Vertebrate Acyl CoA synthetase family member 4 (ACSF4-U26) is a β-alanine-activating enzyme homologous to bacterial non-ribosomal peptide synthetase.
Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for β-alanine, and a KM of ~ 5 μm was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that β-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.
Department of Metabolic Regulation, Institute of Biochemistry, Faculty of Biology, University of Warsaw, Poland.
Jakub
J
Drozak
Maria
M
Veiga-da-Cunha
Beata
B
Kadziolka
Emile
E
Van Schaftingen
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015091:beta-Alanine / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C116191:e protein, Drosophila; D015091:beta-Alanine; C588320:Aasdh protein, mouse; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D003066:Coenzyme A Ligases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
AASDH; ACSF4-U26; adenylation domain; non-ribosomal peptide synthetase; β-alanine