotide (NADH)-dependent enzyme in mitochondria, may function as the major component of photorespiration. Its deletion causes severe photorespiratory defects. CrHPR2 takes part in the cytosolic bypass of photorespiration as the compensatory pathway of CrHPR1 for the reduction of hydroxypyruvate. CrHPR4, with NADH as the cofactor, may participate in photorespiration by acting as the chloroplastidial glyoxylate reductase in glycolate-quinone oxidoreductase system. Therefore, the results reveal that CrHPRs are far more complex than previously recognized and provide a greatly expanded knowledge base for studies to understand how CrHPRs perform their functions in photorespiration. These will facilitate both modification of photorespiration and genetic engineering for crop improvement by synthetic biology.

Photorespiration plays an important role in maintaining normal physiological metabolism in higher plants and other oxygenic organisms, such as algae. The unicellular eukaryotic organism Chlamydomonas is reported to have a photorespiration system different from that in higher plants, and only two out of nine genes encoding photorespiratory enzymes have been experimentally characterized. Hydroxypyruvate reductase (HPR), which is responsible for the conversion of hydroxypyruvate into glycerate, is poorly understood and not yet explored in Chlamydomonas. To identify the candidate genes encoding hydroxypyruvate reductases in Chlamydomonas (CrHPR) and uncover their elusive functions, we performed sequence comparison, enzyme activity measurement, subcellular localization, and analysis of knockout/knockdown strains. Together, we identify five proteins to be good candidates for CrHPRs, all of which are detected with the activity of hydroxypyruvate reductase. CrHPR1, a nicotinamide adenine dinucleotide (NADH)-dependent enzyme in mitochondria, may function as the major component of photorespiration. Its deletion causes severe photorespiratory defects. CrHPR2 takes part in the cytosolic bypass of photorespiration as the compensatory pathway of CrHPR1 for the reduction of hydroxypyruvate. CrHPR4, with NADH as the cofactor, may participate in photorespiration by acting as the chloroplastidial glyoxylate reductase in glycolate-quinone oxidoreductase system. Therefore, the results reveal that CrHPRs are far more complex than previously recognized and provide a greatly expanded knowledge base for studies to understand how CrHPRs perform their functions in photorespiration. These will facilitate both modification of photorespiration and genetic engineering for crop improvement by synthetic biology. [ABSTRACT FROM AUTHOR]

Abstract Background Primary hyperoxaluria type 2 is a rare monogenic disorder inherited in an autosomal recessive pattern. It results from the absence of the enzyme glyoxylate reductase/hydroxypyruvate reductase (GRHPR). As a consequence of deficient enzyme activity, excessive amounts of oxalate and L-glycerate are excreted in the urine, and are a source for the formation of calcium oxalate stones that result in recurrent nephrolithiasis and less frequently nephrocalcinosis. Case presentation We report a case of a 10-month-old patient diagnosed with urolithiasis. Screening of inborn errors of metabolism, including the performance of GC/MS urine organic acid profiling and HPLC amino acid profiling, showed abnormalities, which suggested deficiency of GRHPR enzyme. Additional metabolic disturbances observed in the patient led us to seek other genetic determinants and the elucidation of these findings. Besides the elevated excretion of 3-OH-butyrate, adipic acid, which are typical marks of ketosis, other metabolites such as 3-aminoisobutyric acid, 3-hydroxyisobutyric acid, 3-hydroxypropionic acid and 2-ethyl-3-hydroxypropionic acids were observed in increased amounts in the urine. Direct sequencing of the GRHPR gene revealed novel mutation, described for the first time in this article c.454dup (p.Thr152Asnfs*39) in homozygous form. The frequent nucleotide variants were found in AGXT2 gene. Conclusions The study presents metabolomic and molecular-genetic findings in a patient with PH2. Mutation analysis broadens the allelic spectrum of the GRHPR gene to include a novel c.454dup mutation that causes the truncation of the GRHPR protein and loss of its two functional domains. We also evaluated whether nucleotide variants in the AGXT2 gene could influence the biochemical profile in PH2 and the overproduction of metabolites, especially in ketosis. We suppose that some metabolomic changes might be explained by the inhibition of the MMSADH enzyme by metabolites that increase as a consequence of GRHPR and AGXT2 enzyme deficiency. Several facts support an assumption that catabolic conditions in our patient could worsen the degree of hyperoxaluria and glyceric aciduria as a consequence of the elevated production of free amino acids and their intermediary products.

Hydroxypyruvate reductase (HPR; EC 1.1.1.81) activity is integral to the photorespiratory pathway. Within photorespiration, HPR catalyzes the reduction of hydroxypyruvate, a product of the serine:glyoxylate aminotransferase reaction to glycerate, a substrate for glycerate kinase, using NADH as cofactor. Here we detail a spectrophotometric assay for measuring HPR activity in vitro by following the consumption of NADH at 340 nm., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Photorespiration, an essential metabolic component, is a classic example of interactions between the intracellular compartments of a plant cell: the chloroplast, peroxisome, mitochondria, and cytoplasm. The photorespiratory pathway is often modulated by abiotic stress and is considered an adaptive response. Monitoring the patterns of key enzymes located in different subcellular components would be an ideal approach to assessing the modulation of the photorespiratory metabolism under abiotic stress. This chapter describes the procedures for assaying several individual enzyme activities of key photorespiratory enzymes and evaluating their response to oxidative/photooxidative stress. It is essential to ascertain the presence of stress in the experimental material. Therefore, procedures for typical abiotic stress induction in leaves by highlighting without or with menadione (an oxidant that targets mitochondria) are also included., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

To measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells. They can be rapidly purified as histidine-tagged recombinant proteins by immobilized metal affinity chromatography using Ni 2+ -NTA-agarose. This chapter describes protocols to purify several Arabidopsis thaliana His-tagged recombinant photorespiratory enzymes (phosphoglycolate phosphatase, glycolate oxidase, and hydroxypyruvate reductase) from Escherichia coli cell cultures using two bacterial strain-plasmid systems: BL21(DE3)-pET and LMG194-pBAD., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Photorespiration is an integral component of plant primary metabolism. Accordingly, it has been often observed that impairing the photorespiratory flux negatively impacts other cellular processes. In this study, the metabolic acclimation of the Arabidopsisthaliana wild type was compared with the hydroxypyruvate reductase 1 (HPR1; hpr1) mutant, displaying only a moderately reduced photorespiratory flux. Plants were analyzed during development and under varying photoperiods with a combination of non-targeted and targeted metabolome analysis, as well as 13C- and 14C-labeling approaches. The results showed that HPR1 deficiency is more critical for photorespiration during the vegetative compared to the regenerative growth phase. A shorter photoperiod seems to slowdown the photorespiratory metabolite conversion mostly at the glycerate kinase and glycine decarboxylase steps compared to long days. It is demonstrated that even a moderate impairment of photorespiration severely reduces the leaf-carbohydrate status and impacts on sulfur metabolism. Isotope labeling approaches revealed an increased CO2 release from hpr1 leaves, most likely occurring from enhanced non-enzymatic 3-hydroxypyruvate decarboxylation and a higher flux from serine towards ethanolamine through serine decarboxylase. Collectively, the study provides evidence that the moderate hpr1 mutant is an excellent tool to unravel the underlying mechanisms governing the regulation of metabolic linkages of photorespiration with plant primary metabolism.

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed. [ABSTRACT FROM AUTHOR]

Background: Primary hyperoxaluria type 2 is a rare monogenic disorder inherited in an autosomal recessive pattern. It results from the absence of the enzyme glyoxylate reductase/hydroxypyruvate reductase (GRHPR). As a consequence of deficient enzyme activity, excessive amounts of oxalate and L-glycerate are excreted in the urine, and are a source for the formation of calcium oxalate stones that result in recurrent nephrolithiasis and less frequently nephrocalcinosis. Case presentation: We report a case of a 10-month-old patient diagnosed with urolithiasis. Screening of inborn errors of metabolism, including the performance of GC/MS urine organic acid profiling and HPLC amino acid profiling, showed abnormalities, which suggested deficiency of GRHPR enzyme. Additional metabolic disturbances observed in the patient led us to seek other genetic determinants and the elucidation of these findings. Besides the elevated excretion of 3-OH-butyrate, adipic acid, which are typical marks of ketosis, other metabolites such as 3- aminoisobutyric acid, 3-hydroxyisobutyric acid, 3-hydroxypropionic acid and 2-ethyl-3-hydroxypropionic acids were observed in increased amounts in the urine. Direct sequencing of the GRHPR gene revealed novel mutation, described for the first time in this article c.454dup (p.Thr152Asnfs*39) in homozygous form. The frequent nucleotide variants were found in AGXT2 gene. Conclusions: The study presents metabolomic and molecular-genetic findings in a patient with PH2. Mutation analysis broadens the allelic spectrum of the GRHPR gene to include a novel c.454dup mutation that causes the truncation of the GRHPR protein and loss of its two functional domains. We also evaluated whether nucleotide variants in the AGXT2 gene could influence the biochemical profile in PH2 and the overproduction of metabolites, especially in ketosis. We suppose that some metabolomic changes might be explained by the inhibition of the MMSADH enzyme by metabolites that increase as a consequence of GRHPR and AGXT2 enzyme deficiency. Several facts support an assumption that catabolic conditions in our patient could worsen the degree of hyperoxaluria and glyceric aciduria as a consequence of the elevated production of free amino acids and their intermediary products. [ABSTRACT FROM AUTHOR]

Mutations in the photorespiration pathway display a lethal phenotype in atmospheric air, which can be fully recovered by elevated CO2. An exception is that mutants of peroxisomal hydroxypyruvate reductase (HPR1) do not have this phenotype, indicating the presence of cytosolic bypass in the photorespiration pathway. In this study, we constructed overexpression of the OsHPR1 gene and RNA interference plants of OsHPR1 and OsHPR2 genes in rice (Oryza sativa L. cv. Zhonghua 11). Results from reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and enzyme assays showed that HPR1 activity changed significantly in corresponding transgenic lines without any effect on HPR2 activity, which is the same for HPR2. However, metabolite analysis and the serine glyoxylate aminotransferase (SGAT) activity assay showed that the metabolite flux of photorespiration was disturbed in RNAi lines of both HPR genes. Furthermore, HPR1 and HPR2 proteins were located to the peroxisome and cytosol, respectively, by transient expression experiment. Double mutant hpr1-hpr2 was generated by crossing individual mutant of hpr1 and hpr2. The phenotypes of all transgenic lines were determined in ambient air and CO2-elevated air. The phenotype typical of photorespiration mutants was observed only where activity of both HPR1 and HPR2 were downregulated in the same line. These findings demonstrate that two hydroxypyruvate reductases encoded by OsHPR1 and OsHPR2 are involved in photorespiratory metabolism in rice. [ABSTRACT FROM AUTHOR]

Abstract: NADPH-dependent glyoxylate reductases from Arabidopsis thaliana (AtGLYR) convert both glyoxylate and succinic semialdehyde into their corresponding hydroxyacid equivalents. The primary sequence of cytosolic AtGLYR1 reveals several sequence elements that are consistent with the β-HAD (β-hydroxyacid dehydrogenase) protein family, whose members include 3-hydroxyisobutyrate dehydrogenase, tartronate semialdehyde reductase and 6-phosphogluconate dehydrogenase. Here, site-directed mutagenesis was utilized to identify catalytically important amino acid residues for glyoxylate reduction in AtGLYR1. Kinetic studies and binding assays established that Lys170 is essential for catalysis, Phe231, Asp239, Ser121 and Thr95 are more important in substrate binding than in catalysis, and Asn174 is more important in catalysis. The low activity of the mutant enzymes precluded kinetic studies with succinic semialdehyde. The crystal structure of AtGLYR1 in the absence of substrate was solved to 2.1Å by molecular replacement using a previously unrecognized member of the β-HAD family, cytokine-like nuclear factor, thereby enabling the 3-D structure of the protein to be modeled with substrate and co-factor. Structural alignment of AtGLYR1 with β-HAD family members provided support for the essentiality of Lys170, Phe173, Asp239, Ser121, Asn174 and Thr95 in the active site and preliminary support for an acid/base catalytic mechanism involving Lys170 as the general acid and a conserved active-site water molecule. This information established that AtGLYR1 is a member of the β-HAD protein family. Sequence and activity comparisons indicated that AtGLYR1 and the plastidial AtGLYR2 possess structural features that are absent in Arabidopsis hydroxypyruvate reductases and probably account for their stronger preference for glyoxylate over hydroxypyruvate. [Copyright &y& Elsevier]

Abstract: Background: Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported. Methods: We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches. Results: Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H2O2, NO, GSH and peroxynitrite showed that the ONOO− molecule caused the highest inhibition of activity (51% at 5mM SIN-1), with 5mM H2O2 having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite. Conclusion: These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function. General significance: This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions. [Copyright &y& Elsevier]

Recycling of carbon by the photorespiratory pathway involves enzymatic steps in the chloroplast, mitochondria, and peroxisomes. Most of these reactions are essential for plants growing under ambient CO concentrations. However, some disruptions of photorespiratory metabolism cause subtle phenotypes in plants grown in air. For example, Arabidopsis thaliana lacking both of the peroxisomal malate dehydrogenase genes ( pmdh1pmdh2) or hydroxypyruvate reductase ( hpr1) are viable in air and have rates of photosynthesis only slightly lower than wild-type plants. To investigate how disruption of the peroxisomal reduction of hydroxypyruvate to glycerate influences photorespiratory carbon metabolism we analyzed leaf gas exchange in A. thaliana plants lacking peroxisomal HPR1 expression. In addition, because the lack of HPR1 could be compensated for by other reactions within the peroxisomes using reductant supplied by PMDH a triple mutant lacking expression of both peroxisomal PMDH genes and HPR1 ( pmdh1pmdh2hpr1) was analyzed. Rates of photosynthesis under photorespiratory conditions (ambient CO and O concentrations) were slightly reduced in the hpr1 and pmdh1pmdh2hpr1 plants indicating other reactions can help bypass this disruption in the photorespiratory pathway. However, the CO compensation points (Γ) increased under photorespiratory conditions in both mutants indicating changes in photorespiratory carbon metabolism in these plants. Measurements of Γ*, the CO compensation point in the absence of mitochondrial respiration, and the CO released per Rubisco oxygenation reaction demonstrated that the increase in Γ in the hpr1 and pmdh1pmdh2hpr1 plants is not associated with changes in mitochondrial respiration but with an increase in the non-respiratory CO released per Rubisco oxygenation reaction. [ABSTRACT FROM AUTHOR]

Methylobacterium sp. MB200 capable of producing glyoxylate from methanol was obtained by enrichment culture using a medium containing methanol as the sole carbon source. A hpr gene that encodes a hydroxypyruvate reductase (HPR) was cloned from this strain and was ligated into the vector pLAFR3 to obtain the recombinant plasmid pLAFRh, which was transferred into M. sp. MB200 to generate an recombinant strain MB201. Homologous expression of hpr under the control of the lacZ promoter led to the enhanced glyoxylate accumulation in cultures of Methylobacterium sp MB201. The yield of glyoxylate reached 14.38 mg/mL, representing nearly a twofold increase when compared with the wild-type strain. [ABSTRACT FROM AUTHOR]

Abstract: Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 Å resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme+NADPH+reduced substrate) complex, and the other a binary (enzyme+NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2. [Copyright &y& Elsevier]

• Background and Aims Photorespiration occurs in C4 plants, although rates are small compared with C3 plants. The amount of glycine decarboxylase in the bundle sheath (BS) varies among C4 grasses and is positively correlated with the granal index (ratio of the length of appressed thylakoid membranes to the total length of all thylakoid membranes) of the BS chloroplasts: C4 grasses with high granal index contained more glycine decarboxylase per unit leaf area than those with low granal index, probably reflecting the differences in O2 production from photosystem II and the potential photorespiratory capacity. Thus, it is hypothesized that the activities of peroxisomal enzymes involved in photorespiration are also correlated with the granal development.• Methods The granal development in BS chloroplasts was investigated and activities of the photorespiratory enzymes assayed in 28 C4 grasses and seven C3 grasses.• Key Results The NADP–malic enzyme grasses were divided into two groups: one with low granal index and the other with relatively high granal index in the BS chloroplasts. Both the NAD–malic enzyme and phosphoenolpyruvate carboxykinase grasses had high granal index in the BS chloroplasts. No statistically significant differences were found in activity of hydroxypyruvate reductase between the C3 and C4 grasses, or between the C4 subtypes. The activity of glycolate oxidase and catalase were smaller in the C4 grasses than in the C3 grasses. Among the C4 subtypes, glycolate oxidase activities were significantly smaller in the NADP–malic enzyme grasses with low granal index in the BS chloroplasts, compared with in the C4 grasses with substantial grana in the BS chloroplasts.• Conclusions There is interspecies variation in glycolate oxidase activity associated with the granal development in the BS chloroplasts and the O2 production from photosystem II, which suggests different potential photorespiration capacities among C4 grasses. [ABSTRACT FROM AUTHOR]

The effects of decreased flux in the glycolate pathway on photoinhibition was investigated in transgenic tobacco (Nicotiana tabacum L. cv. SR1) plants. These plants harbored a pumpkin cDNA for glycolate oxidase (GO), an enzyme in the glycolate pathway, under the control of the cauliflower mosaic virus 35S promoter. Some transformants showed both reduced amounts and reduced activities of GO. The decrease of GO was enhanced at a later growth stage of these transformants, whereas no changes were observed in the amounts of other enzymes in the glycolate pathway, such as hydroxypyruvate reductase and serine glyoxylate aminotransferase. The phenotype grown under a low light condition (30 µE s–1 m–2) resembled that of the wild type. Transformants with about 35% lower GO activity than wild type, had a lower Fv/Fm under 500 µE s–1 m–2 irradiation for 8 h. After 60 µE s–1 m–2 irradiation for 8 h, Fv/Fm was lowered in some transformants with less than 20% of the GO activity of the wild type. These results suggest that photosynthesis was susceptible to photoinhibition with reduction to below threshold levels of GO activities and that higher activities of GO are required under a higher irradiation. The increase in the electron transport rate (ETR) with increased irradiation was suppressed only in transformants that had GO activity one-third less than the wild type, suggesting that the regeneration of the substrate for the Calvin cycle was decreased only when there was an extreme reduction of GO. These results also suggest that the photosystem was disturbed when the concentration of the substrate for the Calvin cycle decreased until it became insufficient to receive the excess photon energy generated in each light environment. [ABSTRACT FROM PUBLISHER]

The significance of photorespiration in drought-stressed plants was studied by withholding water from wild-type barley (Hordeum vulgare L.) and from heterozygous mutants with reduced activities of chloroplastic glutamine synthetase (GS-2), glycine decarboxylase (GDC) or serine: glyoxylate aminotransferase (SGAT). Well-watered plants of all four genotypes had identical rates of photosynthesis. Under moderate drought stress (leaf water potentials between –1 and –2 MPa), photosynthesis was lower in the mutants than in the wild type, indicating that photorespiration was increased under these conditions. Analysis of chlorophyll a fluorescence revealed that, in the GDC and SGAT mutants, the lower rates of photosynthesis coincided with a decreased quantum efficiency of photosystem II and increased non-photochemical dissipation of excitation energy. Correspondingly, the de-epoxidation state of xanthophyll-cycle carotenoids was increased several-fold in the drought-stressed GDC and SGAT mutants compared with the wild type. Accumulation of glycine in the GDC mutant was further evidence for increased photorespiration in drought-stressed barley. The effect of drought on the photorespiratory enzymes was determined by immunological detection of protein abundance. While the contents of GS-2 and P- and H-protein of the GDC complex remained unchanged as drought stress developed, the content of NADH-dependent hydroxypyruvate reductase increased. Enzymes of the Benson–Calvin cycle, on the other hand, were either not affected (ribulose-1,5-bisphosphate carboxylase-oxygenase and plastidic fructose-1,6-bisphosphatase) or declined (sedoheptulose- 1,7-bisphosphatase and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase). These data demonstrate that photorespiration was enhanced during drought stress in barley and that the control exerted by photorespiratory enzymes on the rate of photosynthetic electron transport and CO2 fixation was increased. [ABSTRACT FROM AUTHOR]

In rye leaves (Secale cereale L. cv. Petkus "Kustro") bleached in the presence of the chlorosis-inducing herbicides aminotriazole, haloxidine, San 6706 or difunone in white light of 54.2 W -2 (5000 lx), catalase activity was very low. In addition, the activities of glycolate oxidase and hydroxypyruvate reductase were strongly diminished in treatments with San 6706 and difunone. The lowering of the peroxisomal enzyme activities was observed in red, but not in blue light and did not occur after treatment with the non-bleaching pyridazinone derivative San 9785. The deficiencies of the peroxisomal enzymes did not appear to be involved in the initiation of the chlorosis. Instead they are probably produced as secondary consequences of the bleaching. Low peroxisomal enzyme activities were also obtained without herbicide treatment by growing the leaves in an atmosphere of 2% O2 and 3% CO2, but in this case were not accompanied by an increased sensitivity of the Chl to photooxidative bleaching. The peroxisomal enzymes reached as high activities as in untreated controls when the herbicide-treated leaves were grown at a low light intensity of 0.106 W m-2 (10 lx). After transfer of herbicide-treated leaves grown under 0.106 W m-2 to 306 W m-2 (30000 lx), catalase was strongly inactivated, even at 0°C. In treatments with San 6706 and difunone the increase of the activities of glycolate oxidase and hydroxypyruvate reductase was either stopped, remaining unchanged, or the enzymes were slightly inactivated after exposure to 306 W m-2 (30000 lx). The observations suggest that the inactivation of peroxisomal enzymes results from photooxidative events in the chloroplasts. [ABSTRACT FROM AUTHOR]

Leaf extracts from seven monocotyledonous and dicotyledonous species contained considerable levels of NADPH‐dependent glyoxylate‐ and hydroxypyruvate reductase activities. These activities ranged from 0.02 to 0.22 μmol (mg protein)−1 min−1. For all plants tested, the glyoxylate reductase (GR) activity, assayed with either NADPH or NADH, was sensitive to inhibition by acetohydroxamate, a glycine analogue. Hydroxypyruvate reductase (HPR) activities were unaffected by acetohydroxamate. Differential precipitation of soluble leaf proteins of spinach, pea and barley by ammonium sulfate (0–45% and 45–60% saturation) indicated the presence of at least three distinct reductases, which differed in their specificities for glyoxylate, hydroxypyruvate and NAD(P)H. For all species, the NADH‐dependent HPR‐activity was almost completely precipitated by low ammonium sulfate concentration (45%), while precipitation of the NADPH‐GR, NADH‐GR and, to some extent, NADPH‐HPR activities required 60% ammonium sulfate. The NADPH‐dependent GR and HPR activities had high affinity for glyoxylate and hydroxypyruvate, respectively, as indicated by low apparent Km values of 40–120 μM. The occurrence of at least three distinct reductases utilizing hydroxypyruvate and/or glyoxylate as substrate was supported by antibody‐precipitation studies using antibodies prepared against NADH(NADPH)‐HPR, the well‐known peroxisomal enzyme that also shows non‐specific GR activity. These data are discussed with respect to recent reports on the purification and characterization of NADPH(NADH)‐GR, and NADPH (NADH)‐HPR, two cytosolic reductases, and the role is assessed for these enzymes in reducing hydroxypyruvate and glyoxylate that may be leaked from peroxisomes. [ABSTRACT FROM AUTHOR]

Seven enzymes participating in glycolate metabolism were demonstrated to be present in crude extract of the brown alga Spatoglossum pacificum Yendo. These were phosphoglycolate phosphatase, glycolate oxidase, glutamate-glyoxylate aminotransferase, serine hydroxymethyltransferase, amino acid-hydroxy-pyruvate aminotransferase, hydroxypyruvate reductase and catalase. Malate synthase, which is involved in glycolate metabolism in the xanthophycean alga, could not be detected. On demonstration of subcellular distribution of glycolate oxidizing enzymes by linear sucrose density gradient centrifugation, glycolate oxidase was detected in the same traction at a density of 1.23 g cm[sup3] with catalase: that is, the marker enzyme of peroxisome and serine hydroxymethyltransferase was found in the same fraction at a density of 1.21 g cm[sub3] with isocitrate dehydrogenase, the marker of mitochondria. From the present data, it is proposed that the brown alga Spatoglossum possesses the ability to metabolize glycolate to glycerate via the pathway which may be similar to that of higher plants. [ABSTRACT FROM AUTHOR]

Two full-length cDNAs encoding hydroxypyruvate reductase were isolated from a cDNA library constructed with poly(A) RNA from pumpkin green cotyledons. One of the cDNAs, designated HPR1, encodes a polypeptide of 386 amino acids, while the other cDNA, HPR2 encodes a polypeptide of 381 amino acids. Although the nucleotide and deduced amino acid sequences of these cDNAs are almost identical, the deduced HPR1 protein contains Ser-Lys-Leu at its carboxy-terminal end, which is known as a microbody-targeting signal, while the deduced HPR2 protein does not. Analysis of genomic DNA strongly suggests that HPR1 and HPR2 are produced by alternative splicing. [ABSTRACT FROM AUTHOR]

Several clones corresponding to the gene encoding NADH-dependent hydroxypyruvate reductase have been isolated from a cucumber genomic library. Restriction mapping indicates the presence of two HPR genes, hpr-A and hpr-B, in the cucumber genome. Examination of the DNAs of individual plants suggests that hpr-A and hpr-B are most likely alleles at a single locus. The sequence of a 6.7 kb genomic fragment that includes the entire transcribed region, 2.2 kb of 5′ flanking sequence, and about 0.8 kb of 3′ flanking sequence reveals the presence of 12 introns in hpr-A. These introns are AT-rich relative to the exons. The donor sequence at the 5′ end of the sixth intron contains an unusual dinucleotide, GC, rather than the nearly invariant GT. Primer extension analysis maps the transcription initiation site to 61 nucleotides upstream of the translation initiation codon. An AT-rich stretch is centered at position −31 with respect to the transcription initiation site, and a potential CCAAT box is centered at position −138. Several elements that are homologous to regulatory elements of other plant genes have been identified in the flanking regions of hpr-A. [ABSTRACT FROM AUTHOR]

A full-length cDNA encoding NADH-dependent hydroxypyruvate reductase (HPR), a photorespiratory enzyme localized in leaf peroxisomes, was isolated from a λgt11 cDNA library made by reverse transcription of poly(A) RNA from cucumber cotyledons. In vitro transcription and translation of this clone yielded a major polypeptide which was identical in size, 43 kDA, to the product of in vitro translation of cotyledonary poly(A) RNA and subsequent immunoprecipitation with HPR antiserum. Escherichia coli cultures transformed with a plasmid construct containing the cDNA insert were induced to express HPR enzyme activity. RNA blot analysis showed that HPR transcript levels rise significantly in the first eight days of light-grown seedling development. This closely resembles the pattern seen for HPR-specific translatable mRNA. DNA blot analysis indicated that a single HPR gene is likely present per haploid genome. Nucleotide sequence analysis revealed an open reading frame of 1146 bases which encodes a polypeptide with a calculated molecular weight of 41.7 kDa. The derived amino acid sequence from this open reading frame is 26% identical and 50% similar to the amino acid sequence of the E. coli enzyme phosphoglycerate dehydrogenase, which catalyzes a similar reaction and functions in a related pathway. Statistical analyses show that this similarity is significant ( z>10). The derived amino acid sequence for HPR also contains the characteristics of an NAD-binding domain. [ABSTRACT FROM AUTHOR]

The development of peroxisomal enzymes in cotyledons of cucumber seedlings is strongly dependent on light. In light-grown seedlings, activities of two peroxisomal enzymes, hydroxypyruvate reductase (HPR) and serine: glyoxylate aminotransferase (SGAT), were barely detectable until three days postimbibition, after which time both activities increased rapidly and linearly for at least three days. In the dark, the activities of these enzymes increased slightly over the same time period, but only to about 5% to 10% of 7-day light-induced levels. When 51/2-day dark-grown seedlings were transferred into white light, activities of HPR and SGAT began to increase after approximately 8 h. HPR protein was shown by an immunoprecipitation assay to increase concurrently with enzymatic activity in both light- and dark-grown cotyledons. Immunoblotting results suggested that the amounts of SGAT-A and SGAT-B, the two subunits of SGAT, also developed along with SGAT activity. The relative levels of translatable mRNAs encoding HPR, SGAT-A, and SGAT-B were also light-dependent, and increased with a developmental pattern similar to enzyme activity and protein levels in light- and dark-grown cotyledons. In 51/2-day dark-grown cotyledons that were transferred to the light, translatable mRNAs for SGAT-A and SGAT-B began to increase within 1 h of illumination and continued of increase rapidly and linearly for the next 24 h in the light to a new steady-state level that was 45 times that of dark controls. Translatable HPR mRNA exhibited a biphasic pattern of accumulation, with a three-fold increase during the first 6 h of illumination, followed by an additional six-fold increase between 8 and 24 h. The accumulation of translationally active mRNA for both enzymes preceded the accumulation of the corresponding protein and enzyme activity by about 8 h. Our data suggest that the rise in enzyme activity depends on an increase in translatable mRNA for these enzymes and is regulated at a pretranslational level, most likely involving transcription of new mRNA. [ABSTRACT FROM AUTHOR]