Glycolate Metabolism and Excretion by Chlamydomonas reinhardtii

The flux of glycolate through the C₂ pathway in Chlamydomonas reinhardtii was estimated after inhibition of the pathway with aminooxyacetate (AOA) or aminoacetonitrile (AAN) by measurement of the accumulation of glycolate and glycine. Cells grown photoautotrophically in air excreted little glycolate except in the presence of 2 mm AOA when they excreted 5 micromoles glycolate per hour per milligram clorophyll. Cells grown on high CO₂ (1-5%) when transferred to air produced three times as much glycolate, with half of the glycolate metabolized and half excreted. The lower amount of glycolate produced by the air-grown cells reflects the presence of a CO₂ concentrating mechanism which raises the internal CO₂ level and decreases the ribulose-1,5-bisP oxygenase reaction for glycolate production. Despite the presence of the CO₂ concentrating mechanism, there was still a significant amount of glycolate produced and metabolized by air-grown Chlamydomonas. The capacity of these cells to metabolize between 5 and 10 micromoles of glycolate per hour per milligram chlorophyll was confirmed by measuring the biphasic uptake of added labeled glycolate. The initial rapid (<10 seconds) phase represented uptake of glycolate; the slow phase represented the metabolism of glycolate. The rates of glycolate metabolism were in agreement with those determined using the C₂-cycle inhibitors during CO₂ fixation.     

Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

Serological Characterization of the Glycolate-oxidizing Enzymes from Tobacco, Euglena gracilis, and a Yellow Mutant of Chlorella vulgaris

An antiserum to tobacco glycolate oxidase has been prepared by injection of the purified enzyme into rabbits. Double gel diffusion tests between the antiserum and purified antigen and also with a crude tobacco preparation gave a single immunoprecipitation band. Crude extracts of Euglena gracilis Z Klebs, containing glycolate dehydrogenase, and of Chlorella vulgaris 211-11h/20, containing glycolate oxidase, also formed single bands with the tobacco antiserum. The algal bands were identical and showed partial identity with the tobacco band. The antiserum inhibited the glycolate oxidase activities of the tobacco and Chlorella extracts but did not affect Euglena glycolate dehydrogenase activity.     

Effect of glycidate on glycolate formation and photosynthesis in isolated spinach chloroplasts

Glycidate (2,3-epoxypropionate) increased CO₂ photoassimilation in intact spinach (Spinacia oleracea L.) chloroplasts in the presence of various inhibitors of photosynthesis, including O₂, arsenite, azide, iodo-acetamide, and carbonylcyanide 3-chlorophenylhydrazone. Although the mechanism by which glycidate enhances photosynthesis is obscure, the stimulatory effect cannot be ascribed to either an inhibition of glycolate formation, a specific interaction with the O₂ inhibition of photosynthesis, or a direct effect on the ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) reaction. The lack of a differential effect of glycidate on photosynthesis and glycolate formation in the isolated chloroplast was confirmed in whole leaf studies by the CO₂ compensation concentration assay. These results are at variance with the report that glycidate stimulates net photosynthesis in tobacco leaf disks by irreversibly inhibiting glycolate formation and thus photorespiration (Zelitch, I., 1974, Arch. Biochem. Biophys. 163: 367-377).     

Inhibition of glutamate:glyoxylate aminotransferase activity in tobacco leaves and callus by glycidate, an inhibitor of photorespiration

The effect of glycidate (2,3-epoxypropionate), an inhibitor of glycolate synthesis and photorespiration in leaf tissue, was studied on glutamate:glyoxylate and serine:glyoxylate aminotransferases and glycine decarboxylase activities in particulate preparations obtained from tobacco (Nicotiana tabacum L.) callus and leaves. Glycidate specifically and effectively inhibited glutamate:glyoxylate aminotransferase. The inhibition was dependent on glycidate concentration and, to a lesser extent, on substrate concentration. The enzyme was not protected by either substrate. Even with saturating substrate concentrations the glycidate inhibition was only partially reversed. Under the in vitro assay conditions, glycidate inhibition of the aminotransferase was reversible. Glutamate:glyoxylate aminotransferase is the only enzyme of the glycolate pathway thus far examined which is severely inhibited by glycidate. However, in leaf discs, pretreatment with glycidate decreased both glutamate:glyoxylate and serine:glyoxylate aminotransferase activities suggesting binding by glycidate in vivo.

Glycidate increased the pool sizes of both glutamate and glyoxylate in leaf discs. It has been shown that increases in concentration of either of these metabolites decrease photorespiration and glycolate synthesis and increase net photosynthesis. It is proposed that glycidate inhibits photorespiration indirectly by increasing the internal concentrations of glutamate and glyoxylate, as a consequence of the inhibition of glutamate:glyoxylate aminotransferase activity.

     

On the mechanism of glycolate synthesis by Chromatium and Chlorella

When cultures of the photosynthetic bacterium, Chromatium vinosum, capable of photosynthesizing glycolate at about 10 μmol/mg of bacteriochlorophyll/h were exposed to atmospheres enriched with ¹⁸O₂, one atom of oxygen-18 was incorporated into the car?yl group of glycolate. Allowing for the small (3–5%) loss of oxygen-18 during the manipulations leading up to the mass spectrometric determination of the oxygen-18 content of the glycolate, the isotopic enrichment of the¹⁸O-labeled glycolate synthesized by Chromatium was substantially (at least 94%) the same as the isotopic enrichment of the ¹⁸O₂. Similar results were obtained with the green alga, Chlorella fusca. The close agreement between the isotopic enrichments of the glycolate and the oxygen with which it was synthesized was independent of the oxygen concentration. The major pathway of glycolate synthesis by Chromatium therefore involves reaction(s) which bring about the incorporation of one atom of molecular oxygen into the car?yl group of glycolate. The in vitro rate of ribulose 1,5-bisphosphate oxygenase in extracts of Chromatium, previously thought to be too low to account for the rates of glycolate synthesis in vivo, was shown to be adequate for this purpose when precautions were taken to fully activate the enzyme. Similarly, the activity of phosphoglycolate phosphatase, when assayed under optimal conditions, was also adequate to sustain the rates of glycolate formation observed i vivo. It is concluded that the oxygenolytic cleavage of ribulose 1,5-bisphosphate represents the major pathway of glycolate synthesis by Chromatium.     

Construction of a carbon-conserving pathway for glycolate production by synergetic utilization of acetate and glucose in Escherichia coli

Glycolate is a bulk chemical which has been widely used in textile, food processing, and pharmaceutical industries. Glycolate can be produced from sugars by microbial fermentation. However, when using glucose as the sole carbon source, the theoretical maximum carbon molar yield of glycolate is 0.67 mol/mol due to the loss of carbon as CO₂. In this study, a synergetic system for simultaneous utilization of acetate and glucose was designed to increase the carbon yield. The main function of glucose is to provide NADPH while acetate to provide the main carbon backbone for glycolate production. Theoretically, 1 glucose and 5 acetate can produce 6 glycolate, and the carbon molar yield can be increased to 0.75 mol/mol. The whole synthetic pathway was divided into two modules, one for converting acetate to glycolate and another to utilize glucose to provide NADPH. After engineering module I through activation of acs, gltA, aceA and ycdW, glycolate titer increased from 0.07 to 2.16 g/L while glycolate yields increased from 0.04 to 0.35 mol/mol-acetate and from 0.03 to 1.04 mol/mol-glucose. Module II was then engineered to increase NADPH supply. Through deletion of pfkA, pfkB, ptsI and sthA genes as well as upregulating zwf, pgl and tktA, glycolate titer increased from 2.16 to 4.86 g/L while glycolate yields increased from 0.35 to 0.82 mol/mol-acetate and from 1.04 to 6.03 mol/mol-glucose. The activities of AceA and YcdW were further increased to pull the carbon flux to glycolate, which increased glycolate yield from 0.82 to 0.92 mol/mol-acetate. Fed-batch fermentation of the final strain NZ-Gly303 produced 73.3 g/L glycolate with a productivity of 1.04 g/(L·h). The acetate to glycolate yield was 0.85 mol/mol (1.08 g/g), while glucose to glycolate yield was 6.1 mol/mol (2.58 g/g). The total carbon molar yield was 0.60 mol/mol, which reached 80% of the theoretical value.     

Engineering Pichia pastoris for biocatalysis: co-production of two active enzymes

High levels of active glycolate oxidase from spinach (GO) and active catalase T from Saccharomyces cerevisiae (catT) have been co-produced in the methylotrophic yeast Pichia pastoris (Pp). In sequential rounds of transformation using two selectable markers, multiple copies of the genes encoding GO and catT were integrated into the Pp chromosome under control of the methanol inducible alcohol oxidase I promoter, resulting in a strain designated MSP8.6. MSP8.6 is a second-generation biocatalyst used for the conversion of glycolate to glyoxylate in the presence of a reaction component which inhibits endogenous Pp catalase. This work demonstrates a significant advance in the utility of recombinant Pp for commercial bioprocess development.     

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C₃ and C₄ plant species, we analyzed kinetic parameters of purified recombinant C₃ (Arabidopsis thaliana) and C₄ (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The ¹²C/¹³C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the ¹³C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C₃ and C₄ enzymes.     

Evidence for the presence in tobacco leaves of multiple enzymes for the oxidation of glycolate and glyoxylate

The enzymic oxidation of glycolate to glyoxylate and glyoxylate to oxalate by preparations purified from tobacco (Nicotiana tabacum var Havana Seed) leaves was studied. The K_m values for glycolate and glyoxylate were 0.26 and 1.0 millimolar, respectively. The ratio of glycolate to glyoxylate oxidation was 3 to 4 in crude extracts but decreased to 1.2 to 1.5 on purification by (NH₄)₂SO₄ fractionation and chromatography on agarose A-15 and hydroxylapatite. This level of glyoxylate oxidation activity was higher than that previously found for glycolate oxidase (EC 1.1.3.1). The ratio of the two activities was changed by reaction with the substrate analog 2-hydroxy-3-butynoate (HBA) which at all concentrations inhibited glyoxylate oxidation to a greater extent than glycolate oxidation. The ratio of the two activities could also be altered by changing the O₂ concentration. Glycolate oxidation increased 3.6-fold when the O₂ atmosphere was increased from 21 to 100%, whereas glyoxylate oxidation increased only 1.6-fold under the same conditions. These changes in ratio during purification, on inhibition by HBA, and under varying O₂ concentrations imply that tobacco leaves contain at least two enzymes capable of oxidizing glycolate and glyoxylate.     

Enhanced Incorporation of Tritium into Glycolate during Photosynthesis by Tobacco Leaf Tissue in the Presence of Tritiated Water

Tobacco (Nicotiana tabacum var. Havana Seed) leaf discs were allowed to photosynthesize for 3 to 20 minutes in the presence of ¹⁴CO₂ and ³H₂O. Several metabolites of the Calvin cycle and photorespiratory pathway were isolated and purified and the ³H:¹⁴C values measured. Glycolate had a 5- to 10-fold higher ³H:¹⁴C than the Calvin cycle intermediate 3-phosphoglyceric acid, or its end product sucrose. The glycolate oxidase inhibitor α-hydroxy-2-pyridinemethanesulfonic acid caused glycolate to accumulate in the tissue and lowered the ³H:¹⁴C in glycolate to a value similar to that in 3-phosphoglyceric acid. Phosphoglycolate, a possible precursor of glycolate arising from the Calvin cycle, exhibited a ³H:¹⁴C value similar to 3-phosphoglyceric acid under all conditions. The finding of a ³H enrichment in glycolate suggests that another source of glycolate, possibly the reduction of glyoxylate, exists in leaf tissue. Analyses of incorporation of ³H into the pro-2R and pro-2S hydrogens of glycolate, in the presence and absence of α-hydroxy-2-pyridinemethanesulfonic acid, suggest an alternative source of glycolate. Biochemical mechanisms to account for ³H enrichment into glycolate are evaluated.     

Identity of aliphatic L- -hydroxyacid oxidase and glycolate oxidase from rat livers

l-α-Hydroxyacid oxidase and glycolate oxidase have been partially purified from rat livers and found to be identical, judging by substrate specificities, K_m values for certain substrates and coenzyme (FMN), activation energy, inhibition rates by various reagents and pH optimum. K_m values are as follows; glycolate, 2.4 × 10^?4m; l-α-hydroxyisocaproate, 1.26 × 10^?3; glyoxylate, 1.41 × 10^?4m; and FMN, 1.13 × 10^?6m. K_m values for glycolate and FMN are one-tenth and one-twentieth the literature values for hepatic glycolate oxidase. Sucrose density gradient centrifugation establishes that this enzyme is located in hepatic peroxisomes.     

Glycolate Formation and Excretion by Chlorella pyrenoidosa and Netrium digitus

Conditions are described whereby suspensions of Chlorella pyrenoidosa and Netrium digitus photosynthetically biosynthesize and excrete glycolate continuously in high yields. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate-linked enzymes, increased the excretion of glycolate approximately 4-fold in 1 hour (8 millimolar) and 20-fold in 4 hours (40 millimolar) in the presence of 0.2% CO₂ in air. The amount of glycolate excreted in the presence of aminooxyacetate and an atmosphere of 0.2% CO₂ in air equaled or exceeded the amount excreted in 0.2% CO₂ in O₂ minus aminooxyacetate. CO₂ and light were required for glycolate excretion. Aminooxyacetate also stimulated photosynthetic glycolate excretion in an atmosphere of 0.2% CO₂ in nitrogen or helium, although the stimulation was not as great as when air or O₂ was present.

The excreted glycolate was converted to H₂ and CO₂ by the combined action of glycolic oxidase and the formic hydrogenlyase complex found in Escherichia coli in total conversion yields of 80%.

     

Does glycolate decarboxylation occur in intact leaf peroxisomes?

Release of ¹⁴CO₂ during metabolism of [1-¹⁴C]glycolate was studied in purified, intact and Triton X-100-solublized peroxisomes isolated from leaves of Secale cereale. Decarboxylation, apparently resulting from H₂O₂ attack on glyoxylate, was stimulated in both intact and solubilized peroxisomal preparations by the catalase inhibitors aminotriazole and sodium azide. CO₂ evolution was also observed in solubilized peroxisomes in the absence of inhibitor when an amino donor was not provided for conversion of glyoxylate to glycine. Loss of CO₂ from labelled glycolate in the absence of amino donors was reduced by addition of exogenous catalase to the reaction medium.Intact peroxisomes showed no glycolate decarboxylation whether or not amino donors were supplied. It appears that the CO₂ release from glycolate observed in previous investigations may be an artifact of the peroxisomal preparations and the assay systems used, and not a significant factor in photorespiratory metabolism in vivo.     

Effects of aminoacetonitrile on net photosynthesis, ribulose-1,5-bisphosphate levels, and glycolate pathway intermediates

The effects of aminoacetonitrile (a competitive inhibitor of glycine oxidation) on net photosynthesis, glycolate pathway intermediates, and ribulose-1,5-bisphosphate (RuBP) levels have been investigated at different O₂ and CO₂ concentrations with soybean (Glycine max)[L] Merr. cv Pioneer 1677) leaf discs floated on 25 millimolar aminoacetonitrile (AAN) for 50 minutes prior to assay.

At 2% O₂ and 200 or 330 microliters per liter CO₂, the inhibitor had no effect on the rate of net photosynthesis and RuBP levels when compared with the control levels. At 11% to 60% O₂, AAN caused a decrease in net photosynthesis in addition to the inhibition by O₂. This extra inhibition ranged from 22% to 59% depending on the O₂ and CO₂ concentrations. The levels of RuBP, however, were 1.3 to 2.7 times higher than in the control plants at the same O₂ concentrations. At 40% O₂ and 200 microliters per liter CO₂, the inhibitor caused a 6-fold increase in glycine and more than 2-fold increase in glyoxylate levels, whereas those of glycolate decreased by approximately one-half.

The decrease in net photosynthesis observed with AAN is not the result of the depletion of the RuBP pool due to the lack of recycling of carbon from the glycolate pathway to the Calvin cycle. The higher levels of RuBP caused by AAN in photorespiratory conditions, suggest that RuBP carboxylase was inhibited. Glyoxylate could be a possible candidate for the inhibition of the enzyme but what is known so far about its inhibitory properties in vitro may not fit the existing in vivo conditions. An alternative explanation for the inhibition is proposed.

     

Formation of glycolate by a reconstituted spinach chloroplast preparation

A reconstituted preparation requiring fructose 6-phosphate, transketolase, triphosphopyridine nucleotide, ferredoxin, fragmented spinach chloroplasts, and light capable of forming glycolate at rates of about 10 micromoles per milligram of chlorophyll per hour has been characterized. The glycolaldehyde-transketolase addition product could be substituted for fructose 6-phosphate and transketolase. The stoichiometry of the reaction was: 1 mole of fructose 6-phosphate consumed for each mole of glycolate and of reduced triphosphopyridine nucleotide produced. Evidence was presented indicating that glycolate formation was coupled to the photosystems of the photosynthetic electron transport chain. Synthesis of glycolate is envisaged as the result of either (a) a reaction between the upper two carbon atoms derived from fructose 6-phosphate and an uncharacterized oxidant generated by photosystem 2 or (b) hydrogen peroxide produced by the reoxidation of reduced triphos-phopyridine nucleotide or reduced ferredoxin by molecular oxygen.     

Sequence of Formation of Phosphoglycolate and Glycolate in Photosynthesizing Chlorella pyrenoidosa

In Chlorella pyrenoidosa which have been photosynthesizing in either 1.5% ¹⁴CO₂ or 0.05% ¹⁴CO₂ in air, gassing with 100% O₂ results in rapid formation of phosphoglycolate which is apparently converted to glycolate. However, only about one-third to one-half of the rate of glycolate formation can be accounted for by this route. The remaining glycolate formation may be the result of the oxidation of sugar monophosphates. The rates of formation of both glycolate and phosphoglycolate are about four times greater with algae that have been photosynthesizing in 1.5% ¹⁴CO₂ than with algae which have been photosynthesizing with air, when the algae are then gassed with 100% O₂.     

Effect of butyl 2-hydroxy-3-butynoate on sunflower leaf photosynthesis and photorespiration

Detached leaves and whole plants of sunflower were supplied with butyl 2-hydroxy-3-butynoate (BHB), a competitive inactivator of glycolate oxidase, to evaluate the possibility of inhibiting photorespiration and increasing photosynthetic efficiency. In all treatments in vivo and in vitro, BHB inhibited glycolate oxidase. With partially purified glycolate oxidase from spinach leaves, the apparent K_i for BHB was 13.2 micromolar.     

Photosynthetic and Photorespiratory Carbon Metabolism in Mesophyll Protoplasts and Chloroplasts Isolated from Isogenic Diploid and Tetraploid Cultivars of Ryegrass (Lolium perenne L.)

Photosynthetic ¹⁴CO₂ fixation, [¹⁴C]glycolate formation, and the decarboxylation of [1-¹⁴C]glycolate and [1-¹⁴C]glycine by leaf mesophyll protoplasts isolated from isogenic diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) were examined. The per cent O₂ inhibition of photosynthesis in protoplasts from the tetraploid cultivar was less than that of the diploid line at both 21 and 49% O₂. Kinetic studies revealed that the K_m (CO₂) for photosynthesis by the diploid protoplasts was about twice that of the tetraploid line. In contrast, the K_i (O₂) for protoplast photosynthesis was similar in both cultivars, as was the potential for oxidizing glycolate and glycine to CO₂ via the photorespiratory carbon oxidation cycle. Although the maximal rates of glycolate accumulation by the isolated protoplasts in the presence of 21% O₂ and a glycolate oxidase inhibitor were similar in the two cultivars, the percentage of total fixed ¹⁴C entering the [¹⁴C]glycolate pool and the ratio of the rate of [¹⁴C]glycolate formation to ¹⁴CO₂ fixation at 21% O₂ and low pCO₂ were about two times greater in protoplasts and intact chloroplasts isolated from the diploid line compared to the tetraploid. These results fully support the recent observation that a doubling of ploidy in various ryegrass cultivars reduced the K_m (CO₂) of purified ribulose bisphosphate carboxylase-oxygenase by about one-half without affecting the K_i (O₂) (Garrett 1978 Nature 274: 913-915).     

The occurrence of glycolate dehydrogenase and glycolate oxidase in green plants: an evolutionary survey

Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O₂ uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance.