Cystathionine β-synthase mutations: effect of mutation topology on folding and activity

Abstract

Misfolding of mutant enzymes may play an important role in the pathogenesis of cystathionine β-synthase (CBS) deficiency. We examined properties of a series of 27 mutant variants, which together represent 70% of known alleles observed in patients with homocystinuria due to CBS deficiency. The median amount of SDS-soluble mutant CBS polypeptides in the pellet after centrifugation of bacterial extracts was increased by 50% compared to the wild type. Moreover, mutants formed on average only 12% of tetramers and their median activity reached only 3% of the wild-type enzyme. In contrast to the wild-type CBS about half of mutants were not activated by S-adenosylmethionine. Expression at 18°C substantially increased the activity of five mutants in parallel with increasing the amounts of tetramers. We further analyzed the role of solvent accessibility of mutants as a determinant of their folding and activity. Buried mutations formed on average less tetramers and exhibited 23 times lower activity than the solvent exposed mutations. In summary, our results show that topology of mutations predicts in part the behavior of mutant CBS, and that misfolding may be an important and frequent pathogenic mechanism in CBS deficiency. Hum Mutat 31:1–11, 2010. © 2010 Wiley-Liss, Inc.

Surrogate Genetics and Metabolic Profiling for Characterization of Human Disease Alleles

“…Dual remedial alleles favored a global mechanism for cofactor rescue over the simpler model that increasing the cofactor concentration overcomes mutations that decrease the Km of cofactor binding (Ames et al 2002;Wittung-Stafshede 2002). Since many characterized disease-causing mutations alter protein function via folding/stability (Yue et al 2005;Kozich et al 2010), alleles encoding unstable proteins may benefit from the binding energy provided by protein-cofactor interaction. Rescue of CBS function by biological or chemical chaperones is consistent with this hypothesis (Singh et al 2007(Singh et al , 2010Majtan et al 2010).…”

The role of surface electrostatics on the stability, function and regulation of human cystathionine β-synthase, a complex multidomain and oligomeric protein

“…hCBS displays a complex architecture, forming functional homotetramers, and each monomer containing three structural domains: i) a short heme-binding N-terminal domain (residues 1-70), which is thought to modulate CBS activity and proper folding; ii) a catalytic domain (CD; residues 71-413) which contains the catalytically active PLP molecule bound to Lys119 through a Schiff base; iii) a C-terminal regulatory domain (RD), which contains two types of SAM binding sites involved in SAM mediated enzyme activation and kinetic stabilization of hCBS [1, 3, 4]. Over 160 mutations in the CBS gene have been found to cause classical homocystinuria (HCU) ([5] and http://medschool.ucdenver.edu/krauslab). Missense mutations often lead to protein instability and impaired catalytic activity and regulation [1, 4, 6-9].…”

Folding and activity of mutant cystathionine β-synthase depends on the position and nature of the purification tag: Characterization of the R266K CBS mutant

“…Thus, most of the data supporting the positive effect of chemical chaperones on mutant CBS folding, assembly and catalytic activity were obtained by studying the overexpressed CBS in crude extracts [15, 17, 21]. We showed that eight CBS mutants, following expression in the presence of a chemical chaperone in the growth medium, were amenable to purification to homogeneity and thus allowed for detailed characterization [16].…”