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Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL) share a common structure and several active-site residues, but catalyze distinct side-chain rearrangements in the two-step transsulfuration pathway that converts cysteine to homocysteine, the precursor of methionine. A series of 12 chimeric variants of Escherichia coli CGS (eCGS) and CBL (eCBL) was constructed to probe the roles of two structurally distinct, ~25-residue segments situated in proximity to the amino and carboxy termini and located at the entrance of the active-site. In vivo complementation of methionine-auxotrophic E. coli strains, lacking the genes encoding eCGS and eCBL, demonstrated that exchange of the targeted regions impairs the activity of the resulting enzymes, but does not produce a corresponding interchange of reaction specificity. In keeping with the in vivo results, the catalytic efficiency of the native reactions is reduced by at least 95-fold, and α,β versus α,γ-elimination specificity is not modified. The midpoint of thermal denaturation monitored by circular dichroism, ranges between 59 and 80°C, compared to 66°C for the two wild-type enzymes, indicating that the chimeric enzymes adopt a stable folded structure and that the observed reductions in catalytic efficiency are due to reorganization of the active site. Alanine-substitution variants of residues S32 and S33, as well as K42 of eCBL, situated in proximity to and within, respectively, the targeted amino-terminal region were also investigated to explore their role as determinants of reaction specificity via positioning of key active-site residues. The catalytic efficiency of the S32A, S33A and the K42A site-directed variants of eCBL is reduced by less than 10-fold, demonstrating that, while these residues may participate in positioning S339, which tethers the catalytic base, their role is minor. Copyright © 2013 Elsevier B.V. All rights reserved.


Adrienne L Manders, Allison F Jaworski, Mohammed Ahmed, Susan M Aitken. Exploration of structure-function relationships in Escherichia coli cystathionine γ-synthase and cystathionine β-lyase via chimeric constructs and site-specific substitutions. Biochimica et biophysica acta. 2013 Jun;1834(6):1044-53

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PMID: 23470500

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