Structural insight into the inactivation of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> non-classical transpeptidase Ldt<Subscript>Mt2</Subscript> by biapenem and tebipenem

Background

The carbapenem subclass of β-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of β-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 → 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb).

Results

Here, we report the X-ray crystal structures of Mtb L,D-transpeptidase-2 (Ldt_Mt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of Ldt_Mt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by β-lyases.

Conclusion

The results presented here demonstrate biapenem and tebipenem bind to the outer cavity of Ldt_Mt2, covalently inactivate the enzyme, and subsequently degrade via an S-conjugate elimination mechanism. We discuss structure based drug design based on the findings and propose that the S-conjugate elimination can be leveraged to design novel agents to deliver and locally release antimicrobial factors to act synergistically with the carbapenem carrier.

     

Hydrogen bonding in the mechanism of GDP-mannose mannosyl hydrolase

GDP-mannose mannosyl hydrolase (GDPMH) from E. coli catalyzes the hydrolysis of GDP-α-d-sugars to GDP and β-d-sugars by nucleophilic substitution with inversion at the anomeric C1 of the sugar, with general base catalysis by His-124. The 1.3 Å X-ray structure of the GDPMH-Mg²⁺-GDP complex was used to model the complete substrate, GDP-mannose into the active site. The substrate is linked to the enzyme by 12 hydrogen bonds, as well as by the essential Mg²⁺. In addition, His-124 was found to participate in a hydrogen bonded triad: His-124-NδHTyr-127-OHPro-120(CO). The contributions of these hydrogen bonds to substrate binding and to catalysis were investigated by site-directed mutagenesis. The hydrogen bonded triad detected in the X-ray structure was found to contribute little to catalysis since the Y127F mutation of the central residue shows only 2-fold decreases in both k_cat and K_m. The GDP leaving group is activated by the essential Mg²⁺ which contributes at least 10⁵-fold to k_cat, and by nine hydrogen bonds, including those from Tyr-103, Arg-37, Arg-52, and Arg-65 (via an intervening water), each of which contribute factors to k_cat ranging from 24- to 309-fold. Both Arg-37 and Tyr-103 bind the β-phosphate of the leaving GDP and are only 5.0 Å apart. Accordingly, the R37Q/Y103F double mutant shows partially additive effects of the two single mutants on k_cat, indicating cooperativity of Arg-37 and Tyr-103 in promoting catalysis. The extensive activation of the GDP leaving group suggests a mechanism with dissociative character with a cationic oxocarbenium-like transition state and a half-chair conformation of the sugar ring, as found with glycosidase enzymes. Accordingly, Asp-22 which contributes 10^2.1- to 10^2.6-fold to k_cat, is positioned to both stabilize a developing cationic center at C1 and to accept a hydrogen bond from the C2–OH of the mannosyl group, and His-88, which contributes 10^2.3-fold to k_cat, is positioned to accept a hydrogen bond from the C3–OH of the mannose facilitating its distortion to a half-chair conformation. Also, the fluorinated substrate GDP-2-fluoro-α-d-mannose, for which the oxocarbenium ion-like transition state centered at C1 would be destabilized by electron withdrawal, shows a 16-fold lower k_cat and a 2.5-fold greater K_m than does GDP-α-d-mannose. The product of the contributions to catalysis of Arg-37 and Tyr-103 (taking their cooperativity into account), Arg-52, Arg-65, Mg²⁺, Asp-22, His-124, and His-88 is ≥10¹⁹, which exceeds the 10¹²-fold rate acceleration produced by GDPMH by a factor ≥10⁷. Hence, additional pairs or groups of catalytic residues must act cooperatively to promote catalysis.     

Use of bacterial cellulose in the textile industry and the wettability challenge—a review

Bacterial cellulose (BC) has been studied as an alternative material in several segments of the food, pharmaceutical, materials and textile industries. The importance of BC is linked to sustainability goals, since it is an easily degradable biomaterial of low toxicity to the environment and is a renewable raw material. For use in the textile area, bacterial cellulose has attracted great interest from researchers, but it presents some challenges notably to its hydrophilic structure. This integrative review article brings together studies and methods related to minimizing the hydrophilicity of bacterial cellulose, in order to expand its applicability in the textile industry in its dry state. The databases consulted were Scopus, ScienceDirect, ProQuest and Web of Science, the documents investigated were scientific articles and the time period investigated was between 2015 and 2021. The results showed that although there are methods to make the BC membrane more hydrophobic, future studies in this regard and on other properties must continue so that bacterial cellulose can be commercially introduced in the textile sector.