11 research outputs found
A Gene Encoding l-Methionine Ī³-Lyase Is Present in Enterobacteriaceae Family Genomes: Identification and Characterization of Citrobacter freundii l-Methionine Ī³-Lyase
Citrobacter freundii cells produce l-methionine Ī³-lyase when grown on a medium containing l-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded l-methionine Ī³-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3ā²-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found
Crystallographic Snapshots of Tyrosine Phenol-lyase Show That Substrate Strain Plays a Role in CāC Bond Cleavage
The key step in the enzymatic reaction catalyzed by tyrosine phenol-lyase (TPL) is reversible cleavage of the CĪ²āCĪ³ bond of l-tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbonācarbon bond. As for most other pyridoxal 5ā²-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-l-tyrosine followed by flash-cooling. The X-ray structures, determined at ā¼2.0 Ć
resolution, reveal two quinonoid geometries: ārelaxedā in the open and ātenseā in the closed state of the active site. The ātenseā state is characterized by changes in enzyme contacts made with the substrateās phenolic moiety, which result in significantly strained conformation at CĪ² and CĪ³ positions. We also captured, at 2.25 Ć
resolution, the X-ray structure for the state just after the substrateās CĪ²āCĪ³ bond cleavage by preparing the ternary complex between TPL, alanine quinonoid and pyridine <i>N</i>-oxide, which mimics the Ī±-aminoacrylate intermediate with bound phenol. In this state, the enzymeāligand contacts remain almost exactly the same as in the ātenseā quinonoid, indicating that the strain induced by the closure of the active site facilitates elimination of phenol. Taken together, structural observations demonstrate that the enzyme serves not only to stabilize the transition state but also to destabilize the ground state