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The gene encoding the serine cycle hydroxypyruvate reductase of Methylobacterium extorquens AM1 was isolated by using a synthetic oligonucleotide with a sequence based on a known N-terminal amino acid sequence. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced a serine cycle hydroxypyruvate reductase null mutant. This mutant had lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds, showing that the hydroxypyruvate reductase operating in the serine cycle is not involved in the conversion of acetyl coenzyme A to glycine as previously proposed. A second hydroxypyruvate-reducing enzyme with a low level of activity was found in M. extorquens AM1; this enzyme was able to interconvert glyoxylate and glycollate. The gene encoding hydroxypyruvate reductase was shown to be located about 3 kb upstream of two other serine cycles genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase.

Oxalate was found to be a potent uncompetitive, or mixed, inhibitor of the partially purified NADPH-preferring hydroxypyruvate reductase (HPR-2) from a mutant of barley which lacks the NADH-preferring HPR (HPR- 1). Both hydroxypyruvate and glyoxylate-dependent rates of the HPR-2 enzyme were inhibited by oxalate, with the experimentally determined K i values of 3–5 μM, depending on variable substrate. Oxalate did not affect the activity ofbarley NADPH-preferring glyoxylate-specific reductase (GR-1), and had only a negligible effect on HPR-1 from wild-type barley. The selective effect of oxalate, as seen for barley HPR-2, was confirmed in studies on partially purified reductases from leaves of maize, pea, soybean and spinach. In all cases, oxalate acted independently from acetohydroxamate, a selective inhibitor of GR-1. Inhibition by oxalate provides an accurate measure of the contribution of HPR-2 to the overall activities of reductases in leaf extracts and partially purified preparations.

PH1 (primary hyperoxaluria type 1) is a severe inborn disorder of glyoxylate metabolism caused by a functional deficiency of the peroxisomal enzyme AGXT (alanine-glyoxylate aminotransferase), which converts glyoxylate into glycine using L-alanine as the amino-group donor. Even though pre-genomic studies indicate that other human transaminases can convert glyoxylate into glycine, in PH1 patients these enzymes are apparently unable to compensate for the lack of AGXT, perhaps due to their limited levels of expression, their localization in an inappropriate cell compartment or the scarcity of the required amino-group donor. In the present paper, we describe the cloning of eight human cytosolic aminotransferases, their recombinant expression as His6-tagged proteins and a comparative study on their ability to transaminate glyoxylate, using any standard amino acid as an amino-group donor. To selectively quantify the glycine formed, we have developed and validated an assay based on bacterial GO (glycine oxidase); this assay allows the detection of enzymes that produce glycine by transamination in the presence of mixtures of potential amino-group donors and without separation of the product from the substrates. We show that among the eight enzymes tested, only GPT (alanine transaminase) and PSAT1 (phosphoserine aminotransferase 1) can transaminate glyoxylate with good efficiency, using L-glutamate (and, for GPT, also L-alanine) as the best amino-group donor. These findings confirm that glyoxylate transamination can occur in the cytosol, in direct competition with the conversion of glyoxylate into oxalate. The potential implications for the treatment of primary hyperoxaluria are discussed. Abbreviations: AGXT, alanine-glyoxylate aminotransferase; CCBL1, glutamine transaminase K; GO, glycine oxidase; GOT1, glutamic-oxaloacetic transaminase 1; GPT, alanine transaminase; GRHPR, glyoxylate reductase/hydroxypyruvate reductase; LDH, lactate dehydrogenase; PH1, primary hyperoxaluria type 1; PLP, pyridoxal 5′-phosphate; PSAT, phosphoserine aminotransferase

Publisher Summary The primary hyperoxalurias (1 and 2) are genetic disorders in glyoxylate metabolism that lead to systemic overproduction of oxalate. This endogenous production of oxalate cannot be managed by restriction of dietary oxalate, although it may be possible to restrict dietary glyoxylate precursors, such as glycolate and hydroxyproline to limit endogenous oxalate production. Glyoxylate produced in the mitochondria or present in the cytoplasm can be converted to glycolate by the glyoxylate reductase (GR) activity of the GRHPR enzyme. This enzyme is present both in the cytoplasm and in the mitochondria. In the absence of GR activity (PH2 patients) the excess glyoxylate is converted to oxalate, presumably in the cytoplasm by lactate dehydrogenase (LDH). In PH2 patients, who lack both GR and hydroxypyruvate reductase (HPR) enzymatic activities, the lack of HPR results in an accumulation of hydroxypyruvate, which is converted to L-glycerate by LDH. Thus, the diagnostic biochemical features of PH2 are high urinary L-glycerate in conjunction with hyperoxaluria. The diagnosis and screening of PH, and the further classification of PH into PH1 and PH2 utilizes biochemical analyte measurements of urine, genetic screening for mutations, and measurements of tissue enzyme functions. A definitive diagnosis of PH1 and PH2 can be obtained by assessments of glyoxylate aminotransferase (AGT) and GRHPR enzymatic activities and immunoreactivity in hepatic tissue (minimum of 2 mg), respectively. Alternatively, the diagnosis can be established at the molecular level in the majority of patients with minimal invasiveness and associated morbidity. Measurement of GR and DGDH activity in liver can be used for definitivediagnosis of PH2.

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.

The genus Flaveria shows evidence of evolution in the mechanism of photosynthesis as its 21 species include C(3), C(3)-C(4), C(4)-like, and C(4) plants. In this study, several physiological and biochemical parameters of photosynthesis and photorespiration were measured in 18 Flaveria species representing all the photosynthetic types. The 10 species classified as C(3)-C(4) intermediates showed an inverse continuum in level of photorespiration and development of the C(4) syndrome. This ranges from F. sonorensis with relatively high apparent photorespiration and lacking C(4) photosynthesis to F. Among the intermediates, the photosynthetic CO(2) compensation points at 30 degrees C and 1150 micromoles quanta per square meter per second varied from 9 to 29 microbars. The values for the three C(4)-like species varied from 3 to 6 microbars, similar to those measured for the C(4) species. The activities of the photorespiratory enzymes glycolate oxidase, hydroxypyruvate reductase, and serine hydroxymethyltransferase decreased progressively from C(3) to C(3)-C(4) to C(4)-like and C(4) species. On the other hand, most intermediates had higher levels of phosphenolpyruvate carboxylase and NADP-malic enzyme than C(3) species, but generally lower activities compared to C(4)-like and C(4) species. The levels of these C(4) enzymes are correlated with the degree of C(4) photosynthesis, based on the initial products of photosynthesis. Another indication of development of the C(4) syndrome in C(3)-C(4)Flaveria species was their intermediate chlorophyll a/b ratios. The chlorophyll a/b ratios of the various Flaveria species are highly correlated with the degree of C(4) photosynthesis suggesting that the photochemical machinery is progressively altered during evolution in order to meet the specific energy requirements for operating the C(4) pathway. In the progression from C(3) to C(4) species in Flaveria, the CO(2) compensation point decreased more rapidly than did the decrease in O(2) inhibition of photosynthesis or the increase in the degree of C(4) photosynthesis. These results suggest that the reduction in photorespiration during evolution occurred initially by refixation of photorespired CO(2) and prior to substantive reduction in O(2) inhibition and development of the C(4) syndrome. However, further reduction in O(2) inhibition in some intermediates and C(4)-like species is considered primarily due to the development of the C(4) syndrome. Thus, the evolution of C(3)-C(4) intermediate photosynthesis likely occurred in response to environmental conditions which limit the intercellular CO(2) concentration first via refixation of photorespired CO(2), followed by development of the C(4) syndrome.

Peroxisomes are single-membrane-bound organelles found in virtually all eukaryotes. In plants, there are several classes of peroxisomes. Glyoxysomes are found in germinating seedlings and contain enzymes specific for the glyoxylate cycle, including isocitrate lyase and malate synthase. After seedlings become photosynthetic, leaf peroxisomes participate in reactions of the photorespiration pathway and contain characteristic enzymes such as glycolate oxidase and hydroxypyruvate reductase. As leaves begin to senesce, leaf peroxisomes are transformed back into glyoxysomes. Root peroxisomes in the nodules of legumes, for example, sequester enzymes such as allantoinase and uricase, which contribute to nitrogen metabolism in these tissues. Thus, peroxisomes participate in many metabolic pathways and contain specific enzyme complements, depending on the tissue source. All peroxisomes contain catalase to degrade hydrogen peroxide and enzymes to accomplish β-oxidation of fatty acids. Glyoxysomes can be isolated from pumpkin cotyledons by standard differential centrifugation and density separation, as described in this article. Keywords: glyoxysomes; peroxisomes; protein import

By probing total RNA blots with an NADH-dependent hydroxypyruvate reductase (HPR) cDNA clone, we have found that HPR transcript abundance is highly regulated in developing cucumber (Cucumis sativus) seedlings. HPR transcript levels in light-grown seedlings are 50-fold more abundant in leaves and cotyledons than in roots. When 12-day light-grown seedlings are shifted to the dark for 4 days, the HPR transcript level in leaves drops 20-fold. Upon return of these dark-adapted plants to the light, HPR transcript levels rise to 50% of the previous light-grown level within 2 hours.

The relationship between the thermal dependence of the reappearance of chlorophyll variable fluorescence following illumination and temperature dependence of the apparent Michaelis constant (K(m)) of NADH hydroxypyruvate reductase for NADH was investigated in cool and warm season plant species. Brancker SF-20 and SF-30 fluorometers were used to evaluate induced fluorescence transients from detached leaves of wheat (Triticum aestivum L. cv TAM-101), cotton (Gossypium hirsutum L. cv Paymaster 145), tomato (Lycopersicon esculentum cv Del Oro), bell pepper (Capsicum annuum L. cv California Wonder), and petunia (Petunia hybrida cv. Red Sail). Following an illumination period at 25 degrees C, the reappearance of variable fluorescence during a dark incubation was determined at 5 degrees C intervals from 15 degrees C to 45 degrees C. Variable fluorescence recovery was normally distributed with the maximum recovery observed at 20 degrees C in wheat, 30 degrees C in cotton, 20 degrees C to 25 degrees C in tomato, 30 to 35 degrees C in bell pepper and 25 degrees C in petunia. Comparison of the thermal response of fluorescence recovery with the temperature sensitivity of the apparent K(m) of hydroxypyruvate reductase for NADH showed that the range of temperatures providing fluorescence recovery corresponded with those temperatures providing the minimum apparent K(m) values (viz. the thermal kinetic window).

Hydroxypyruvate reductase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity, crystallized and characterized, the first time for an enzyme from a methylotroph. The enzyme was found to be a dimer composed of identical subunits (38 kDa), the molecular mass of the enzyme being about 70 kDa. The enzyme was stable against heating at 25 degrees C for 10 min at pH values between 5 and 9. Optimal activity was observed at pH 6.8 and around 45 degrees C. The enzyme catalyzed the reduction of hydroxypyruvate with the oxidation of only NADH. Other than hydroxypyruvate, only glyoxylate served as a substrate. The Km values were found to be 0.175 mM for hydroxypyruvate and 10.8 mM for glyoxylate. Taking advantage of the high substrate specificity of this enzyme, a means of enzymatic determination of hydroxypyruvate was established.

The amino acid L-lysine was produced from homoserine auxotrophic and S-(2-aminoethyl)-L-cysteine-resistant mutants of a newly isolated gram-positive methylotrophic bacterium, capable of growth on methanol at 60 degrees C. The temperature optimum for growth was between 50 and 53 degrees C. These aerobic, gram-positive, endospore-forming, rod-shaped bacteria required biotin and vitamin B12 for growth. Extracts of the bacteria grown on methanol lacked hydroxypyruvate reductase and contained hexulose 6-phosphate synthase activity. Therefore, these bacteria were considered to be type I methylotrophic bacteria of the genus Bacillus. Fed-batch fermentations resulted in cell densities of 50 g of cell dry weight per liter. Biomass yields on carbon, nitrogen, phosphate, and sulfate were determined. Generation of homoserine auxotrophic and amino acid analog-resistant mutants resulted in L-lysine concentrations of nearly 20 g/liter in fed-batch fermentations.

Excised leaves of spinach were induced to senescence in continuous darkness. In the course of 8 days marked and progressive losses of chlorophyll and protein were observed. Marker enzymes of photo-respiratory metabolism, glycolate-oxidase and hydroxypyruvate reductase, were initially retained for 2–3 days and subsequently began to decline. Activities of markers of the glyoxylate-cycle, malate-synthase and isocitrate-lyase, which were hardly detectable in pre-senescent leaves appeared soon after induction of senescence and increased dramatically up to day 6–7 of the senescence period. Photorespiratory as well as glyoxisomal enzyme activities were localized in glyoxisome-like organelles isolated from senescent spinach leaves. It is concluded that foliar senescence is associated with the transition of leaf peroxisomes into a glyoxisome-like type of microbody.

Agrobacterium sp. M3C, previously isolated from canal-water for its ability to grow on monomethyl sulphate, degraded this ester with stoichiometric liberation of inorganic sulphate. In contrast with the biodegradation of monomethyl sulphate in Hyphomicrobium sp., and of other longer-chain alkyl sulphates in Pseudomonas spp., the pathway in Agrobacterium appeared not to involve a sulphatase enzyme capable of catalysing ester-bond hydrolysis. No such sulphatase was detectable under a range of conditions of bacterial culture, or using various methods for preparing cell-extracts, or different assay conditions. There was no incorporation of 18O-label from H2(18O) into the liberated inorganic sulphate. No methanol was detectable during biodegradation, and the organism was incapable of growth on methanol, and did not produce methanol dehydrogenase activity when grown on monomethyl sulphate. Tracer studies using mono[14C]-methyl sulphate indicated that formate serine and glycine were produced during the biodegradation. The presence of these amino acids, together with high activity of hydroxypyruvate reductase, indicated the operation of the serine pathway common in methylotrophs. Use of an oxygen electrode in conjunction with monomethyl[35S]sulphate showed that release of 35SO2(-4) was dependent on availability of O2, and that there was equimolar stoichiometry among monomethyl sulphate degraded, O2 consumed and 35SO2(-4) released. A proposed pathway for the degradation involved an initial mono-oxygenation to methanediol monosulphate with subsequent elimination of SO2(-4) and concomitant formation of formaldehyde. The pathway was compared with degradation mechanisms for other C1 compounds and for other sulphate esters.

10 bacterial strains utilizing methylacetate as a source of carbon and energy were isolated. Two of the most active strains (Pseudomonas spp. 27RD and 24RA) completely consumed this substrate at a concentration of 0.2% (v/v) during 24 and 36 h, respectively. Both strains possessed non-specific inducible carboxyl esterase that hydrolyzed methylacetate to methanol and acetate. The latter was then metabolized through the Krebs cycle and glyoxylate shunt. Pseudomonas 27RD had inducible dehydrogenases of methanol (PMS), formaldehyde (NAD) and formate (PMS) as well as hexulosephosphate synthase. The organism assimilated methanol via the ribulose monophosphate cycle (FBP-variant, TA/TK). Alternatively, Pseudomonas 24RA had no methanol dehydrogenase (PMS or NAD) and enzymes of the RuMP- and RuBP-pathways, but possessed the low alcohol oxidase activity as well as inducible dehydrogenases of formaldehyde (NAD, GSH) and formate (PMS), hydroxypyruvate reductase, serine-glyoxylate transaminase and malte lyase.

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.

Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of peroxisomal enzyme alanine:glyoxylate-aminotransferase (AGT) which catalyzes the conversion of glyoxylate to glycine. In PH1 patients, glyoxylate cannot be converted into glycine and is oxidized to oxalate resulting in hyperoxaluria. The excess of oxalate causes deposition of insoluble calcium oxalate in the kidney and other tissues leading to nephrolithiasis, nephrocalcinosis, kidney failure, and systemic tissue damage. Combined liver/kidney transplantation is the only available therapeutic strategy for disease treatment. Gene therapy is an attractive option to provide a definitive cure for PH1. Towards this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. We injected PH1 mice with an HDAd encoding AGT under the control of a liver-specific promoter and observed normalization of urinary oxalate at the doses of 5×10e12 and 1×10e13 vector particles (vp)/kg and partial correction with 1×10e12 vp/kg. Following challenge with Ethylene Glycol (EG), a precursor of glyoxylate, we observed reduced elevations of urinary oxalate in HDAd-injected mice compared to saline controls.Next, we hypothesized that overexpression of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) or glutamate-pyruvate transaminase (GPT) results in reduction of hyperoxaluria in PH1 by steering glyoxylate towards glycolate synthesis or transamination, respectively. To test this hypothesis, we injected PH1 mice with HDAd vectors expressing GRHPR or GPT. Both vectors resulted in significant reduction of hyperoxaluria and co-injection of the two vectors resulted in long-term normalization of oxalate excretion. Therefore, metabolic diversion towards non-toxic metabolites has potential for treatment of hyperoxaluria and vector-mediated GRHPR and/or GPT overexpression may be an alternative or adjunctive strategy to enhance efficiency of gene replacement therapy for PH1.We have recently developed a minimally invasive method to improve the therapeutic index of HDAd based on balloon occlusion catheter to achieve preferential delivery of the vector to the liver (Brunetti-Pierri et al., 2009 and 2012). This method may permit in humans correction of PH1 using clinically relevant lower doses of HDAd. Based on risk:benefit assessment, PH1 is an attractive disease for clinical application of this method.

Primary hyperoxaluria is a name given to a group of hereditary disorders characterized by increased synthesis and excretion of the metabolic end-product oxalate, and deposition of insoluble calcium oxalate (CaOx) in the kidney and urinary tract [1]. Only two of the primary hyperoxalurias have been well characterized—type 1 (PH1, MIM 259900) and type 2 (PH2, MIM260000). PH1 is caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT, EC 2.6.1.44), and PH2 is caused by a deficiency of the cytosolic/mitochondrial enzyme glyoxylate/hydroxypyruvate reductase (GR/HPR, EC 1.1.1.26/79) [2]. Although the mutant genes and the dysfunctional enzymes are different in PH1 and PH2, the clinical outcome is very similar (i.e. hyperoxaluria, urolithiasis and/or nephrocalcinosis) (Figure 1). Especially in the case of PH1, renal CaOx deposition is likely to lead to kidney failure, after which the combined effects of increased oxalate synthesis and failure to remove it from the body allow widespread deposition of CaOx throughout the body. In addition to hyperoxaluria, PH1 patients often have hyperglycolic aciduria and PH2 patients hyper-l-glyceric aciduria. However, the calcium salts of glycolate and l-glycerate are soluble and, therefore, do not result in any pathology. Genotype–phenotype correlations

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor alpha (PPARalpha) of the mouse GRHPR gene in liver. Mice fed with a PPARalpha ligand or in which PPARalpha activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARalpha present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARalpha ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARalpha-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARalpha regulation. Overall, these data indicate a species-specific regulation by PPARalpha of GRHPR, a key gene of the glyoxylate cycle.

Primary hyperoxaluria type 2, an inherited autosomal recessive disorder of endogenous oxalate overproduction, is caused by mutations in the GRHPR gene encoding the glyoxylate/hydroxypyruvate reductase enzyme. The GRHPR genes from nineteen unrelated patients with PH2 were analysed for mutations using a combination of PCR-SSCP and sequence analysis of genomic and cDNA. Eleven mutations were identified, seven of which are novel. The mutations included five point mutations: c.84-2A>G, c.295C>T (R99X), c.494G>A (G165D), and c.904C>T (R302C) as well as six minor deletions: c.103delG, c.375delG, c.403_405+2 delAAGT, c.540delT, c.608_609delCT and a more complex mutation in intron 1: c.84-13_c.84-12del; c.84-8_c.84-5del. Aberrant transcripts were demonstrated in hepatic mRNA as a result of the c.403_405+2 delAAGT and c.84-2A>G mutations. In addition, a splice variant lacking 28 bp of exon 1 was expressed in a number of tissues but is of unknown function. Two polymorphisms, c.579A>G in exon 6 and a (CT)(n) microsatellite in intron 8 were identified. Expression studies showed that the G165D and R302C mutants had glyoxylate reductase activity 1.5 and 5.6% respectively of the wild type protein. Both mutant proteins were unstable on purification. Although there is wide expression of the GRHPR mRNA demonstrated by northern blot analysis, our study shows that GRHPR protein distribution is predominantly hepatic and concludes that PH2, like the related type 1 disease, is primarily a disorder affecting hepatic glyoxylate metabolism.

Chara fragilis possesses microbodies with a remarkably large size of up to 2 micro m in diameter. Many of the organelles contain huge nucleoids of amorphous material or paracrystalline inclusions. After isolation of the organelles by gradient centrifugation the specific density of the microbodies was determined to be 1.25 g cm-3. Catalase, glycolate oxidase and hydroxypyruvate reductase as well as enzymes of the fatty acid beta-oxidation pathway were demonstrated to be constituents of the microbodies in Chara indicating that they are similar to those in green leaves. The data obtained are in agreement with the view that the Charophyceae and especially the algae in the subgroup of Charales are very closely related to the land plants.

Photorespiration is a light-dependent process that results in the uptake of O2 and the release of CO2. Photorespiration is linked to photosynthesis by the dual function of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) which uses the mutually competitive substrates CO2 and O2 for reaction with ribulose-1.5-bisphosphate (Rul.5BP). Oxygenation of ribulose-1.5-bisphosphate leads to the production of 2-P-glycolate, which is salvaged in the photorespiratory pathway (C2 cycle). Several enzymes of the photorespiratory pathway are localized in leaf peroxisomes and the recycling of 2-P-glycolate is the main function of leaf peroxisomes known so far. Apart from the photorespiratory C2 cycle, leaf peroxisomes have an important physiological role in the metabolism of active oxygen species (see Chapter 7), mediate fatty acid β-oxidation (Chapter 1 this volume; Gerhardt, 1981, 1983; Guhnemarm-Schafer and Kindl, 1995), and are involved in the biosynthesis of glycine betaine.

The primary hyperoxalurias are a group of inherited disorders of endogenous oxalate overproduction. Diagnosis of the two best-characterized disorders, primary hyperoxaluria (PH) Types 1 and 2, is achieved by sequential measurement of alanine:glyoxylate aminotransferase and glyoxylate reductase enzyme activity in a single needle liver biopsy. While genetic analysis of PH2 is still at a relatively early stage, the AGXT gene defective in the Type 1 disorder is well characterized, and a number of mutations have been identified.To determine whether mutation analysis could replace enzymatic analysis for the diagnosis of PH1, DNA samples from 127 consecutive unrelated patients in whom there was a high clinical suspicion of primary hyperoxaluria were analyzed for the presence of the G630A and T853C mutations, which together account for approximately 34% of the mutant alleles in our patient cohort.The sensitivity of mutation detection was 47% in those patients with enzymologically confirmed Type 1 disease, showing that mutation analysis cannot effectively replace enzymology at the present time. However, there is little doubt of the value of genetic methods (mutation and linkage analysis) for diagnosing PH1 (and eventually PH2) in other family members and for prenatal diagnosis and carrier testing.

Primary hyperoxaluria Type II (PH2) is a rare monogenic disease characterized by excessive urinary oxalate and L-glycerate excretion. The severity of clinical complications in PH2 patients can range from none to end-stage renal failure secondary to massive deposits of calcium oxalate crystals in the kidney. The disease is a result of the absence of an enzyme with glyoxylate reductase and hydroxypyruvate reductase activities (GRHPR). Recent breakthroughs have occurred in our understanding of the molecular basis of PH2. In this article, we briefly review the literature concerning the clinical and biochemical characteristics of the disease and the enzyme associated with it. We describe the identification of the cDNA for the GRHPR enzyme using the expressed sequence tag database, the characterization of the human GRHPR gene, and the identification of mutations in patients with PH2. Insights gained from the molecular biology underlying this disease as they relate to relevant clinical issues such as potential therapeutic strategies are discussed.

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 microE 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 microE s(-1) m(-2) irradiation for 8 h. After 60 microE 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.

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.

Purified spinach (Spinacia oleracea) NADPH-preferring hydroxypyruvate reductase (HPR-2) was potently and selectively inhibited by oxalate, an end product of metabolism in plants. Both hydroxypyruvate- and glyoxylate-dependent rates of the HPR-2 enzyme were affected. Oxalate acted as an uncompetitive inhibitor of the enzyme, with Ki values of 7 and 36 microM for the NADPH/hydroxypyruvate and NADPH/glyoxylate pairs of reactants respectively. Oxalate, at millimolar levels, caused less than 10% inhibition of purified spinach NADH-preferring HPR (HPR-1) and had no effect on purified spinach NADPH-preferring glyoxylate-specific reductase (GR-1). The inhibition of spinach HPR-2 by oxalate is by far the strongest for any known inhibitor of leaf HPR and GR activities. In photosynthetic tissues, oxalate could potentially act as a primary regulator of extraperoxisomal metabolism of hydroxypyruvate and glyoxylate.

Hydroxypyruvate reductase (HPR) has been partially purified from human liver and can be separated into at least two forms by chromatofocusing; these forms therefore differ in their pI values. Both forms, one with a pI of7.2 (peak A) and the other with a pI between pH 6.5 and 5.5 (peak B), use NADPH as a cofactor. However, only peak B was able to reduce hydroxypyruvate and glyoxylate, with a Km of 2.3 mM for the latter substrate. Peak A coeluted with lactate dehydrogenase and could represent lactate dehydrogenase (which is known to reduce hydroxypyruvate) alone or a mixture of proteins with HPR activity. The Km for hydroxypyruvate of the enzyme(s) in peak A (8 mM) was 80 times greater than that of peak B (0.1 mM), suggesting that the HPR enzyme contained in peak B may be more important physiologically, where the hydroxypyruvate concentrations are in the micromolar range. The data presented provide a biochemical explanation for the previously observed differences in the tissue distribution of HPR and glyoxylate reductase activities in human subjects and support the claim that diagnoses of primary hyperoxaluria type 2 should be made by measurement of glyoxylate reductase activity in the liver.

Primary hyperoxaluria type II (PH2) is a rare monogenic disorder that is characterized by a lack of the enzyme that catalyzes the reduction of hydroxypyruvate to D-glycerate, the reduction of glyoxylate to glycolate and the oxidation of D-glycerate to hydroxypyruvate. The disease is characterized by an elevated urinary excretion of oxalate and L-glycerate. The increased oxalate excretion can cause nephrolithiasis and nephrocalci-nosis and can, in some cases, result in renal failure and systemic oxalate deposition. We identified a glyoxylate reductase/hydroxypyruvate reductase (GRHPR) cDNA clone from a human liver expressed sequence tag (EST) library. Nucleotide sequence analysis identified a 1198 nucleotide clone that encoded a 984 nucleotide open reading frame. The open reading frame encodes a predicted 328 amino acid protein with a mass of 35 563 Da. Transient transfection of the cDNA clone into COS cells verified that it encoded an enzyme with hydroxy-pyruvate reductase, glyoxylate reductase and D-glycerate dehydrogenase enzymatic activities. Database analysis of human ESTs reveals widespread tissue expression, indicating that the enzyme may have a previously unrecognized role in metabolism. The genomic structure of the human GRHPR gene was determined and contains nine exons and eight introns and spans approximately 9 kb pericentromeric on chromosome 9. Four PH2 patients representing two pairs of siblings from two unrelated families were analyzed for mutations in GRHPR by single strand conformation polymorphism analysis. All four patients were homozygous for a single nucleotide deletion at codon 35 in exon 2, resulting in a premature stop codon at codon 45. The cDNA that we have identified represents the first characterization of an animal GRHPR sequence. The data we present will facilitate future genetic testing to confirm the clinical diagnosis of PH2. These data will also facilitate heterozygote testing and prenatal testing in families affected with PH2 to aid in genetic counseling.

WOS: 000081089900008 A methylotrophic hydroxypyruvate reductase was partially purified and characterized from Methylophilus spp. using the biomimetic dye, Cibacron Blue F3FA attached to poly(HEMA-EGDMA) microspheres. The absorption capacities of the dye-affinity microspheres were determined by changing pH and the concentration of the proteins in the adsorption medium. Hydroxypyruvate reductase was desorbed from the dye-affinity support specifically with 2 mM NADH solution. The enzyme was purified 10.4-fold with 47% yield. The molecular mass and subunit molecular mass of the enzyme was estimated to be 75 kDa and 37 kDa on the basis of its mobility in polyacrylamide and SDS-polyacrylamide gels, respectively. This suggested a homogeneous dimer structure. The optimal pH was between 5.0 and 7.0, and the maximum enzyme activity was obtained at 50 degrees C. The K-m values of hydroxpyruvate reductase were 0.222 mM for hydroxpyruvate and 0.067 mM for NADH. (C) 1999 Elsevier Science Ltd. All rights reserved.

Glycolate oxidase and hydroxypyruvate reductase, two key enzymes of glycolate metabolism, are present in the brown alga Egregia menziesii (Turn.) Aresch. The pH optimum and Km for the partially purified glycolate oxidase are similar to the enzyme from higher plants, Charophyta and Xanthophyta, whereas the substrate specificity is different from the higher plant enzyme

Transcription of the cucumber hpr-A gene is responsive to cytokinin and light. To investigate the molecular basis for transcriptional regulation by cytokinin, we have identified DNA sequences and proteins that may be involved in the regulation of hpr-A gene expression. Transient expression assays in etiolated cucumber cotyledons indicate that the 315 bp fragment (-382 to -67) contains sequences necessary for cytokinin responsiveness of the luciferase reporter gene. Band shift assays detected cytokinin-enhanced and -reduced protein binding sites in a 97 bp fragment (-382 to -285) upstream of the hpr-A gene. DNase I footprinting identified two protein-protected sites, a 15 bp sequence, 5'-AAATGACGAAAATGC-3', that contains an as-1 TGACG motif found in other plant promoters, and a 13 bp sequence, 5'-AAGATTGATTGAG-3', of unknown function. Two-dimensional band shift analysis of the cytokinin-responsive DNA protein complex revealed the presence of six DNA protein interactions. Band shift assays showed that cytokinin and light have different effects on the interaction of nuclear proteins to the 97 bp fragment of the hpr-A gene. These data suggest that cytokinin and light do not share identical signal transduction pathways in regulating hpr-A gene expression.

Constructs containing the cDNAs encoding the primary leaf catalase in Nicotiana or subunit 1 of cottonseed (Gossypium hirsutum) catalase were introduced in the sense and antisense orientation into the Nicotiana tabacum genome. The N. tabacum leaf cDNA specifically overexpressed CAT-1, the high catalatic [corrected] form, activity. Antisense constructs reduced leaf catalase specific activities from 0.20 to 0.75 times those of wild type (WT), and overexpression constructs increased catalase specific activities from 1.25 to more than 2.0 times those of WT. The NADH-hydroxypyruvate reductase specific activity in transgenic plants was similar to that in WT. The effect of antisense constructs on photorespiration was studied in transgenic plants by measuring the CO2 compensation point (gamma) at a leaf temperature of 38 degrees C. A significant linear increase was observed in gamma with decreasing catalase (at 50% lower catalase activity gamma increased 39%). There was a significant temperature-dependent linear decrease in gamma in transgenic leaves with elevated catalase compared with WT leaves (at 50% higher catalase gamma decreased 17%). At 29 degrees C, gamma also decreased with increasing catalase in transgenic leaves compared with WT leaves, but the trend was not statistically significant. Rates of dark respiration were the same in WT and transgenic leaves. Thus, photorespiratory losses of CO2 were significantly reduced with increasing catalase activities at 38 degrees C, indicating that the stoichiometry of photorespiratory CO2 formation per glycolate oxidized normally increases at higher temperatures because of enhanced peroxidation.

We examined the effects of CO2 concentration on the white-light-stimulated expression of the cucumber (Cucumis sativus L.) Hpr gene. Hpr encodes hydroxypyruvate reductase, an enzyme important in the photorespiratory glycolate pathway, which plays an integral role in carbon allocation in C3 plants. Because CO2 is an end product of this pathway and because increased CO2 concentrations lessen the need for photorespiration, we tested whether exposure of plants to elevated CO2 would affect white-light-stimulated Hpr gene expression. Exposure of dark-adapted cucumber seedlings to elevated CO2 (2 to 3 times ambient) during a 4-h white-light irradiation significantly inhibited the accumulation of Hpr mRNA. Increasing the CO2 concentration during irradiation to 6 or 9 times ambient did not further inhibit Hpr mRNA accumulation. The depressing effect of high CO2 on Hpr mRNA accumulation was seen in both high and low light, but was more pronounced in higher light. These results suggest that maximum sensitivity to CO2 occurs in conditions near those normally encountered by the plant (high light, CO2 concentration near ambient) and support a model in which white-light-regulated Hpr expression is modulated in part by environmental CO2 concentration.

To understand the mechanisms by which the expression of a specific gene is modulated by cytokinin, the regulation of hydroxypyruvate reductase (HPR) transcript levels by N6-benzyladenine (BA) in etiolated pumpkin (Cucurbita pepo L. cv. Halloween) cotyledons was investigated. A pumpkin HPR cDNA was generated by reverse transcriptase-polymerase chain reaction and its nucleotide sequence was determined. An antisense HPR RNA was prepared for RNase protection analysis of HPR-mRNA expression patterns in the cotyledons of dark-grown pumpkin seedlings. Treatment of the cotyledons with BA was shown to modulate HPR mRNA levels in a dose- and time-dependent manner. Similarly, nuclear run-on studies showed that the rate of transcription was also enhanced by BA treatment of the cotyledons. These results suggest that the enhancement of HPR mRNA by cytokinin is, at least in part, at the level of transcription.

The presence of isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) was investigated in detached pumpkin fruits (Cucurbita pepo L., var. Alberello di Sarzana). After incubation of pumpkin fruit slices in permanent darkness, the enzyme activities appeared after only 1 day and then declined to zero at day 6. Catalase (EC 1.11.1.6) specific activity increased during the first 2 days of incubation and decreased thereafter. Hydroxypyruvate reductase (EC 1.1.1.81) and 3-hydroxyacyl CoA dehydrogenase (E.C. 1.1.1.35) specific activities changed very little, but NADP+-dependent isocitrate dehydrogenase (E.C. 1.1.1.42) and fumarase (E.C. 4.2.1.2) specific activities increased during the first day and declined thereafter. Sucrose density gradient fractionation of cell organelles showed that isocitrate lyase and malate synthase are localized in peroxisomal fractions. The presence of the two key enzymes of the glyoxylate cycle was confirmed by immunoblotting, both in crude extracts and in peroxisomal fractions. It is concluded that following detachment and dark incubation, a transition from peroxisomes to glyoxysomes occurs in pumpkin fruit.

Peroxisomes were isolated by gradient centrifugation from two different diatoms: Nitzschia laevis (subgroup of Pennales) and Thalassiosira fluviatilis (subgroup of Centrales). In neither of these organelles could catalase or any H2O2-forming oxidase be demonstrated. The glycolate-oxidizing enzyme present in the peroxisomes is a dehydrogenase capable of oxidizing l-lactate as well. The peroxisomes also contain the glyoxysomal markers isocitrate lyase and malate synthase. However, enzymes of the fatty-acid β-oxidation pathway are located exclusively in the mitochondria. The mitochondria additionally possess glutamate-glyoxylate aminotransferase and a glycolate dehydrogenase which differs from the peroxisomal glycolate dehydrogenase since it preferably utilizes d-lactate as an alternative substrate. Hydroxypyruvate reductase and glyoxylate carboligase were not found in the cells of either diatom. By culturing Nitzschia laevis it could be demonstrated that decreasing the CO2 concentration in the aeration mixture from 2% to 0.03% and increasing the irradiance from 40 to 250 μmol quanta · m−2 · s−1 resulted in an increase of all peroxisomal enzyme activities. In addition, enzyme activities of the β-oxidation pathway were increased. However, mitochondrial glycolate dehydrogenase and aminotransferase did not alter their activities under these conditions. Summarizing all results, it is postulated that there are two different pathways for the metabolism of glycolate in the diatoms.

In a previous paper, we reported identification of the 5' part of hprA of Methylobacterium extorquens AM1, which encodes the serine cycle enzyme hydroxypyruvate reductase (L. V. Chistoserdova and M. E. Lidstrom, J. Bacteriol. 174:71-77, 1992). Here we present the complete sequence of hprA and partial sequence of genes adjacent to hprA. Upstream of hprA, the 3' part of an open reading frame was discovered, separated from hprA by 263 bp. This open reading frame was identified as the gene encoding another serine cycle enzyme, serine glyoxylate aminotransferase (sgaA). Cells containing an insertion mutation into sgaA were unable to grow on C1 compounds, demonstrating that the gene is required for C1 metabolism. Sequencing downstream of hprA has revealed the presence of another open reading frame (mtdA), which is probably cotranscribed with hprA. This open reading frame was identified as the gene required for the synthesis of 5,10-methylenetetrahydrofolate dehydrogenase. Our data suggest that this enzyme plays an integral role in methylotrophic metabolism in M. extorquens AM1, either in formaldehyde oxidation or as part of the serine cycle.

The synthesis of glycerate by isolated intact spinach (Spinacia oleracea L.) leaf peroxisomes upon the addition of glycolate, serine, and glutamate, with either NADH or malate as reductant, has been measured. Measurement of the concentration dependence of NADH-and malate-dependent glycerate synthesis, and the exclusion of various artefacts, clearly demonstrate that under in vivo conditions the transfer of reducing equivalents into the peroxisomes required for the reduction of hydroxypyruvate to glycerate, occurs exclusively via a malate shuttle. The results indicate that a direct uptake of NADH into the peroxisomes does not occur under invivo conditions to any appreciable extent. As these results have been observed with intact as well as with osmotically shocked peroxisomes, it is concluded that the specificity of redox transfer into the peroxisomes is not due to a selectivity of the peroxisomal boundary membrane, but to a multi-enzyme structure of the peroxisomal matrix.

We have investigated the regulation of cucumber (Cucumis sativus) hydroxypyruvate reductase mRNA abundance in response to white-, red-, and far-red-light treatments. Following irradiation of dark-adapted cucumber seedlings with 15 min to 4 h of either white or red light and return to darkness, the mRNA level for the gene encoding hydroxypyruvate reductase (Hpr) in cotyledons peaks in the darkness 16 to 20 h later. The response of the Hpr mRNA level to total fluence of white light depends more directly on irradiation time than on fluence rate. In addition to this time-dependent component, a phytochrome-dependent component is involved in Hpr regulation in dark-adapted green cotyledons as shown by red-light induction and partial far-red-light reversibility. Parallel measurements of mRNA levels for the ribulose bisphosphate carboxylase/oxygenase small subunit and for the chlorophyll a/b-binding protein show that Hpr is the most responsive to short (about 60 min) white- and red-light treatments and that each mRNA has a characteristic pattern of accumulation in dark-adapted cotyledons in response to light.

Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium that uses the serine pathway for formaldehyde incorporation as its assimilation pathway during growth on one-carbon compounds. A DNA region from M. extorquens AM1 previously shown to contain genes for the serine pathway enzymes malyl coenzyme A (CoA) lyase and hydroxypyruvate reductase has been characterized in more detail. Insertion mutagenesis revealed an additional region required for growth on one-carbon compounds, and all of the insertion mutants in this region lacked activity for another serine pathway enzyme, the acetyl-CoA-independent phosphoenolpyruvate (PEP) carboxylase. Expression analysis with Escherichia coli of DNA fragments that included the malyl-CoA lyase and PEP carboxylase regions identified five polypeptides, all transcribed in the same direction. Three of these polypeptides were expressed from the region necessary for the acetyl-CoA-independent PEP carboxylase, one was expressed from the region containing the malyl-CoA lyase gene, and the fifth was expressed from a region immediately downstream from the gene encoding hydroxypyruvate reductase. All six genes are transcribed in the same direction, but the transposon insertion data suggest that they are not all cotranscribed.

An easy, rapid, and accurate enzymatic assay method for L-serine was established involving two enzymes, serine-glyoxylate aminotransferase (SGAT, EC 2.6.1.45) and hydroxypyruvate reductase (HPR, EC 1.1.1.81), in the serine pathway of the methylotrophic bacterium, Hyphomicrobium methylovorum (IFO 14180), from which they were purified. This method consists of two reaction steps: the first is the nearly irreversible transamination of L-serine and glyoxylate by SGAT, and the second is the HPR reaction involving NADH, which comprises the absolutely irreversible reduction of hydroxypyruvate derived from L-serine by SGAT. The amounts of L-serine were determined spectrophotometrically as the decrease in the amount of NADH. When the values determined with the present enzymatic method were compared with those obtained with an amino acid analyzer, the correlation coefficient was found to be 0.9963. This method can also be applied to the assaying of glyoxylate.

A glutamine synthetase (GS) cDNA isolated from an alfalfa cell culture cDNA library was found to represent a cytoplasmic GS. The full-length alfalfa GS1 coding sequence, in both sense and antisense orientation and under the transcriptional control of the cauliflower mosaic virus 35S promoter, was introduced into tobacco. Leaves of tobacco plants transformed with the sense construct contained greatly elevated levels of GS transcript and GS polypeptide which assembled into active enzyme. Leaves of the plants transformed with the antisense GS1 construct showed a significant decrease in the level of both GS1 and GS2 polypeptides and GS activity, but did not show any significant decrease in the level of endogenous GS mRNA. We have proposed that antisense inhibition using a heterologous antisense GS RNA occurs at the level of translation. Our results also suggest that the post-translational assembly of GS subunits into a holoenzyme requires an additional factor(s) and is under regulatory control.

The long-term effects of an enriched CO2 atmosphere on the primary carbon metabolism of four year old spruce trees (Picea abies L. Karst) were examined. Six key enzymes were studied in one year old needles of trees exposed for two years in open-top chambers to three CO2 levels (350, 480 and 570 ppmv). The specific activity and quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39) and the specific activities of the photorespiratory enzymes, glycolate oxidase (EC 1.1.3.15) and hydroxypyruvate reductase (HPR, EC 1.1.1.29) showed significant decreases in the CO2-enriched atmospheres. In contrast, a net increase was found for the specific activities of the mitochondrial enzymes, NAD-malic enzyme (NAD-ME, EC 1.1.1.39) and especially fumarase (EC 4.2.1.2). The carboxylating enzyme, phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), showed a marked decrease in activity. These results clearly demonstrate increases in the activities of enzymes linked to the respiratory process and decreases in CO2-fixing enzymes as a result of long-term exposure to less than double the present ambient level of CO2.

Glyoxylate and hydroxypyruvate are metabolites involved in the pathway of carbon in photorespiration. The chief glyoxylate-reducing enzyme in leaves is now known to be a cytosolic glyoxylate reductase that uses NADPH as the preferred cofactor but can also use NADH. Glyoxylate reductase has been isolated from spinach leaves, purified to homogeneity, and characterized kinetically and structurally. Chloroplasts contain lower levels of glyoxylate reductase activity supported by both NADPH and NADH, but it is not yet known whether a single chloroplastic enzyme catalyzes glyoxylate reduction with both cofactors. The major hydroxypyruvate reductase activity of leaves has long been known to be a highly active enzyme located in peroxisomes; it uses NADH as the preferred cofactor. To a lesser extent, NADPH can also be used by the peroxisomal enzyme. A second hydroxypyruvate reductase enzyme is located in the cytosol; it preferentially uses NADPH but can also use NADH as cofactor. In a barley mutant deficient in peroxisomal hydroxypyruvate reductase, the NADPH-preferring cytosolic form of the enzyme permits sufficient rates of hydroxypyruvate reduction to support continued substrate flow through the terminal stages of the photosynthetic carbon oxidation (glycolate/glycerate) pathway. The properties and metabolic significance of the cytosolic and organelle-localized glyoxylate and hydroxypyruvate reductase enzymes are discussed.

The use of LaPr 88/29 mutant of barley (Hordeum vulgare), which lacks NADH-preferring hydroxypyruvate reductase (HPR-1), allowed for an unequivocal demonstration of at least two related NADPH-preferring reductases in this species: HPR-2, reactive with both hydroxypyruvate and glyoxylate, and the glyoxylate specific reductase (GR-1). Antibodies against spinach HPR-1 recognized barley HPR-1 and partially reacted with barley HPR-2, but not GR-1, as demonstrated by Western immunoblotting and immunoprecipitation of proteins from crude leaf extracts. The mutant was deficient in HPR-1 protein. In partially purified preparations, the activities of HPR-1, HPR-2, and GR-1 could be differentiated by substrate kinetics and/or inhibition studies. Apparent K(m) values of HPR-2 for hydroxypyruvate and glyoxylate were 0.7 and 1.1 millimolar, respectively, while the K(m) of GR-1 for glyoxylate was 0.07 millimolar. The K(m) values of HPR-1, measured in wild type, for hydroxypyruvate and glyoxylate were 0.12 and 20 millimolar, respectively. Tartronate and P-hydroxypyruvate acted as selective uncompetitive inhibitors of HPR-2 (K(i) values of 0.3 and 0.4 millimolar, respectively), while acetohydroxamate selectively inhibited GR-1 activity. Nonspecific contributions of HPR-1 reactions in assays of HPR-2 and GR-1 activities were quantified by a direct comparison of rates in preparations from wild-type and LaPr 88/29 plants. The data are evaluated with respect to previous reports on plant HPR and GR activities and with respect to optimal assay procedures for individual HPR-1, HPR-2, and GR-1 rates in leaf preparations.

Photorespiratory mutants have been obtained in five different C3 species (1, 2, 3, 4), each deficient in enzyme activities associated with phosphoglycolate metabolism. These mutants survive only in atmospheres in which photorespiration is very slow, i.e. either enriched with CO2 or depleted of O2. In air, the rate of photosynthesis soon drops and these plants cease to thrive.

Background The presence of the branched Entner-Doudoroff (ED) pathway in two hyperthermophilic Crenarchaea, the anaerobe Thermoproteus tenax and the aerobe Sulfolobus solfataricus, was suggested. However, so far no enzymatic information of the non-phosphorylative ED branch and especially its key enzyme – glycerate kinase – was available. In the T. tenax genome, a gene homolog with similarity to putative hydroxypyruvate reductase/glycerate dehydrogenase and glycerate kinase was identified. Results The encoding gene was expressed in E. coli in a recombinant form, the gene product purified and the glycerate kinase activity was confirmed by enzymatic studies. The enzyme was active as a monomer and catalyzed the ATP-dependent phosphorylation of D-glycerate forming exclusively 2-phosphoglycerate. The enzyme was specific for glycerate and highest activity was observed with ATP as phosphoryl donor and Mg2+ as divalent cation. ATP could be partially replaced by GTP, CTP, TTP and UTP. The enzyme showed high affinity for D-glycerate (Km 0.02 ± 0.01 mM, Vmax of 5.05 ± 0.52 U/mg protein) as well as ATP (Km of 0.03 ± 0.01 mM, Vmax of 4.41 ± 0.04 U/mg protein), although at higher glycerate concentrations, substrate inhibition was observed. Furthermore, the enzyme was inhibited by its product ADP via competitive inhibition. Data bank searches revealed that archaeal glycerate kinases are members of the MOFRL (multi-organism fragment with rich leucine) family, and homologs are found in all three domains of life. Conclusion A re-evaluation of available genome sequence information as well as biochemical and phylogenetic studies revealed the presence of the branched ED pathway as common route for sugar degradation in Archaea that utilize the ED pathway. Detailed analyses including phylogenetic studies demonstrate the presence of three distinct glycerate kinase classes in extant organisms that share no common origin. The affiliation of characterized glycerate kinases with the different enzyme classes as well as their physiological/cellular function reveals no association with particular pathways but a separate phylogenetic distribution. This work highlights the diversity and complexity of the central carbohydrate metabolism. The data also support a key function of the conversion of glycerate to 2- or 3-phosphoglycerate via glycerate kinase in funneling various substrates into the common EMP pathway for catabolic and anabolic purposes.