The Chlamydomonas reinhardtii gtr gene encoding the tetrapyrrole biosynthetic enzyme glutamyl-trna reductase: structure of the gene and properties of the expressed enzyme.

摘要: 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 first committed step in this pathway, which is reduction tRNA-bound to produce 1-semialdehyde. Chlamydomonas reinhardtii mRNA encoding gtr was sequenced cDNA genomic libraries. 3179-bp contains 1566-bp open reading frame that encodes 522-amino polypeptide. After removal predicted transit peptide, mature 480-residue GTR has calculated molecular weight 52,502. deduced C. amino sequence more than 55% identity Arabidopsis thaliana, significant similarity proteins plants prokaryotes. Southern blot analysis DNA indicates only one gene. Genomic sequencing revealed presence small intron near putative peptide cleavage site. Expression constructs for full-length initial translation product, protein after removal, coding second exon were cloned into expression vector also introduced C-terminal His6 tag. All these expressed E. coli, both 2 product complemented hemA mutation. purified Ni-affinity column chromatography yield active GTR. Purified not inhibited heme, but heme inhibition restored upon addition soluble proteins.