STRUCTURAL AND FUNCTIONAL APPROACHES TOWARD ACTIVITY RESTORATION OF NOVEL CYSTATHIONINE BETA-SYNTHASE MUTATIONS

Abstract

Homocystinuria, a rare disorder stemming from mutations in the CBS gene, results in cystathionine β-synthase (CBS) deficiency. CBS is a pivotal enzyme whose activity is enhanced by the allosteric regulator S-adenosylmethionine (SAM) and mediates the conversion of serine and homocysteine to cystathionine in the transsulfuration pathway. ,. Beyond its enzymatic role, CBS contributes to hydrogen sulfide (H2S) production, a gaseous signaling molecule with diverse regulatory functions within the vascular, nervous, and immune systems. This study investigates the impact of five pathogenic CBS missense mutations [c.1006C>T (p.R336C), c.689T>A (p.L230Q), 215A>T (p.K72I), c.707C>A (p.T236N), and c.457G>A (p.G153R)] identified in homocystinuria patients and appeared to be resistant to pyridoxine treatment on the biochemical and biophysical properties of CBS protein. Our hypothesis posits that novel pathogenic missense mutations within critical functional domains of CBS protein will exert varying effects on protein structure, stability and eventually function. We anticipate that mutations affecting conserved residues or cofactor binding sites will lead to pronounced disruptions in enzymatic activity and protein stability and allow us to elucidate the underlying molecular mechanisms of disease pathogenesis for each CBS-associated missense mutation. Utilizing a multidisciplinary approach, including site-directed mutagenesis and DNA cloning, recombinant protein expression, affinity chromatography purification, circular dichroism spectroscopy, enzymatic assays, and in silico modeling, we assessed the impact of these mutations on CBS stability and function. Only one mutation (p.K72I) showed no discernible impact on the spectroscopic and catalytic properties of the full-length CBS enzyme. In contrast, all the other CBS mutation studies did not fully retain heme and, when compared to the wild-type enzyme, exhibited more significant impairments in both the canonical cystathionine synthesis and the alternative H2S-producing reactions. This reduced activity is consistent with both in vitro and in silico evidence, which indicates that the studied CBS mutations, except p.K72I, significantly decrease the overall protein's stability, which may represent the underlying cause of their pathogenicity. In addition to molecular and biochemical characterization, crystallographic studies are underway to provide further insight into the structural alterations induced by the identified CBS missense mutations. Furthermore, screening for potential chemical and molecular chaperones is being initiated to explore therapeutic avenues for mitigating the deleterious effects of these mutations on CBS protein stability and function.