Comp. Biochem. Physiol. Vol. 72B, No. 4, pp. 663 to 667, 1982 0305-0491/82/080663-05503.00/0 Printed in Great Britain © 1982 Pergamon Press Ltd PORPHYRIN BIOSYNTHESIS IN PARASITIC HEMOFLAGELLATES: FUNCTIONAL AND DEFECTIVE ENZYMES IN TRYPANOSOMA CRUZI TERESA A. SALZMAN, 1 ANA M. STELLA, 1 EVA A. WIDER DE XIFRA 1, ALCIRA M. DEL C. BATLLE 1, ROBERTO DOCAMPO 2 and ANDRES O. M. STOPPANI 2 lCentro de Investigaciones sobre Porfirinas y Porfirias (CIPYP), CONICET and FCEN, UBA, Ciudad Universitaria, Pab. II. 1428 Buenos Aires, Argentina, and 2Centro de Investigaciones Bioenerg6ticas (CIBIERG), CONICET and FCM, UBA, Paraguay 2155, 1121 Buenos Aires, Argentina Abstract 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 support- ing evidence for this hypothesis, an extensive study of porphyrin biosynthesis in the epimastogote form of T. cruzi (Tulahu6n strain) was carried out. 2. Low levels of endogenous 6-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 6-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. ~-Aminolevulinic acid dehydratase (ALA-D) activity was almost negligible and both porphobilino- genase (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. INTRODUCTION Porphyrins are involved in two essential processes of life, with major bioenergetic functions: photosynthesis and cellular respiration. That is why, except for cer- tain prokaryotes and viruses, organisms are normally capable of synthesizing tetrapyrroles. A singular nu- tritional characteristic of parasitic hemoflagellates however, is, that they need heme compounds as a growth factor in vitro, generally supplied to them as hemoglobin, hematin or hemin (Lwoff, 1951). The reasons for this porphyrin requirement in different parasitic protozoa are not yet elucidated, and might vary from one to another. It is commonly accepted that it must be due to their inability to synthesize heme, with either the whole pathway being non-func- tional or some part of it being defective. However, supporting experimental evidence for this assumption is fragmentary and very little is known about the ability of these parasites to synthesize their essential heme compounds, Cytochromes (aa3, b and c558) are essential con- stituents of the Trypanosoma cruzi respiratory mech- anism (Boiso and Stoppani, 1973; Docampo et al., 1978; Stoppani et al., 1980). Consequently, we con- sidered that an extensive study of the porphyrin bio- synthesis in this heme flagellate was warranted with the aim of determining its capacity for making these vital pigments. Since the chemotherapy of Chagas' desease is still an unsolved problem, the elucidation of the functionality of the porphyrin pathway in T. cruzi might be of importance in the development of new trypanocidal agents, which selectively block either the formation or utilization of heme by the parasite. Taking into account the currently accepted por- phyrin pathway (Reaction 1), Suc + CoASH + ATP DOVA DOVA-T Suc. Coa-S ALA-S ALA-D Suc. CoA + Glycine ~ ALA - , PBG ~UROGEN I PBG .jPBGase Urogen Dec.ase , UROGEN III ~ ~ ~ ----, COPROGEN III Reaction 1. Porphyrin pathway. C.B.P. 72/4B L 663 CPGase , PROTOGEN IX Ppgen-ox ,L PROTO + Fe Herne-S ,~ HEME