Protein-structure-based prediction of animal model suitability for pharmacodynamic studies of subtype-selective estrogens

Subtype-selective estrogens are of increasing importance as tools used to unravel physiological roles of the estrogen receptors, ERalpha and ERbeta, in various species. Although human ERalpha and ERbeta differ by only two amino acids within the binding pockets, we and others recently succeeded in generating subtype-selective agonists. We have proposed that the selectivity of the steroidal compounds 16alpha-lactone-estradiol (16alpha-LE(2), hERalpha selective) and 8beta-vinyl-estradiol (8beta-VE(2), hERbeta selective) is based on the interaction of certain substituents of these compounds with essentially one amino acid in the respective ER binding pockets. For in vitro and ex vivo pharmacological experiments with these compounds we intended to use bovine tissues available from slaughterhouses in larger quantities. Using homology modeling techniques we determined that the amino acid conferring high hERbeta-selectivity to 8beta-VE(2) is not exchanged between human and bovine ERalpha and bovine ERbeta. Thus, we predicted our steroidal hERbeta-selective compound to exhibit only weak agonistic activity at bERbeta and that bovine tissue is therefore not suited for investigation of ERbeta functions. The situation is presumably identical for pig, sheep, and the common marmoset, whereas rats, mice, and rhesus macaques are appropriate animal models to study pharmacological effects of 8beta-VE(2) in vivo. This prediction was confirmed in transactivation studies assessing estradiol (E(2)) and the two subtype-selective ligands on bovine ERbeta and on a series of hERalpha and hERbeta with mutations in their respective ligand-binding pockets. We have shown that the detailed understanding of the interactions of a compound with its target protein enables the identification of relevant species for pharmacological studies.     

The Alkylquinolone Repertoire of Pseudomonas aeruginosa is Linked to Structural Flexibility of the FabH‐like 2‐Heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) Biosynthesis Enzyme PqsBC

Pseudomonas aeruginosa is a bacterial pathogen that causes life‐threatening infections in immunocompromised patients. It produces a large armory of saturated and mono‐unsaturated 2‐alkyl‐4(1H)‐quinolones (AQs) and AQ N‐oxides (AQNOs) that serve as signaling molecules to control the production of virulence factors and that are involved in membrane vesicle formation and iron chelation; furthermore, they also have, for example, antibiotic properties. It has been shown that the β‐ketoacyl‐acyl‐carrier protein synthase III (FabH)‐like heterodimeric enzyme PqsBC catalyzes the last step in the biosynthesis of the most abundant AQ congener, 2‐heptyl‐4(1H)‐quinolone (HHQ), by condensing octanoyl‐coenzyme A (CoA) with 2‐aminobenzoylacetate (2‐ABA), but the basis for the large number of other AQs/AQNOs produced by P. aeruginosa is not known. Here, we demonstrate that PqsBC uses different medium‐chain acyl‐CoAs to produce various saturated AQs/AQNOs and that it also biosynthesizes mono‐unsaturated congeners. Further, we determined the structures of PqsBC in four different crystal forms at 1.5 to 2.7 Å resolution. Together with a previous report, the data reveal that PqsBC adopts open, intermediate, and closed conformations that alter the shape of the acyl‐binding cavity and explain the promiscuity of PqsBC. The different conformations also allow us to propose a model for structural transitions that accompany the catalytic cycle of PqsBC that might have broader implications for other FabH‐enzymes, for which such structural transitions have been postulated but have never been observed.     

Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4‐Oxalocrotonate Tautomerase More Effectively than Distant Mutations

The enzyme 4‐oxalocrotonate tautomerase (4‐OT), which catalyzes enol–keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon–carbon bond‐forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4‐OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000‐fold improvement in catalytic efficiency (k_cat/K_m) and a >10⁷‐fold change in reaction specificity, by exploring small libraries in which only “hotspots” are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4‐OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site.     

Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

Arginine methylation is a prevalent post‐translational modification in eukaryotic cells. Two significant debates exist within the field: do these enzymes dimethylate their substrates in a processive or distributive manner, and do these enzymes operate using a random or sequential method of bisubstrate binding? We revealed that human protein arginine N‐methyltransferase 1 (PRMT1) enzyme kinetics are dependent on substrate sequence. Further, peptides containing an Nη‐hydroxyarginine generally demonstrated substrate inhibition and had improved K_M values, which evoked a possible role in inhibitor design. We also revealed that the perceived degree of enzyme processivity is a function of both cofactor and enzyme concentration, suggesting that previous conclusions about PRMT sequential methyl transfer mechanisms require reassessment. Finally, we demonstrated a sequential ordered Bi–Bi kinetic mechanism for PRMT1, based on steady‐state kinetic analysis. Together, our data indicate a PRMT1 mechanism of action and processivity that might also extend to other functionally and structurally conserved PRMTs.     

The Synechocystis sp. PCC6803 Sfp‐Type Phosphopantetheinyl Transferase Does Not Possess Characteristic Broad‐Range Activity

The cyanobacterium Synechocystis sp. PCC6803 harbours one phosphopantetheinyl transferase (PPTase), Sppt. Protein modelling supported previous bioinformatics analyses, which suggested that Sppt is a Sfp‐type PPTase with the potential to phosphopantetheinylate a broad range of carrier proteins from both primary and secondary metabolism. However, no natural products are synthesised by this species, which raises interesting evolutionary and functional questions. Phosphopantetheinylation assays and kinetic data demonstrate that Sppt was able to activate its cognate fatty acid synthesis carrier protein, SACP, but was unable to effectively activate various cyanobacterial carrier proteins from secondary metabolism or glycolipid biosynthesis pathways. To our knowledge, this is the first example of a PPTase with a Sfp‐type structure, but with activity more closely resembling AcpS‐type enzymes. The broad‐range PPTase from Nodularia spumigena NSOR10 was introduced into Synechocystis sp. PCC6803 and was shown to activate a noncognate carrier protein, in vivo. This engineered strain could provide a future biotechnological platform for the heterologous expression of cyanobacterial biosynthetic gene clusters.