Acyl-CoA synthetase family member 3 (ACSF3) is a mitochondrial enzyme encoded by the ACSF3 gene. It is required for the degradation of malonic acid and methylmalonic acid and provides the precursor for mitochondrial fatty acid synthesis (mtFAS) and mitochondrial lysine malonylation. The enzyme belongs to the acyl-CoA synthetase family.

The ACSF3 gene is located on chromosome 16, at locus q24.3. It comprises 14 exons and produces four alternatively spliced mRNAs that encode two isoforms of the ACSF3 protein:

Three transcript variants encode a 576-amino acid protein of about 64.1 kDa with a predicted N-terminal mitochondrial targeting sequence (MTS) comprising the first 58 or 83 residues depending on the prediction method. Experimental studies confirmed that the mature protein localizes to the mitochondrial matrix.

Observed post-translational modifications:

A single transcript variant encodes a shorter 311-amino-acid protein that begins translation at a downstream start codon relative to isoform 1.

ACSF3 differs from acyl-CoA synthetases that activate fatty acids by preferring the dicarboxylic acids malonate and methylmalonate, which it converts into the thioesters malonyl-CoA and methylmalonyl-CoA. Lignoceric acid (C24) has been reported as an additional substrate, although in vitro studies provide conflicting results. Despite this substrate specificity, it follows the same two-step ATP-dependent mechanism as other acyl-CoA synthetases via an acyl-adenylate intermediate:

Accordingly, ACSF3 catalyzes the following overall reactions:

ACSF3 most efficiently converts malonate and processes methylmalonate at about 70% of that rate. Like other acyl-CoA synthetases, ACSF3 activity is subject to feedback inhibition by its products.