Disease-causing cystathionine β-synthase linker mutations impair allosteric regulation

Homocystinuria is a genetic disorder linked to mutations in the CBS gene. Prior research has shown that CBS is essential for transsulfuration, which clears homocysteine and produces cysteine and H2S. The enzyme’s structure includes a catalytic core and a regulatory domain connected by a linker. This gap motivated investigation into how linker mutations affect CBS function. No prior work had resolved how these mutations disrupt allosteric regulation. Established knowledge includes the role of AdoMet as an activator. This paper's contribution focuses on three clinical variants in the linker region. The study addresses how these mutations alter CBS structure and activity. Understanding this could clarify the molecular basis of homocystinuria.

The aim is to characterize three clinical variants in the CBS linker region. These variants are known to cause homocystinuria, but their specific effects on enzyme function are unclear. The study tests whether these mutations destabilize the native fibrillar form of CBS. Researchers also examine how the mutations affect allosteric regulation by AdoMet. The problem is that prior work did not address the role of the linker in CBS architecture. This study uses structural and kinetic approaches to explore the variants' effects. The motivation is to determine how these mutations impair CBS function. The findings may help explain the pathogenesis of homocystinuria.

The study uses cryo-EM to analyze the fibrillar structure of CBS. Limited proteolysis is used to detect conformational changes in the variants. Crystallography is applied to the truncated K384N variant. Pre-steady state kinetics are used to assess enzyme activity. Researchers compare the variants to the wild-type enzyme. The experiments focus on how the linker region influences CBS structure. The study also evaluates how AdoMet binding is affected by the mutations. The approach combines structural and functional analyses to explore the variants' effects.

The K384E/N and M391I variants destabilize the native fibrillar structure of CBS. Limited proteolysis reveals conformational changes in the variants. The catalytic core of the K384N variant remains structurally intact. M391I CBS shows a 1.4-fold decrease in basal activity. The K384E/N variants show an 8-fold decrease in activity. AdoMet either has no effect or inhibits these variants. Pre-steady state kinetics show that the K384E/N variants affect the second half reaction. The mutations impair homocysteine binding and reaction with the aminoacrylate intermediate.

The linker region is important for stabilizing the higher-order structure of CBS. The study shows that linker mutations disrupt allosteric regulation by AdoMet. The findings suggest that the linker is essential for CBS architecture. The K384E/N variants have a more severe effect than M391I. The mutations affect the second half of the catalytic reaction. The results support a role for the linker in enabling AdoMet-dependent regulation. The study confirms that the catalytic core remains intact in the variants. These conclusions align with the authors' claim that the linker is critical for CBS function.