Exploration of structure–function relationships in Escherichia coli cystathionine γ-synthase and cystathionine β-lyase via chimeric constructs and site-specific substitutions

Cystathionine β-Lyase-Like Protein with Pyridoxal Binding Domain Characterized in Leishmania major by Comparative Sequence Analysis and Homology Modelling

“…Cystathionine -lyase (CBL, EC 4.4.1.8) found in plants and microbes is a pyridoxal-5 -phosphate (PLP)-dependent homotetrameric enzyme of approximately 35-40 kDa and catalyses the penultimate step of methionine biosynthesis, that is, , -elimination reaction, where cystathionine is hydrolysed into pyruvate, ammonia, and homocysteine ( Figure 1) [1,2]. Subsequently, homocysteine gets methylated to methionine.…”

Exploration of Active-Site Residues of Escherichia Coli Cystathionine β-Lyase by Site-Directed Mutagenesis

“…It is an useful model system for exploring cofactor regulation by the protein component of PLP enzymes because of its structural similarity to other enzymes catalyzing side chain rearrangements of sulfur-containing amino acids, the availability of several crystal structures and earlier work by our lab characterizing the role of several key active-site residues. These studies, and those of the related enzymes E. coli cystathionine γ-synthase (eCGS) and yeast cystathionine γ-lyase (yCGL), have identified the roles of several active-site residues in substrate binding and catalysis as well as part of the basis for regulation of specificity (Clausen et al, 1996;Messerschmidt et al, 2003;Ejim et al, 2007;Farsi et al, 2009;Lodha, Jaworski and Aitken, 2010;Lodha and Aitken 2011;Jaworski et al, 2012;Jaworski and Aitken, 2013;Manders et al, 2013;Hopwood, Ahmed and Aitken, 2014). This study extends the network of characterized active-site residues by characterization of 20 sitedirected variants of 8 eCBL active-site residues that influence the positioning of catalytic base and/or PLP cofactor.…”

“…As such, they provide an ideal model system to characterize and compare the roles of active-site residues and to investigate determinants of substrate and reaction specificity. Our lab has investigated the active sites of three of the enzymes of this family: cystathionine β-lyase, cystathionine γ-lyase and cystathionine γ-synthase (Farsi et al, 2009;Lodha et al, 2010;Lodha and Aitken, 2011;Jaworski et al, 2012;Manders et al, 2013;Jaworski and Aitken, 2013;Hopwood, Ahmed and Aitken, 2014). Building on this work, additional studies exploring the roles of both conserved and distinct active-site residues in the context of these enzymes are required to further our understanding of the structure-function relationships underlying their specificity.…”

“…Clausen et al (1997) and Ejim et al (2007) showed that only minor structural changes in the active site occur upon binding of either L-aminoethoxyvinylglycine (AVG) and Nhydrazinocarbonylmethyl-2-Nitro-Benzamide. Contacts formed by these inhibitors have been useful in identifying amino acids to target for site-directed mutagenesis studies probing the role of amino acids lining the active site (Farsi et al, 2009;Lodha, Jaworski and Aitken, 2010;Lodha and Aitken, 2011;Manders et al, 2013).…”

“…As such they are primary determinants of reaction specificity . The positioning of S339 may in turn be influenced by other active site residues, such as S32, S33 and K42 of eCBL, which were explored by Manders et al (2013).…”