Determinants of Enzymatic Specificity in the Cys-Met-Metabolism PLP-Dependent Enzyme Family: Crystal Structure of Cystathionine γ-Lyase from Yeast and Intrafamiliar Structure Comparison

  title={Determinants of Enzymatic Specificity in the Cys-Met-Metabolism PLP-Dependent Enzyme Family: Crystal Structure of Cystathionine $\gamma$-Lyase from Yeast and Intrafamiliar Structure Comparison},
  author={Albrecht Messerschmidt and Michael Worbs and Clemens Steegborn and Markus C. Wahl and Robert Huber and Bernd Laber and Tim Clausen},
  booktitle={Biological chemistry},
Abstract The crystal structure of cystathionine γ-lyase (CGL) from yeast has been solved by molecular replacement at a resolution of 2.6 å. The molecule consists of 393 amino acid residues and one PLP moiety and is arranged in the crystal as a tetramer with D2 symmetry as in other related enzymes of the CysMetmetabolism PLP-dependent family like cystathionine β-lyase (CBL). A structure comparison with other family members revealed surprising insights into the tuning of enzymatic specificity… 

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii

The results suggest that CGL is an important functional enzyme in T. gondii, likely implying that the reverse transsulfuration pathway is operative in the parasite; the roles of active-site architecture and substrate binding conformations as determinants of reaction specificity in transsolfuration enzymes are probed.

Structure of the antitumour enzyme L-methionine gamma-lyase from Pseudomonas putida at 1.8 A resolution.

The three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method to an R-factor of 20.4% at 1.8 A resolution and it is suggested that electrostatic interactions at the subunit interface are involved in the stabilization of the structural conformation.

Interconversion of a pair of active-site residues in Escherichia coli cystathionine gamma-synthase, E. coli cystathionine beta-lyase, and Saccharomyces cerevisiae cystathionine gamma-lyase and development of tools for the investigation of their mechanisms and reaction specificity.

The D BeltametB and DeltametC strains, the optimized CBL and CGL assay conditions, and the efficient expression and affinity purification systems described provide the necessary tools to enable the continued exploration of the determinants of reaction specificity in the enzymes of the transsulfuration pathways.

Catalytic specificity of the Lactobacillus plantarum cystathionine γ-lyase presumed by the crystallographic analysis

It is found that the enzyme has the high γ-lyase activity toward cystathionine to generate l-cysteine, together with the β-ly enzyme activity toward l-Cystine to generated l- Cysteine persulfide.

Role of active‐site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C‐S lyase

Spect spectral data and 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.

The Role of Amino Acid Residues in the Active Site of L-Methionine γ-lyase from Pseudomonas putida

It is suggested that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methionine-biosynthetic pathway of Wolinella succinogenes.

The crystal structure of Wolinella succinogenes OAHS (MetY) determined at 2.2 Å resolution provides insight into the mechanism of sulfur transfer to a small molecule via a protein thiocarboxylate intermediate.



The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum reveals its substrate and reaction specificity.

General insight regarding the reaction specificity of transsulphuration enzymes is gained by the comparison to cystathionine beta-lyase from E. coli, indicating the mechanistic importance of a second substrate binding site for L-cysteine which leads to different chemical reaction types.

Crystal structure of the pyridoxal-5'-phosphate dependent cystathionine beta-lyase from Escherichia coli at 1.83 A.

The crystal structure of CBL from E. coli has been solved using MIR phases in combination with density modification and suggests that Lys210, the PLP-binding residue, mediates the proton transfer between C alpha and S gamma.

The three-dimensional structure of cystathionine beta-lyase from Arabidopsis and its substrate specificity.

The three-dimensional structure of cystathionine beta-lyase from Arabidopsis was determined by Patterson search techniques, and the overall structure is very similar to other pyridoxal 5'-phosphate-dependent enzymes of the gamma-family.

Crystal structure of Escherichia coli cystathionine γ‐synthase at 1.5 Å resolution

The transsulfuration enzyme cystathionine γ‐synthase (CGS) catalyses the pyridoxal 5′‐phosphate (PLP)‐dependent γ-replacement of O‐succinyl‐L‐homoserine and L‐cysteine, yielding L-cystathioneine, helping in the understanding of the chemical versatility of PLP.

A hydrogen-bonding network modulating enzyme function: asparagine-194 and tyrosine-225 of Escherichia coli aspartate aminotransferase.

The kinetic studies showed that Asn194 is not essential for AspAT catalysis, although the Kd values for the substrates were increased by 10- to 50-fold upon the replacement of Asn193, and the analyses of the pH-pKd curves for the wild-type and mutant enzymes showed that the hydrogen bond between O(3') of PLP and Asn 194 is weakened by the binding of maleate to Asp AT.

Cystathionine γ‐lyase of Saccharomyces cerevisiae: Structural gene and cystathionine γ‐synthase activity

N‐terminal amino acid sequence analysis indicated that CYS3 is the structural gene for γ‐CTLase and that the contaminant is O‐ acetylserine/O‐acetylhomoserine sulfhydrylase (OAS/OAH SHLase).

Cloning and bacterial expression of the CYS3 gene encoding cystathionine gamma-lyase of Saccharomyces cerevisiae and the physicochemical and enzymatic properties of the protein

The possibility that this gene rescuing the cysteine requirement in a "cys1" strain of Saccharomyces cerevisiae has a physiological role as cystathionine Beta-lyase and cystATHionine gamma-synthase in addition to its previously described role as CYI1, as well as the enzymatic properties of this enzyme are discussed.

Evolutionary relationships among pyridoxal‐5′‐phosphate‐dependent enzymes

A comprehensive comparison of amino acid sequences has shown that most pyridoxal-5'-phosphate-dependent enzymes can be assigned to one of three different families of homologous proteins, and their homology confirmed by profile analysis.

Slow-binding inhibition of Escherichia coli cystathionine beta-lyase by L-aminoethoxyvinylglycine: a kinetic and X-ray study.

The interaction of Escherichia coli CBL with AVG and methoxyvinylglycine (MVG) is characterized by a combination of kinetic methods and X-ray crystallography, which suggests a binding mode for rhizobitoxine and explains the failure of MVG to inhibit CBL.

Reaction mechanism of Escherichia coli cystathionine gamma-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent gamma-elimination and gamma-replacement reactions.

The results establish that the partitioning intermediate is an alpha-imino beta,gamma-unsaturated pyridoxamine derivative with lambda max congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.