Porphyrin biosynthesis in parasitic hemoflagellates: functional and defective enzymes in Trypanosoma cruzi.

1. Heme compounds are necessary as a growth factor for Trypanosoma cruzi in culture, this porphyrin requirement being due to the inability of the parasite to synthesize heme. To obtain supporting evidence for this hypothesis, an extensive study of porphyrin biosynthesis in the epimastogote form of T. cruzi (Tulahuén strain) was carried out. 2. Low levels of endogenous delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) were found in extracts of T. cruzi. Free porphyrins and heme contents were practically nil. 3. The activity of succinyl CoA synthetase (Suc. CoA-S) was rather high and therefore non-limiting. 4. Both delta-aminolevulinic acid synthetase (ALA-S) and 4.5, dioxovaleric transaminase (DOVA-T), the two enzymes forming ALA, were readily detected and their activities, although low, were of the same order. 5. delta-Aminolevulinic acid dehydratase (ALA-D) activity was almost negligible and both porphobilinogenase (PBGase) and deaminase were absent or inactive. 6. Heme-Synthetase (Heme-S) was totally functional. 7. It is concluded that T. cruzi has lost part of its heme biosynthetic pathway, possibly due to mutations of several genes involved in the synthesis of the soluble enzymes ALA-D, PBGase, deaminase and probably others preceding Heme-S; while the particulate enzymes Suc CoA-S, ALA-S, DOVA-T and Heme-S are functional. As a consequence, the host should supply the parasite with the porphyrin substrate to form its essential heme compounds.

[Bacteriochlorophyll a synthesis in Rhodopseudomonas palustris]. / Biosíntesis de bacterioclorofila a en Rhodopseudomonas palustris.

By means of electron microscope it was demonstrated that photosynthetically-grown Rhodopseudomonas palustris exhibits an intracytoplasmic membrane system (Figure 1a), which is not observed in aerobically-dark grown bacteria (Figure 1b). The content of bacteriochlorophyll alpha and the enzyme activity of succinil-CoA-synthetase, ALA-sinthetase and ALA-dehydratase in several media grown Rhodopseudomonas palustris could be measured. Aerobically-dark grown cells do not synthetize bacteriochlorophyll alpha and the activity of ALA-synthetase is lower than in photosynthetically-grown cells (Table 1), suggesting a possible regulatory role for this enzyme in the pigment biosynthesis. Some inhibitors of electron transport and uncouplers of photophosphorylation inhibit both the bacterial growth and bacteriochlorophyll alpha biosynthesis (Table 2), while the levels of ALA-synthetase are not affected. If the incubation in the presence of these kinds of compounds is prolongated, the effects disappear. Although the regulatory role of ALA-synthetase should be very important, apparently it would not be the unique regulatory factor for bacteriochlorophyll alpha biosynthesis in Rhodopseudomonas palustris.

Biosíntesis de bacterioclorofila a en Rhodopseudomonas palustris. / [Bacteriochlorophyll a synthesis in Rhodopseudomonas palustris]

By means of electron microscope it was demonstrated that photosynthetically-grown Rhodopseudomonas palustris exhibits an intracytoplasmic membrane system (Figure 1a), which is not observed in aerobically-dark grown bacteria (Figure 1b). The content of bacteriochlorophyll alpha and the enzyme activity of succinil-CoA-synthetase, ALA-sinthetase and ALA-dehydratase in several media grown Rhodopseudomonas palustris could be measured. Aerobically-dark grown cells do not synthetize bacteriochlorophyll alpha and the activity of ALA-synthetase is lower than in photosynthetically-grown cells (Table 1), suggesting a possible regulatory role for this enzyme in the pigment biosynthesis. Some inhibitors of electron transport and uncouplers of photophosphorylation inhibit both the bacterial growth and bacteriochlorophyll alpha biosynthesis (Table 2), while the levels of ALA-synthetase are not affected. If the incubation in the presence of these kinds of compounds is prolongated, the effects disappear. Although the regulatory role of ALA-synthetase should be very important, apparently it would not be the unique regulatory factor for bacteriochlorophyll alpha biosynthesis in Rhodopseudomonas palustris.

Biosíntesis de bacterioclorofila a en Rhodopseudomonas palustris / [Bacteriochlorophyll a synthesis in Rhodopseudomonas palustris]

By means of electron microscope it was demonstrated that photosynthetically-grown Rhodopseudomonas palustris exhibits an intracytoplasmic membrane system (Figure 1a), which is not observed in aerobically-dark grown bacteria (Figure 1b). The content of bacteriochlorophyll alpha and the enzyme activity of succinil-CoA-synthetase, ALA-sinthetase and ALA-dehydratase in several media grown Rhodopseudomonas palustris could be measured. Aerobically-dark grown cells do not synthetize bacteriochlorophyll alpha and the activity of ALA-synthetase is lower than in photosynthetically-grown cells (Table 1), suggesting a possible regulatory role for this enzyme in the pigment biosynthesis. Some inhibitors of electron transport and uncouplers of photophosphorylation inhibit both the bacterial growth and bacteriochlorophyll alpha biosynthesis (Table 2), while the levels of ALA-synthetase are not affected. If the incubation in the presence of these kinds of compounds is prolongated, the effects disappear. Although the regulatory role of ALA-synthetase should be very important, apparently it would not be the unique regulatory factor for bacteriochlorophyll alpha biosynthesis in Rhodopseudomonas palustris.

Porphyrin biosynthesis: immobilized enzymes and ligands. VI. Studies on succinyl CoA synthetase from cultured soya bean cells.

Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme.