Structural and Dynamic Features of Acinetobacter baumannii OXA-66 ß-Lactamase Explain Its Stability and Evolution of Novel Variants.

OXA-66 is a member of the OXA-51 subfamily of class D ß-lactamases native to the Acinetobacter genus that includes Acinetobacter baumannii, one of the ESKAPE pathogens and a major cause of drug-resistant nosocomial infections. Although both wild type OXA-66 and OXA-51 have low catalytic activity, they are ubiquitous in the Acinetobacter genomes. OXA-51 is also remarkably thermostable. In addition, newly emerging, single and double amino acid variants show increased activity against carbapenems, indicating that the OXA-51 subfamily is growing and gaining clinical significance. In this study, we used molecular dynamics simulations, X-ray crystallography, and thermal denaturation data to examine and compare the dynamics of OXA-66 wt and its gain-of-function variants: I129L (OXA-83), L167V (OXA-82), P130Q (OXA-109), P130A, and W222L (OXA-234). Our data indicate that OXA-66 wt also has a high melting temperature, and its remarkable stability is due to an extensive and rigid hydrophobic bridge formed by a number of residues around the active site and harbored by the three loops, P, Ω, and ß5-ß6. Compared to the WT enzyme, the mutants exhibit higher flexibility only in the loop regions, and are more stable than other robust carbapenemases, such as OXA-23 and OXA-24/40. All the mutants show increased rotational flexibility of residues I129 and W222, which allows carbapenems to bind. Overall, our data support the hypothesis that structural features in OXA-51 and OXA-66 promote evolution of multiple highly stable variants with increased clinical relevance in A. baumannii.

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target.

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC50 0.45 µM), VT1598 (0.25 µM), and VT1161 (0.20 µM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch.

Resistant starch is a prebiotic accessed by gut bacteria with specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses Sas6 (Starch Adherence System member 6), which consists of two starch-specific carbohydrate-binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here, we present the crystal structures of Sas6 and of RbCBM74 bound with a double helical dimer of maltodecaose. The RbCBM74 starch-binding groove complements the double helical α-glucan geometry of amylopectin, suggesting that this module selects this feature in starch granules. Isothermal titration calorimetry and native mass spectrometry demonstrate that RbCBM74 recognizes longer single and double helical α-glucans, while RbCBM26 binds short maltooligosaccharides. Bioinformatic analysis supports the conservation of the amylopectin-targeting platform in CBM74s from resistant-starch degrading bacteria. Our results suggest that RbCBM74 and RbCBM26 within Sas6 recognize discrete aspects of the starch granule, providing molecular insight into how this structure is accommodated by gut bacteria.