Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa

The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H2S) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections. In addition to a canonical l-cystathionine hydrolysis, PaCGL efficiently catalyzes the production of H2S using l-cysteine and/or l-homocysteine as alternative substrates. Comparative analysis with the human enzyme and counterparts from other pathogens revealed distinct structural features within the primary enzyme cavities. Specifically, a distinctly folded entrance loop could potentially modulate the access of substrates and/or inhibitors to the catalytic site. Our findings offer significant insights into the structural evolution of CGL enzymes across different pathogens and provide novel opportunities for developing specific inhibitors targeting PaCGL.

would correspond to a closed conformation of chamber-2.In this state, this cavity significantly restricts its accessibility and internal volume.On the contrary, chamber-2 is more accessible and ready to host small molecules when the loop adopts the extended conformation found in the crystals.
Figure S1.Properties of recombinant PaCGL.(A) Cropped 12% SDS-PAGE gel of purified recombinant wild type PaCGL (original gel is presented in Supplementary Figure S8).Lane M, protein marker.(B) Size exclusion chromatography of PaCGL using Sephacryl S-200 16/60 high resolution column in 20 mM sodium phosphate pH 8.0, 150 mM NaCl, 0.1 mM DTT. (Inset) Calibration curve of molecular weight logarithm versus elution volumes.(C) UV-visible absorption spectrum of 15 μM purified PaCGL in 20 mM sodium phosphate buffer pH 8.0.(D) Emission spectra of 1 μM apo-PaCGL before and after reconstitution with PLP in 20 mM sodium phosphate buffer pH 8. Inset, representative fluorescence titration of apo-PaCGL (1 μM) with PLP (0.01-4 μΜ) monitoring the quenching of intrinsic fluorescence emission at 333 nm upon excitation of the apo-PaCGL at 295 nm.The Kd value is determined by fitting the fraction of bound PLP (fb) to a hyperbolic equation and represents a mean value ± SEM of three independent measurements.(E) Thermal denaturation profile of 0.2 mg/mL apo-(black) and holo-PaCGL (red) recorded following the ellipticity signal at 222 nm in 20 mM sodium phosphate buffer pH 8.0.

Figure S2 .
Figure S2.Effect of pH and temperature on PaCGL γ-elimination of L-Cth.(A) pH-dependent activity profile for PaCGL performed at constant saturating L-Cth concentration in the pH range of 6-10 at 37°C.(B) PaCGL enzyme activity over temperature range of 15-65°C at constant saturating L-Cth concentrations at pH 8.0.Data points correspond to average values of four independent measurements, while error bars represent SEMs.

Figure S3 .
Figure S3.Conformation of the loop 347-370 in PaCGL and homologs.(A) Zoomed view of the loop L347-370 in PaCGL and homolog enzymes obtained from the crystal structure of CGLs from various species, and from the Alphafold-2 predicted model of PaCGL.SaCGL, Staphylococcus aureus (PDB ID 7MCB); TgCGL, Toxoplasma gondii (PDB ID 7NL1); ScCGL, Saccharomyces cerevisiae (PDB ID 1N8P); SmCGL, Stenotrophomonas maltophila (PDB ID 6K1L); XoCGL, Xanthomonas oryzae (PDB ID 4IYO); LpCGL, Lactobacillus plantarum (PDB ID 6LDO); HsCGL, Homo sapiens (PDB ID 2NMP); PaCGL, Pseudomonas aeruginosa-crystal structure (PDB ID 7BA4); PaCGL-AF2 (Pseudomonas aeruginosa, predicted with Alphafold-2).(B) Detailed view of the loop L347-370 of PaCGL observed in the crystals and 2FoFc sweighted electron density map, contoured at 1 s.(C) Alignment of the amino acid sequence from loop 347-370 of PaCGL with the homologous region of the organisms depicted in panel A. The numbering of the corresponding amino acids and the UniProt code of each protein are indicated in the figure.The upper part displays the consensus sequence and the level of conservation resulting from the alignment using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).Residues included in the commonly observed two-turn helix a13 are indicated with a cylinder.

Figure S5 .
Figure S5.Fluctuation plot analysis (A) and B-thermal parameter (B) distribution found in known CGL structures.(A) The structure flexibility of CGLs from different organisms was assessed using the CABSflex tool (https://biocomp.chem.uw.edu.pl/CABSflex2)[27] and the results were presented graphically as Root Mean Square Fluctuation (RMSF) plotted against residues.The regions exhibiting the highest flexibility were denoted by * and **.(B) Visualization of B-thermal parameters extracted from the available crystal structures of diverse CGLs; arrows highlight the location of the

Table S2 Statistics for data collection and refinement
One crystal was used for each data set.Values in parentheses are for highest-resolution shell.