Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp.
Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.
Atteia
Ariane
A
Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.
van Lis
Robert
R
Beale
Samuel I
SI
D000595:Amino Acid Sequence; D000622:Aminolevulinic Acid / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001483:Base Sequence; D018929:Cell Culture Techniques; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism; D048028:DNA, Algal / Q000032:analysis; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D056890:Eukaryota / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D019143:Evolution, Molecular; D005294:Ferrochelatase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D005816:Genetic Complementation Test; D006418:Heme / Q000096:biosynthesis*; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000737:chemistry
D000906:Antibodies; D048028:DNA, Algal; D018076:DNA, Complementary; D011506:Proteins; D006418:Heme; D000622:Aminolevulinic Acid; D044925:Oxidoreductases Acting on CH-CH Group Donors; D005294:Ferrochelatase; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase