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Updated: Jun 25, 2025

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Published on: October 4, 2017
Pathogenic Homocystinuria-Associated T236N Mutation Dramatically Alters the Biochemical Properties of Cystathionine
Duaa W Al-Sadeq1,2, Angelos Thanassoulas2, Maria Theodoridou1
1Biomedical Research Center, Qatar University, Doha P.O. Box 2713, Qatar.
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 CBST236N protein, which we directly compared to those of the wild-type (CBSWT) 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 CBSWT 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 CBSWT and CBST236N mutant protein species. In addition, chemical denaturation experiments further highlighted that the CBSWT protein exhibited greater thermodynamic stability than the CBST236N 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.
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