Pseudomonas aeruginosa 4-Amino-4-Deoxychorismate Lyase: Spatial Conservation of an Active Site Tyrosine and Classification of Two Types of Enzyme

4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp2 to sp3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism

Two high-resolution structures of the human E3 ubiquitin ligase Siah1

Siah1 is an E3 ubiquitin ligase that contributes to proteasome-mediated degradation of multiple targets in key cellular processes and which shows promise as a therapeutic target in oncology. Structures of a truncated Siah1 bound to peptide-based inhibitors have been reported. Here, new crystallization conditions have allowed the determination of a construct encompassing dual zinc-finger subdomains and substrate-binding domains at significantly higher resolution. Although the crystals appear isomorphous, two structures present distinct states in which the spatial orientation of one zinc-finger subdomain differs with respect to the rest of the dimeric protein. Such a difference, which is indicative of conformational freedom, infers potential biological relevance related to recognition of binding partners. The crystallization conditions and improved models of Siah1 may aid future studies investigating Siah1–ligand complexes

Confirmed hit compounds from hit discovery campaign.

<p>Summary of the compounds and series identified through the HTS and their respective potencies and Hill slope values.</p

FolD assay development.

<p>(<b>A</b>) <i>N</i>⁵,<i>N</i>¹⁰-methylene-THF K_m determination in the presence of 1 mM NADP⁺. (<b>B</b>) NADP⁺<i>K</i>_M determination in the presence of 1 mM <i>N</i>⁵,<i>N</i>¹⁰-methylene-THF. All <i>K</i>_M measurement data are presented as mean ± SD (n = 4) (<b>C</b>) Representative IC₅₀ determination for LY374571. Data points are mean ± SD (n = 14). This representative example returns an IC₅₀ for LY374571 of 27±3 nM.</p

Replicate testing.

<p>Correlation between replicate pIC₅₀ values for each of the 24 compounds advanced to potency testing. Linear regression of these data returned a correlation coefficient of 0.92.</p

Structure of <i>Pa</i>FolD.

<p>Cartoon representation of a homodimer of <i>Pa</i>FolD with secondary structure labeled. The interface occurs between α5, α7 and β6 of partner subunits.</p

Ligand docking.

<p>Stereoview of the docking of DDD32388 (cyan) into the active site of <i>Pa</i>FolD. Potentially important residues are highlighted as sticks and with hydrogen bonds shown as dashes.</p