Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine β-synthase fromApis mellifera

Abstract

Cystathionine -synthase (CBS) is a pyridoxal-5 0 -phosphate-dependent enzyme that catalyzes the first step of the transsulfuration pathway, namely the condensation of serine with homocysteine to form cystathionine. Mutations in the CBS gene are the single most common cause of hereditary homocystinuria, a multisystemic disease affecting to various extents the vasculature, connective tissues and central nervous system. At present, the crystal structure of CBS from Drosophila melanogaster is the only available structure of the full-length enzyme. Here we describe a cloning, overexpression, purification and preliminary crystallographic analysis of a full-length CBS from Apis mellifera (AmCBS) which maintains 51 and 46% sequence identity with its Drosophila and human homologs, respectively. The AmCBS yielded crystals belonging to space group P2 1 2 1 2 1 , with unit-cell parameters a = 85.90, b = 95.87, c = 180.33 Å . Diffraction data were collected to a resolution of 3.0 Å . The crystal structure contained two molecules in the asymmetric unit which presumably correspond to the dimeric species observed in solution.

Purification, crystallization and preliminary crystallographic analysis of human cystathionine β-synthase

“…However, all efforts were unsuccessful despite testing more than 10 000 different conditions, which included all available commercial screens as well as in-house-developed recipes. Alternatively, and based on careful analyses of sequence and structural alignments of hCBS versus DmCBS (Koutmos et al, 2010) and AmCBS (Oyenarte et al, 2012), we engineered a protein construct (hCBS 516-525 ) that lacks amino-acid residues 516-525, which presumably form a disordered loop that may impede crystal growth (Fig. 2).…”

Potential Pharmacological Chaperones for Cystathionine Beta-Synthase-Deficient Homocystinuria

“…We answered this question by showing that artificial removal of residues 516-525, within the quite unique extended loop (residues 513-529) connecting the two last β-strands (β15, β16) of the CBS2 motif, resulted in the irreversible disassembly of the tetrameric native protein into dimers (Ereno-Orbea et al 2013b). Interestingly, the length of this loop is significantly shorter in dimeric CBS enzymes from organisms, such as fruit fly or honey bee (Koutmos et al 2010;Oyenarte et al 2012). Undoubtedly, the most important role of the Bateman module is to regulate the CBS activity.…”