ISOLATION OF A CDNA-ENCODING HUMAN HOLOCARBOXYLASE SYNTHETASE BY FUNCTIONAL COMPLEMENTATION OF A BIOTIN AUXOTROPH OF ESCHERICHIA-COLI

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of the fo ur biotin-dependent carboxylases in human cells. Patients with HCS def iciency lack activity of all four carboxylases, indicating that a sing le HCS is targeted to the mitochondria and cytoplasm. We isolated 21 h uman HCS cDNA clones, in four size classes of 2.0-4.0 kb, by complemen tation of an Escherichia coli birA mutant defective in biotin ligase. Expression of the cDNA clones promoted biotinylation of the bacterial biotinyl carboxyl carrier protein as well as a carboxyl-terminal fragm ent of the alpha subunit of human propionyl-CoA carboxylase expressed from a plasmid. The open reading frame encodes a predicted protein of 726 aa and M(r) 80,759. Northern blot analysis revealed the presence o f a 5.8-kb major species and 4.0-, 4.5-, and 8.5-kb minor species of p oly(A)(+) RNA in human tissues. Human HCS shows specific regions of ho mology with the BirA protein of E. coli and the presumptive biotin lig ase of Paracoccus denitrificans. Several forms of HCS mRNA are generat ed by alternative splicing, and as a result, two mRNA molecules bear d ifferent putative translation initiation sites. A sequence upstream of the first translation initiation site encodes a peptide structurally similar to mitochondrial presequences, but it lacks an in-frame ATG co don to direct its translation. We anticipate that alternative splicing most likely mediates the mitochondrial versus cytoplasmic expression, although the elements required for directing the enzyme to the mitoch ondria remain to be confirmed.