Interconversion of a pair of active-site residues inEscherichia colicystathionine γ-synthase,E. colicystathionine β-lyase, andSaccharomyces cerevisiaecystathionine γ-lyase and development of tools for the investigation of their mechanisms and reaction specificity

Abstract

Cystathionine gamma-synthase (CGS) and cystathionine beta-lyase (CBL), which comprise the transsulfuration pathway of bacteria and plants, and cystathionine gamma-lyase (CGL), the second enzyme of the fungal and animal reverse transsulfuration pathway, share approximately 30% sequence identity and are almost indistinguishable in overall structure. One difference between the active site of Escherichia coli CBL and those of E. coli CGS and Saccharomyces cerevisiae CGL is the replacement of a pair of aromatic residues, F55 and Y338, of the former by acidic residues in CGS (D45 and E325) and CGL (E48 and E333). A series of interconverting, site-directed mutants of these 2 residues was constructed in CBL (F55D, Y338E, F55D/Y338E), CGS (D45F, E325Y and D45F/E325Y) and CGL (E48A,D and E333A,D,Y) to probe the role of these residues as determinants of reaction specificity. Mutation of either position results in a reduction in catalytic efficiency, as exemplified by the 160-fold reduction in the kcat/KmL-Cys of eCGS-D45F and the 2850- and 30-fold reductions in the kcat/KmL-Cth of the eCBL-Y338E and the yCGL-E333A,Y mutants, respectively. However, the in vivo reaction specificity of the mutants was not altered, compared with the corresponding wild-type enzymes. The DeltametB and DeltametC strains, the optimized CBL and CGL assay conditions, and the efficient expression and affinity purification systems described provide the necessary tools to enable the continued exploration of the determinants of reaction specificity in the enzymes of the transsulfuration pathways.

Exploration of the active site of Escherichia coli cystathionine γ‐synthase

“…In contrast, replacement of residue D45 with phenylalanine, to mimic the corresponding F55 of eCBL, decreases k cat by 16-fold and causes a 6-fold increase in K l-Cys m . 9 These results suggest that, although this residue does not participate directly in substrate binding, it is situated in proximity to the L-Cys binding site. Similarly, the unique 4-to 6-fold decreases in the K l-OSHS mR and K l-Cys iR values of the alanine and glutamine substitutions of E325 (Table I, Fig.…”

“…Additionally, interconversion of the two residues most distinct between the active sites of the a,c-elimination catalyzing eCGS (D45 and E325) and yCGL (E48 and E333) and eCBL (F55 and Y338), which catalyzes an a,b-elimination reaction, does not result in a corresponding increase in a,c or a,b-elimination activity. 9 Therefore, exploration of the roles of active-site residues in these enzymes, dependent on the versatile PLP cofactor, is necessary to decipher the subtle and complex structure-function relationships of these structurally similar but mechanistically distinct enzymes. The characterization of a series of 16 sitedirected variants of 10 eCGS active-site residues (D45, Y46, R48, R49, Y101, R106, N227, E325, S326, and R361) is the focus of this study.…”

“…The amino-terminal, 6-His tag, and linker encoded by this vector enables affinity purification of the expressed enzymes but, as demonstrated by Farsi et al, it does not alter the kinetic parameters of eCGS. 9 Wild-type and site-directed variants of eCGS were expressed in the E. coli ER1821 metB::aadA strain, in which the gene encoding eCGS is replaced by aadA, encoding resistance to streptomycin, to prevent contamination with the wild-type E. coli enzyme. The wild-type and site-directed mutants of eCGS were expressed and purified, via Ni-nitrilotriacetic acid affinity chromatography, as described by Farsi et al 9 Determination of steady-state kinetic parameters…”

Site-Directed Mutagenesis on Human Cystathionine-γ-Lyase Reveals Insights into the Modulation of H2S Production

“…Recently, a similar mutation had been performed on this residue in yeast CSE to explore its effect on the catalytic efficiency of the enzyme during the α,γ-elimination reaction of L-cystathionine. 25 Interestingly, a 30-fold reduction in catalytic efficiency was observed for the alanine and tyrosine mutants. This inverse correlation between the α,β-elimination or α,γ-elimination reaction of CSE and the hydrophobicity of the 339th residue may imply that residues with a more hydrophobic side chain at the 339th position could possibly favor the α,β-elimination reaction over the α,γ-elimination reaction in CSE.…”

Bacterial methionine biosynthesis

“…In spite of this phylogenetic evidence, however, it has not yet proven possible to interconvert enzymes with these activities by using rational mutagenesis approaches (Farsi et al, 2009;Manders et al, 2013).…”