The Chlamydomonas reinhardtii gtr gene encoding the tetrapyrrole biosynthetic enzyme glutamyl-trna reductase: structure of the gene and properties of the expressed enzyme.
Plants, algae, cyanobacteria and many other bacteria synthesize the tetrapyrrole precursor, delta-aminolevulinic acid (ALA), from glutamate by means of a tRNAGlu-mediated pathway. The enzyme glutamyl-tRNA reductase (GTR) catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. Chlamydomonas reinhardtii mRNA encoding gtr was sequenced from a cDNA and genomic libraries. The 3179-bp gtr cDNA contains a 1566-bp open reading frame that encodes a 522-amino acid polypeptide. After removal of the predicted transit peptide, the mature 480-residue GTR has a calculated molecular weight of 52,502. The deduced C. reinhardtii mature GTR amino acid sequence has more than 55% identity to a GTR sequence of Arabidopsis thaliana, and significant similarity to GTR proteins of other plants and prokaryotes. Southern blot analysis of C. reinhardtii genomic DNA indicates that C. reinhardtii has only one gtr gene. Genomic DNA sequencing revealed the presence of a small intron near the putative transit peptide cleavage site. Expression constructs for the full-length initial gtr translation product, the mature protein after transit peptide removal, and the coding sequence of the second exon were cloned into expression vector that also introduced a C-terminal His6 tag. All of these constructs were expressed in E. coli, and both the mature protein and the exon 2 translation product complemented a hemA mutation. The expressed proteins were purified by Ni-affinity column chromatography to yield active GTR. Purified mature GTR was not inhibited by heme, but heme inhibition was restored upon addition of C. reinhardtii soluble proteins.
Srivastava
Alaka
A
Division of Biology and Medicine, Brown University, 02912, Providence, Rhode Island 02912, USA.
Lake
Vanessa
V
Nogaj
Luiza A
LA
Mayer
Sandra M
SM
Willows
Robert D
RD
Beale
Samuel I
SI
D000042:Absorption; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000622:Aminolevulinic Acid / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002384:Catalysis / Q000187:drug effects; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics*; D048028:DNA, Algal / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D004950:Esterases / Q000378:metabolism; D005816:Genetic Complementation Test; D006418:Heme / Q000032:analysis / Q000494:pharmacology; D006427:Hemin / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry / Q000379:methods; D045725:Tetrapyrroles / Q000096:biosynthesis*
D020418:Algal Proteins; D048028:DNA, Algal; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D045725:Tetrapyrroles; D006418:Heme; D006427:Hemin; D000622:Aminolevulinic Acid; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D004950:Esterases