Heme Biosynthesis in Fish and Land Vertebrates: Enzyme and cDNA Comparisons.

Abstract

Heme is required by all living organisms for vital processes such as electron transport in mitochondrial oxidative phosphorylation and chloroplast photosynthesis, metabolism of hydroperoxides, transport of respiratory oxygen, and metabolism of toxicological and pharmacological chemicals as well as natural hydrophobic materials, e.g., steroid hormones. Each cell has to synthesize the heme needed for those processes. However, the rate of production must be well-regulated because excesses of either free heme or its precursors can have adverse consequences as evidenced by human porphyrias-diseases of heme metabolism that can be caused by inherited gene defects or by environmental pollutants (1). Some regulatory features of heme biosynthesis are known. The biosynthetic pathway includes eight enzymatic reactions, and, in common laboratory species (rat, chicken), the first enzyme-5-aminolevulinate synthase (ALS)-plays the major role in determining the overall rate of heme production (2). Those animals have two forms of ALS: the erythroid type (E-ALS), which is expressed only in differentiating red blood cells, and a mitochondrial, housekeeping form (H-ALS) that occurs in almost all tissues. H-ALS is inducible by drugs and xenobiotics, e.g., barbiturates and polychlorinated biphenyls (3). Most important, heme influences the rate of its own production via feedback regulation of the tissue content of ALS, but the mechanism of that action has not been established. Among the few studies in fish, results with rainbow trout indicated that as much as 30% of total hepatic ALS was cytoplasmic and that the hepatic activity of the biosynthetic pathway’s second enzyme, aminolevulinate dehydratase (ALD), was much lower than that of ALS (4). This implied that fish heme synthesis has regulatory features that differ significantly from those in mammals. That report led us to measure the liver activity and subcellular distribution of ALS and ALD in rainbow trout, Oncorhynchus mykiss, and the marine teleost Opsanus tag (oyster toadfish). Our measurements showed that, in either fish, total liver activity of ALS is about 60-fold less than that of ALD. Subcellular distribution of hepatic ALS, measured with reference to marker enzymes for the cytoplasmic and mitochondrial compartments, indicated that this enzyme is more than 92% mitochondrial in toadfish and 100% mitochondrial in trout. These results show that rates of heme biosynthesis in teleosts, as in mammals, are limited by the activity of the mitochondrial enzyme, ALS. Known inducers of ALS in terrestrial vertebrates include succinyl acetone, an inactivator of ALD (5), and N-methylprotoporphyrin IX and 3,5-diethoxy-carbonyl-1,4-dihydrocollidine, both of which inhibit ferrochelatase, the last of the eight enzymes (3). In cultured toadfish liver cells, the last two compounds increased ALS activity, respectively, by 7and 3-fold. Succinyl acetone is particularly potent; at 0.5 mM it increased ALS activity of cultured toadfish liver cells by almost 20-fold, and that increase was completely blocked by 10 &I’ heme added to the medium. Similar results were obtained with cultured hepatoma cells from the topminnow, Poeciliopsis lucida. These inductions, and the apparent feedback action of heme, resemble regulatory phenomena observed in terrestrial vertebrates. A surprising difference, however, is that northern blots show that the induction of the fish enzyme by succinyl acetone occurs without a concomitant increase in the ALS mRNA. Thus, there probably are important posttranscriptional events in the regulation of heme biosynthesis in fish. ALS is an enzyme of very low abundance in toadfish, as it is in other vertebrates. Consequently, recombinant DNA techniques were employed to obtain information about protein structure. We cloned cDNAs for both the E and H forms of toadfish ALS. Two criteria were used to confirm that the cDNAs encode ALS protein. One was that each cDNA gave rise to enzymatically active ALS when expressed in wild-type E. coli, and the other was that each was able to complement a mutation in the hem A strain, which otherwise cannot grow in the absence of added aminolevulinate, the product of the ALS reaction (6). The cDNAs were sequenced and have open reading frames that encode proteins with molecular weights of 65,000 and 70,000, in good agreement with the ALS subunit sizes in chickens (7) mice (8) rats (9), and humans (10). Amino acid sequence alignments indicate that two-thirds of the ALS protein at the carboxylterminal is highly conserved among vertebrates. That region shows 62 to 77% amino acid identity among E forms, and 80 to 82% identity among H forms of toadfish, birds, and mammals. Further studies, in progress, will define the regulation of heme synthesis in marine fish. Our observations suggest that those studies will also help us to understand this process in humans and other mammals.