title: Role of active-site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C-S lyase.
abstract: In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C-S lyase from Corynebacterium diphtheriae is a pyridoxal 5'-phosphate-dependent enzyme in the transsulfuration pathway that catalyzes the α,β-elimination of sulfur-containing amino acids, such as L-cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L-methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active-site residues, Y55F and Y114F, were characterized by UV-visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady-state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130-fold decrease in K(d)(PLP) at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP-binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of L-cystathionine, and causes a change in substrate specificity towards L-serine and O-acetyl-L-serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C-S lyase, which seems to be regulated by active-site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors.
authors.affiliation: Department of Biotechnology, University of Verona, Strada Le Grazie 15, Verona, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Astegno
authors.forename: Alessandra
authors.initials: A
authors.lastname: Allegrini
authors.forename: Stefano
authors.initials: S
authors.lastname: Piccoli
authors.forename: Alejandro
authors.initials: A
authors.lastname: Giorgetti
authors.forename: Paola
authors.initials: P
authors.lastname: Dominici
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D003353:Corynebacterium diphtheriae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D014443:Tyrosine; D011732:Pyridoxal Phosphate; D008190:Lyases; C033511:carbon-sulfur lyase
keywords: 5′-phosphate; molecular dynamics simulations; protein engineering; pyridoxal; reaction intermediates; α,β-elimination