Vertebrate Acyl CoA synthetase family member 4 (ACSF4‐U26) is a β‐alanine‐activating enzyme homologous to bacterial non‐ribosomal peptide synthetase

Abstract

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 b-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates b-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled b-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 b-alanine, and a K M of~5 lM 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 b-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a b-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.

SETD3 protein is the actin-specific histidine N-methyltransferase

“…Protein methylation was linear for at least 15 min under all of the conditions studied. By analogy to assays of nonribosomal peptide synthetase activity ( Richardt et al, 2003 ; Drozak et al, 2014 ), the incubation was stopped by the addition of 0.1 ml of the reaction mixture to 0.025 ml of bovine serum albumin (BSA) (1 mg) and 0.8 ml of ice-cold 10% (w/v) trichloroacetic acid (TCA). After 10 min on ice, the precipitate was pelleted and washed twice with ice-cold 10% TCA.…”

The biosynthetic diversity of the animal world

“…Unusual C termini are also found in a second group of animal single-module NRPS, where in place of a condensation domain, there is a pyrroloquinoline quinone (PQQ) dehydrogenase-like domain (45). These are mammalian ACS4-U26 proteins and their relatives.…”

“…In contrast to what is found with Ebony, the downstream target of activated ␤-alanine is not known. Because PQQ appears not to be a cofactor for ACS4-U26, it was speculated that the PQQ binding-like domain is actually a protein interaction domain, which may ultimately direct posttranslational modification rather than small-molecule biosynthesis (45).…”

Comprehensive Evaluation of Protein Coding Mononucleotide Microsatellites in Microsatellite-Unstable Colorectal Cancer

“…(aminoadipate-semialdehyde dehydrogenase) encodes an enzyme that has been suggested to activate b-alanine (29). It is located in a region of chromosome 4 that exhibits copy number loss in early onset colorectal cancer (30).…”