Functional characterisation of the N-terminal region of holocarboxylase synthetase.

Abstract

Biotin (vitamin H or B7) is an important micronutrient that is covalently attached to biotin-dependent enzymes by human biotin protein ligase (hBPL) or holocarboxylase synthetase (HCS). Patients with HCS deficiency are treated with oral biotin supplementation, which in most cases is able to reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. The molecular explanation for this is not understood. In this study HCS was investigated to improve our understanding of this key enzyme. The catalytic region of all BPLs is contained in the conserved C-terminal region. HCS contains a long N-terminal extension that is not present in bacterial BPLs. The structure and function of the N-terminal region is yet to be determined. In order to delineate the domain structure of HCS limited proteolysis was performed previously in our laboratory. Two protease-sensitive linker regions were identified, one between residues 151-153, the other at amino acid 314. Of particular importance is the proposed structured domain containing residues 159-314, as amino acid substitutions in this region have been shown to compromise enzyme activity despite being distal to the C-terminally located active site. This thesis provides genetic evidence for a direct interaction between the N-terminal and C-terminal halves of HCS using a yeast two-hybrid assay. This interaction was mapped using a trucation study to the proposed structured domain of the N-terminal region of HCS (159-314 N-HCS). HCS deficiency gives rise to the metabolic disorder multiple carboxylase deficiency (MCD). Mutations within the proposed structured domain 159-314 N-HCS give rise to MCD patients that are poorly responsive to the current therapy (biotin supplementation) and have an extremely poor long-term prognosis. In this thesis, a series of novel mutations in the proposed structured domain 159-314 N-HCS were generated using “error prone” PCR. The catalytically inactive mutants were isolated from the library using an in vivo complementation assay. The mutants that were isolated were identified by DNA sequencing as L166R, L206P, W210R, L246M, L270S, H306R, F321S and the double mutant E181G, E327G. These residues are highly conserved within vertebrate species. These novel HCS mutants, together with the MCD mutants L216R HCS and L237P HCS, were employed to further characterise the function of the proposed structured N-terminal domain. Using the Yeast Two-hybrid assay, it was shown that the interaction between the two halves of HCS was not disrupted by the MCD mutants nor the novel mutants. Conversely, it was shown that the MCD mutants, and the majority of the novel HCS mutants, disrupted the interaction between HCS and its protein substrate the Pyruvate Carboxylase biotin domain hPC107. Surface Plasmon Resonance was then employed to further characterise this observation. This study has demonstrated for the first time that although the association between HCS and its substrate was not compromised by mutation, the MCD mutants had a >15-fold increase in dissociation rate from the substrate compared to wild type HCS. This work provided a novel function for the proposed structural N-terminal domain. Furthermore, these data provide a molecular explanation for the HCS deficient patients that do not respond to biotin therapy.