title: A novel molecular mechanism to explain biotin-unresponsive holocarboxylase synthetase deficiency.
abstract: Biotin (vitamins H and B7) is an important micronutrient as defects in its availability, metabolism or adsorption can cause serious illnesses, especially in the young. A key molecule in the biotin cycle is holocarboxylase synthetase (HLCS), which attaches biotin onto the biotin-dependent enzymes. Patients with congenital HLCS deficiency are prescribed oral biotin supplements that, in most cases, reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. Whilst a small number of mutations in the HLCS gene have been implicated, the molecular mechanisms that lead to the biotin-unresponsive phenotype are not understood. To improve our understanding of HLCS, limited proteolysis was performed together with yeast two-hybrid analysis. A structured domain within the N-terminal region that contained two missense mutations was identified in patients who were refractory to biotin therapy, namely p.L216R and p.L237P. Genetic studies demonstrated that the interaction between the enzyme and the protein substrate was disrupted by mutation. Further dissection of the binding mechanism using surface plasmon resonance demonstrated that the mutations reduced affinity for the substrate through a >15-fold increase in dissociation rate. Together, these data provide the first molecular explanation for HLCS-deficient patients that do not respond to biotin therapy.
authors: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia.
authors: Lungisa
authors: L
authors: Mayende
authors: Rachel D
authors: RD
authors: Swift
authors: Lisa M
authors: LM
authors: Bailey
authors: Tatiana P
authors: TP
authors: Soares da Costa
authors: John C
authors: JC
authors: Wallace
authors: Grant W
authors: GW
authors: Booker
authors: Steven W
authors: SW
authors: Polyak
mesh_terms: D001710:Biotin / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D028922:Holocarboxylase Synthetase Deficiency / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011766:Pyruvate Carboxylase / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases; D011766:Pyruvate Carboxylase