The Three-Dimensional Structure of Cystathionine β-Lyase from Arabidopsis and Its Substrate Specificity

Abstract

The pyridoxal 5'-phosphate-dependent enzyme cystathionine beta-lyase (CBL) catalyzes the penultimate step in the de novo biosynthesis of Met in microbes and plants. Absence of CBL in higher organisms makes it an important target for the development of antibiotics and herbicides. The three-dimensional structure of cystathionine beta-lyase from Arabidopsis was determined by Patterson search techniques, using the structure of tobacco (Nicotiana tabacum) cystathionine gamma-synthase as starting point. At a resolution of 2.3 A, the model was refined to a final crystallographic R-factor of 24.9%. The overall structure is very similar to other pyridoxal 5'-phosphate-dependent enzymes of the gamma-family. Exchange of a few critical residues within the active site causes the different substrate preferences between Escherichia coli and Arabidopsis CBL. Loss of interactions at the alpha-carboxyl site is the reason for the poorer substrate binding of Arabidopsis CBL. In addition, the binding pocket of Arabidopsis CBL is larger than that of E. coli CBL, explaining the similar binding of L-cystathionine and L-djenkolate in Arabidopsis CBL in contrast to E. coli CBL, where the substrate binding site is optimized for the natural substrate cystathionine.

A Peroxisomal Glutathione Transferase of Saccharomyces cerevisiae Is Functionally Related to Sulfur Amino Acid Metabolism

“…This plasmid was used to construct strain MML572, after EcoRV-mediated linearization of pMM401, transformation of wild-type cells, and integration at the chromosomal URA3 site. pMM440 is a centromeric URA3 plasmid that carries the complete coding sequence of GTO1 under the control of the tetO 7 promoter from pCM189 (26), with an N-terminal fusion of the coding sequence to the GFP tag. Plasmid pMM736 is based in YCplac211 (29) and contains the GTO1 gene expressed under its own promoter and TAP tagged at its N terminus; the TAP tag was PCR amplified from plasmid pBS1761 (54).…”

“…Therefore, the growth deficiency in the mutant cannot be attributed to increased toxicity of cysteine (and probably cystathionine) on the mutant but to a defect in conversion of cysteine or cystathionine into homocysteine. The three-dimensional structure of cystathionine ␤-lyase from Arabidopsis thaliana has been determined, and no cysteine residue is present at the enzyme binding site (7). However, the thiol-blocking inhibitor N-ethylmaleimide inhibits the enzyme activity, which points to the importance of some cysteine residues in the molecule (58), maybe for maintaining the functional tertiary structure.…”

A Carbon-Nitrogen Lyase from Leucaena leucocephala Catalyzes the First Step of Mimosine Degradation

“…S3). The high degree of sequence similarity between Arabidopsis CBL and L. leucocephala mimosinase and their conserved active site residues suggest that mimosinase may have a similar catalytic mode of action as described by Breitinger et al (2001) for CBL.…”

“…S3). A previous study of the three-dimensional structure of Arabidopsis CBL identified the key active site residues of the enzyme (Breitinger et al, 2001). Most of the known active site residues of CBL are also present in mimosinase (Supplemental Fig.…”

A New Member of Plant CS-lyases

“…Plant CS-lyases are involved in a range of processes, including the synthesis and degradation of natural products, amino acid metabolism, and generation of precursors for the synthesis of ethylene, polyamines, and particular proteins (2). Although several plant pyridoxal 5-phosphate (PLP) 1 dependent CS-lyases have been characterized comprehensively (3,4), little is still known about the molecular character and biological role of cystine lyases in plant metabolism (1). Cystine lyases have previously been purified from broccoli and characterized with regard to substrate specificities and kinetics in the presence or absence of inhibitors (1,5).…”