Compartmentation studies on spinach leaf peroxisomes : evidence for channeling of photorespiratory metabolites in peroxisomes devoid of intact boundary membrane

In concurrence with earlier results, the following enzymes showed latency in intact spinach (Spinacia oleracea L.) leaf peroxisomes: malate dehydrogenase (89%), hydroxypyruvate reductase (85%), serine glyoxylate aminotransferase (75%), glutamate glyoxylate aminotransferase (41%), and catalase (70%). In contrast, glycolate oxidase was not latent. Aging of peroxisomes for several hours resulted in a reduction in latency accompanied by a partial solubilization of the above mentioned enzymes. The extent of enzyme solubilization was different, being highest with glutamate glyoxylate aminotransferase and lowest with malate dehydrogenase. Osmotic shock resulted in only a partial reduction of enzyme latency. Electron microscopy revealed that the osmotically shocked peroxisomes remained compact, with smaller particle size and pleomorphic morphology but without a continuous boundary membrane. Neither in intact nor in osmotically shocked peroxisomes was a lag phase observed in the formation of glycerate upon the addition of glycolate, serine, malate, and NAD. Apparently, the intermediates, glyoxylate, hydroxypyruvate, and NADH, were confined within the peroxisomal matrix in such a way that they did not readily leak out into the surrounding medium. We conclude that the observed compartmentation of peroxisomal metabolism is not due to the peroxisomal boundary membrane as a permeability barrier, but is a function of the structural arrangement of enzymes in the peroxisomal matrix allowing metabolite channeling.     

Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.     

Metabolism of glycolate and glyoxylate in intact spinach leaf peroxisomes

Intact and broken (osmotically disrupted) spinach (Spinacia oleracea) leaf peroxisomes were compared for their enzymic activities on various metabolites in 0.25 molar sucrose solution. Both intact and broken peroxisomes had similar glycolate-dependent o₂ uptake activity. In the conversion of glycolate to glycine in the presence of serine, intact peroxisomes had twice the activity of broken peroxisomes at low glycolate concentrations, and this difference was largely eliminated at saturating glycolate concentrations. However, when glutamate was used instead of serine as the amino group donor, broken peroxisomes had slightly higher activity than intact peroxisomes. In the conversion of glyoxylate to glycine in the presence of serine, intact peroxisomes had only about 50% of the activity of broken peroxisomes at low glyoxylate concentrations, and this difference was largely overcome at saturating glyoxylate concentrations. In the transamination between alanine and hydroxypyruvate, intact peroxisomes had an activity only slightly lower than that of broken peroxisomes. In the oxidation of NADH in the presence of hydroxypyruvate, intact peroxisomes were largely devoid of activity. These results suggest that the peroxisomal membrane does not impose an entry barrier to glycolate, serine, and O₂ for matrix enzyme activity; such a barrier does exist to glutamate, alanine, hydroxypyruvate, glyoxylate, and NADH. Furthermore, in intact peroxisomes, glyoxylate generated by glycolate oxidase is channeled directly to glyoxylate aminotransferase for a more efficient glycolate-glycine conversion. In related studies, application of in vitro osmotic stress to intact or broken peroxisomes had little effect on their ability to metabolize glycolate to glycine.     

Development of Enzymes of the Glyoxylate Cycle during Senescence of Pumpkin Cotyledons

The presence and activities of isocitrate lyase (EC 4.1.3.1 [EC] )and malate synthase (EC 4.1.3.2 [EC] ) were studied during senescenceof pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). Afterincubation of detached cotyledons in permanent darkness, theactivities appeared and increased up to the eighth day and thendeclined, while the activities of catalase (EC 1.11.1.6 [EC] ), glycolateox-idase (EC 1.1.3.1 [EC] ), and hydroxypyruvate reductase (EC 1.1.1.81 [EC] )decreased dramatically. After fractionation of cell organellesby sucrose density gradient, we detected isocitrate lyase andmalate synthase activities in peroxisomal fractions. The activityof the two key enzymes of the glyoxylate cycle also increasedduring senescence in vivo and we confirmed the presence of thetwo enzymes in the peroxisomal fractions after sucrose gradientcentrifugation. At every point examined, the level of malatesynthase was demonstrated by immunoblotting. It is concludedthat the development of isocitrate lyase and malate synthaseactivities represents the transition from leaf peroxisomes toglyoxysomes and that such a phenomenon is associated with senescence. (Received January 25, 1991; Accepted March 22, 1991)     

Glycolate and glyoxylate metabolism by isolated peroxisomes or chloroplasts

Chloroplasts, mitochondria, and peroxisomes from leaves were separated by isopycnic sucrose density gradient centrifugation. The peroxisomes converted glycolate-¹⁴C or glyoxylate-¹⁴C to glycine, and contained a glutamate: glyoxylate aminotransferase as indicated by an investigation of substrate specificity. The pH optimum for the aminotransferase was between 7.0 and 7.5, and the Km for l-glutamate was 3.6 mm and for glyoxylate, 4.4 mm. The reaction of glutamate plus glyoxylate was not reversible. The isolated peroxisomes did not convert glycine to glyoxylate nor glycine to serine.     

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.

     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demonstrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Identification of mammalian aminotransferases utilizing glyoxylate or pyruvate as amino acceptor. Peroxisomal and mitochondrial asparagine aminotransferase

The subcellular distribution of asparagine:oxo-acid aminotransferase (EC 2.6.1.14) in rat liver was examined by centrifugation in a sucrose density gradient. About 30% of the homogenate activity after the removal of the nuclear fraction was recovered in the peroxisomes, about 56% in the mitochondria, and the remainder in the soluble fraction from broken peroxisomes. The mitochondrial asparagine aminotransferase had identical immunological properties with the peroxisomal one. Glucagon injection to rats resulted in the increase of its activity in the mitochondria but not in the peroxisomes. Immunological evidence was obtained that the enzyme was identical with alanine:glyoxylate aminotransferase 1 (EC 2.6.1.44) which had been reported to be identical with serine:pyruvate aminotransferase (EC 2.6.1.51) (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The same results as described above were obtained with mouse liver. All of alanine:glyoxylate aminotransferase 1 in livers of mammals other than rodents, which cross-react with the antibody against rat liver alanine:glyoxylate aminotransferase 1, had no asparagine aminotransferase activity.     

Isolation of serine:glyoxylate aminotransferase from cucumber cotyledons

Serine:glyoxylate aminotransferase, a marker enzyme for leaf peroxisomes, has been purified to homogeneity from cucumber cotyledons (Cucumis sativus cv Improved Long Green). The isolation procedure involved precipitation with polyethyleneimine, a two-step ammonium sulfate fractionation (35 to 45%), gel filtration on Ultrogel AcA 34, and ion exchange chromatography on diethylaminoethyl-cellulose, first in the presence of pyridoxal-5-phosphate, and then in its absence. The enzyme was purified approximately 690-fold to a final specific activity of 34.4 units per milligram. Electrophoresis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gels revealed two polypeptide bands with apparent molecular weights of approximately 47,000 and 45,000. Both polypeptides coeluted with enzyme activity under all chromatographic conditions investigated, both were localized to the peroxisome, and both accumulated in cotyledons as enzyme activity increased during development. The two polypeptides appear not to be structurally related, since they showed little immunological cross-reactivity and gave rise to different peptide fragments when subjected to partial proteolytic digestion. Antiserum raised against either the denatured enzyme or the 45,000-dalton polypeptide did not react with any other polypeptides present in a crude cotyledonary homogenate. The purified enzyme also had alanine:glyoxylate aminotransferase activity, but was about twice as active with serine as the amino donor.     

An activated-oxygen-mediated role for peroxisomes in the mechanism of senescence of Pisum sativum L. leaves

The possible involvement of peroxisomes and their activated-oxygen metabolism in the mechanism of leaf senescence was investigated in detached pea (Pisum sativum L.) leaves which were induced to senesce by incubation in complete darkness for up to 11 d. At days 0, 3, 8, and 11 of senescence, peroxisomes were purified from leaves and the activities of different peroxisomal and glyoxysomal enzymes were measured. Xanthine-oxidoreductase activity increased with senescence, especially the O ₂ ^{. -} -producing xanthine oxidase (EC 1.1.3.22). The activities of H₂O₂-generating Mn-superoxide dismutase (EC 1.15.1.1) and urate oxidase (EC 1.7.3.3) were also enhanced by senescence, whereas catalase (EC 1.11.1.6) activity was severely depressed. Hydrogen peroxide concentrations increased significantly in senescent leaf peroxisomes. During the progress of senescence, glycollate oxidase (EC 1.1.3.1) and hydroxypyruvate reductase (EC 1.1.1.81), two marker enzymes of photorespiratory metabolism, gradually decreased in activity and disappeared. At the same time, the activities of malate synthase (EC 4.1.3.2) and isocitrate lyase (EC 4.1.3.1), key enzymes of the glyoxylate cycle, which were undetectable in presenescent leaves, increased dramatically upon induction of senescence. Ultrastructural studies of intact leaves showed that the population of peroxisomes and mitochondria increased with senescence. Results indicate that peroxisomes could play a role, mediated by activated oxygen species, in the oxidative mechanism of leaf senescence, and further support the idea, proposed by other authors, that foliar senescence is associated with the transition of leaf peroxisomes into glyoxysomes.Abbreviation Mn-SOD (manganese-containing) superoxide dismutase The authors thank Dr. A.J. Sánchez-Raya (Unidad de Fisiología Vegetal, Estación Experimental del Zaidín, Granada, Spain) for his valuable help in measuring ethylene production, and Dr. G. Barja de Quiroga (Departamento de Biología Animal II, Universidad Complutense, Madrid, Spain) for carrying out the malondialdehyde determinations by HPLC. This work was supported by grant PB87-0404-01 from the DGICYT and the Junta de Andaluc'ia (Research Group # 3315), Spain.     

Subcellular distribution of alanine aminotransferase activity in maize (Zea mays L.) leaves

The intracellular distribution of alanine aminotransferase (AlaAT, EC 2.6.1.2) activity with L-alanine and 2-oxoglutarate as a substrates in maize whole leaf extract and bundle sheath cells was studied. After isolation of the mitochondrial-peroxisomal fraction, mitochondria and peroxisomes were separated by centrifugation on a linear 40–52 % (w/w) sucrose gradient. L-Alanine-2-oxoglutarate transaminating activity of whole leaf extract showed two peaks: first distinctly higher associated with mitochondria and second lower with peroxisomes. In bundle sheath cells only one peak of this activity was found. It corresponded to the mitochondrial region of the gradient. It is proposed that mitochondrial L-alanine — 2-oxoglutarate activity was brought about by AlaAT. Glycine aminotransferase (EC 2.6.1.4) could be responsible for the same activity in peroxisomes. This work was supported by the State Committee for Scientific Research, a grant No. 5PO6A00510     

Development of glutamate:glyoxylate aminotransferase in the cotyledons of cucumber (Cucumis sativus) seedlings.

Glutamate:glyoxylate aminotransferase had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration (Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. Glutamate:glyoxylate aminotransferase activity was already present in the etiolated cotyledons of cucumber (Cucumis sativus) seedlings, and increased during greening. The enzyme was present only in the cytosol of the etiolated cotyledons and appeared in the peroxisomes during greening. The enzyme was purified to homogeneity from the cytosol of the etiolated cotyledons and from the peroxisomes of the green cotyledons of cucumber seedlings. The two enzyme preparations had nearly identical enzymic and physical properties. On the basis of these findings, roles of glutamate:glyoxylate aminotransferase in the glycollate pathway in photorespiration, and the mechanism of its appearance in the peroxisomes during greening, are discussed.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demontrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Alanine:glyoxylate aminotransferase is present as the apoenzyme in the peroxisomes of chicken kidney

The subcellular distribution of alanine:glyoxylate aminotransferase in chicken kidney was examined by centrifugation in a sucrose density gradient. The enzyme was found to be present as the apoform in the peroxisomes and as the holoform in the mitochondria. Alanine:glyoxylate aminotransferase in different mammalian kidneys were all present as the holoenzyme in the mitochondrial and soluble fractions.     

Subcellular Location of NADPH-Dependent Hydroxypyruvate Reductase Activity in Leaf Protoplasts of Pisum sativum L. and Its Role in Photorespiratory Metabolism

Protoplasts purified from pea (Pisum sativum L.) leaves were lysed and fractionated to assess the subcellular distribution of NADPH-dependent hydroxypyruvate reductase (NADPH-HPR) activity. Rate-zonal centrifugation and sucrose-gradient experiments demonstrated that most (about 70%) of the NADPH-HPR activity was located in the supernatant or cytosol fraction. Detectable, but relatively minor activities were associated with the chloroplast fraction (up to 10% on a chlorophyll basis when compared to the lysate) and with peroxisomes. The minor NADPH-HPR activity in the peroxisomes could be fully accounted for by the secondary NADPH-dependent activity of NADH-dependent HPR. The subcellular distribution of NADPH-HPR followed closely that previously determined for NADPH-dependent glyoxylate reductase (NADPH-GR), an enzyme localized predominantly in the cytosol of pea leaf protoplasts (CV Givan et al. 1988 J Plant Physiol 132: 593-599). Low activities of both NADPH-HPR and NADPH-GR were also found in purified chloroplasts prepared by mechanical homogenization of Pisum and Spinacia leaves. In pea and spinach chloroplasts, rates of both NADPH-HPR and NADPH-GR were lower than the activity of the NADH-dependent GR. The results are discussed in relation to a possible role for NADPH-HPR in the oxidative carbon pathway of photorespiration. Both NADPH-HPR and the GRs could function as auxiliary reactions to photorespiration, utilizing hydroxypyruvate and/or glyoxylate `leaked' or otherwise exported from peroxisomes. NADPH-HPR function might be especially significant under conditions of limiting NADH supply to peroxisomes, with extraperoxisomal reduced pyridine nucleotide acting as the reductant.     

Purification and characterization of hydroxypyruvate reductase from cucumber cotyledons

Hydroxypyruvate reductase (HPR), a marker enzyme of peroxisomes, has been purified to homogeneity from cotyledons of light-grown cucumber seedlings (Cucumis sativus var. Improved Long Green). In addition, the peroxisomal location of both HPR and serine-glyoxylate aminotransferase has been confirmed in cucumber cotyledons. The isolation procedure involved Polymin-P precipitation, a two-step precipitation with ammonium sulfate (35 and 50% saturation), affinity chromatography on Cibacron Blueagarose, and ion-exchange chromatography on DEAE-cellulose. HPR was purified 541-fold to a final specific activity of 525 ± 19 micromoles per minute per milligram of protein. Enzyme homogeneity was established by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was 91 to 95 kilodaltons, approximately double the apparent subunit molecular weight of 40,500 ± 1,400. With hydroxypyruvate as substrate, the pH optimum was 7.1 and K_m values were 62 ± 6 and 5.8 ± 0.7 micromolar for hydroxypyruvate and NADH, respectively. With glyoxylate as substrate, the pH optimum was 6.0, and the K_m values for glyoxylate and NADH were 5700 ± 600 and 2.9 ± 0.5 micromolar, respectively. Antibodies to HPR were raised in mice (by the ascites tumor method) and in rabbits, and their monospecificity was demonstrated by a modified Western blot immunodetection technique.     

Metabolism of Glycolate in Isolated Spinach Leaf Peroxisomes : KINETICS OF GLYOXYLATE, OXALATE, CARBON DIOXIDE, AND GLYCINE FORMATION

The flow of glyoxylate derived from glycolate into various metabolic routes in the peroxisomes during photorespiration was assessed. Isolated spinach leaf peroxisomes were fed [¹⁴C] glycolate in the absence or presence of exogenous glutamate, and the formation of radioactive glyoxylate, CO₂, glycine, oxalate, and formate was monitored at time intervals. In the absence of glutamate, 80% of the glycolate was consumed within 2 hours and concomitantly glyoxylate accumulated; CO₂, oxalate, and formate each accounted for less than 5% of the consumed glycolate. In the presence of equal concentration of glutamate, glycolate was metabolized at a similar rate, and glycine together with some glyoxylate accumulated; CO₂, oxalate, and formate each accounted for an even lesser percentage of the consumed glycolate. CO₂ and oxalate were not produced in significant amounts even in the absence of glutamate, unless glycolate had been consumed completely and glyoxylate had accumulated for a prolonged period. These in vitro findings are discussed in relation to the extent of CO₂ and oxalate generated in leaf peroxisomes during photorespiration.     

Presence of amino acid dehydrogenases and transaminases in Myxococcus xanthus during vegetative growth and myxospore formation

The following enzymatic activities were demonstrated in Myxococcus xanthus during growth and myxospore formation: l-alanine dehydrogenase (EC 1.4.1.1), l-glutamic acid dehydrogenase (EC 1.4.1.2), glycine dehydrogenase (EC 1.4.1.5), l-glutamic glyoxylate aminotransferase (EC 2.6.1.4), and l-alanine glyoxylate aminotransferase (EC 2.6.1.12).     

Characterization of peroxisomes in Tetrahymena pyriformis

Peroxisomes from Tetrahymena pyriformis contained catalase, d-amino acid oxidase, cyanide-insensitive fatty acyl-CoA oxidizing system, carnitine acetyltransferase, isocitrate lyase, leucine:glyoxylate aminotransferase and phenylalanine:glyoxylate aminotransferase. These activities, except carnitine acetyltransferase, were found at the highest levels in the light mitochondrial fraction, whereas the highest activity of carnitine acetyltransferase was found in the micotchondrial fraction. Sucrose density gradient centrifugation showed that the density of peroxisomes was approx. 1.228 g/ml and that of mitochondria was approx. 1.213 g/ml. When the light mitochondrial fraction was treated with deoxycholate or by freeze-thawing, most of the activities of catalase and isocitrate lyase were solubilized, whereas about half of the original activity of aminotransferase remained in the pellet fraction. Addition of fatty acid and clofibrate increased the activities of the cyanide-insensitive fatty acyl-CoA oxidizing system and isocitrate lyase in the peroxisomes. The activity of catalase was slightly increased by glucose and clofibrate; leucine:glyoxylate aminotransferase activity was significantly increased by clofibrate treatment.     

Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana

Leaf peroxisomes are present in greening cotyledons and contain enzymes of the glycolate pathway that functions in photorespiration. However, only a few leaf peroxisomal proteins, that is hydroxypyruvate reductase (HPR), glycolate oxidase (GO) and alanine:glyoxylate aminotransferase 1 (AGT1), have been characterized, and other functions in leaf peroxisomes have not been solved. To better understand the functions of leaf peroxisomes, we established a method to isolate leaf peroxisomes of greening cotyledons. We analyzed 53 proteins by MALDI-TOF MS and then identified 29 proteins. Among them, five proteins are related to the glycolate pathway, four proteins function in scavenging of hydrogen peroxide and additionally 20 novel leaf peroxisomal proteins were identified. In particular, protein kinases and protein phosphatase were first identified as peroxisomal proteins suggesting that protein phosphorylation is one of the regulatory mechanisms in leaf peroxisomes. Novel leaf peroxisomal proteins contained five PTS1-like proteins that have sequences where one amino acid is substituted with another one in PTS1 sequences. The PTS1 motif was suggested to have novel PTS1 sequences.