title: Trypanosoma brucei S-adenosylmethionine decarboxylase N terminus is essential for allosteric activation by the regulatory subunit prozyme.
abstract: Human African trypanosomiasis is caused by a single-celled protozoan parasite, Trypanosoma brucei. Polyamine biosynthesis is a clinically validated target for the treatment of human African trypanosomiasis. Metabolic differences between the parasite and the human polyamine pathway are thought to contribute to species selectivity of pathway inhibitors. S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in the production of the polyamine spermidine. We previously showed that trypanosomatid AdoMetDC differs from other eukaryotic enzymes in that it is regulated by heterodimer formation with a catalytically dead paralog, designated prozyme, which binds with high affinity to the enzyme and increases its activity by up to 10(3)-fold. Herein, we examine the role of specific residues involved in AdoMetDC activation by prozyme through deletion and site-directed mutagenesis. Results indicate that 12 key amino acids at the N terminus of AdoMetDC are essential for prozyme-mediated activation with Leu-8, Leu-10, Met-11, and Met-13 identified as the key residues. These N-terminal residues are fully conserved in the trypanosomatids but are absent from other eukaryotic homologs lacking the prozyme mechanism, suggesting co-evolution of these residues with the prozyme mechanism. Heterodimer formation between AdoMetDC and prozyme was not impaired by mutation of Leu-8 and Leu-10 to Ala, suggesting that these residues are involved in a conformational change that is essential for activation. Our findings provide the first insight into the mechanisms that influence catalytic regulation of AdoMetDC and may have potential implications for the development of new inhibitors against this enzyme.
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.
authors.forename: Nahir
authors.initials: N
authors.lastname: Velez
authors.forename: Chad A
authors.initials: CA
authors.lastname: Brautigam
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Phillips
mesh_terms: D012437:Adenosylmethionine Decarboxylase / Q000737:chemistry*; D000409:Alanine / Q000737:chemistry; D000495:Allosteric Site; D000595:Amino Acid Sequence; D002384:Catalysis; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000378:metabolism; D014353:Trypanosomiasis, African / Q000378:metabolism
chemical_list: D010455:Peptides; D011073:Polyamines; D012437:Adenosylmethionine Decarboxylase; D000409:Alanine