Pathogenic Homocystinuria-Associated T236N Mutation Dramatically Alters the Biochemical Properties of Cystathionine Beta-Synthase Protein.

BACKGROUND

Cystathione beta-synthase (CBS) T236N is a novel mutation associated with pyridoxine non-responsiveness, which presents a significant difficulty in the medical treatment of homocystinuria. Reported severe phenotypes in homocystinuria patients highlight the urgent requirement to comprehend the molecular mechanisms underlying mutation pathogenicity for the advancement of the disease.

METHODOLOGY

In this study, we used a multidisciplinary approach to investigate the molecular properties of bacterially expressed and purified recombinant CBS protein, which we directly compared to those of the wild-type (CBS) protein.

RESULTS

Our data revealed a profound impact of the p.T236N mutation on CBS enzymatic activity, with a dramatic reduction of ~96% compared to the CBS protein. Circular dichroism (CD) experiments indicated that the p.T236N mutation did not significantly alter the secondary structure of the protein. However, CD spectra unveiled distinct differences in the thermal stability of CBS and CBS mutant protein species. In addition, chemical denaturation experiments further highlighted that the CBS protein exhibited greater thermodynamic stability than the CBS mutant, suggesting a destabilizing effect of this mutation.

CONCLUSIONS

Our findings provide an explanation of the pathogenicity of the p.T236N mutation, shedding light on its role in severe homocystinuria phenotypes. This study contributes to a deeper understanding of CBS deficiency and may improve the development of targeted therapeutic strategies for affected individuals.