Mechanism of biotin responsiveness in biotin-responsive multiple carboxylase deficiency

Holocarboxylase synthetase (HCS) catalyses the biotinylation of the four biotin-dependent carboxylases found in humans. A deficiency in HCS results in biotin-responsive multiple carboxylase deficiency. We have evaluated the biotin responsiveness associated with six missense mutations previously identified in affected patients by expression of plasmids containing the mutated HCS in an Escherichia coli strain mutated in the corresponding BirA gene. We demonstrate that the mutations identified in the MCD patients are indeed responsible for their reduced HCS activity. Four of the mutations, clustering in the putative biotin binding domain as deduced from the structure of the E. coli enzyme, are consistent with an explanation for biotin responsiveness based on altered affinity for biotin. The remaining mutations, located outside the biotin binding region, were associated with a more limited biotin responsiveness that may be explained by the degree of residual enzyme activity present. The data suggest that the concentration of circulating biotin is as low as 100 times below the Km of the enzyme, so that any increase in biotin concentration through dietary supplementation would result in saturation of the available mutant enzyme. We suggest that these alternative explanations are sufficient to account for the apparent universality of biotin responsiveness in biotin responsive multiple carboxylase deficiency.