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Upon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell division-type tripartite efflux pumps. These include a H + /drug antiporter module that recognizes structurally diverse substances, including antibiotics. Here, we show the 3.5 Å structure of subunit AdeB from the Acinetobacter baumannii AdeABC efflux pump solved by single-particle cryo-electron microscopy. The AdeB trimer adopts mainly a resting state with all protomers in a conformation devoid of transport channels or antibiotic binding sites. However, 10% of the protomers adopt a state where three transport channels lead to the closed substrate (deep) binding pocket. A comparison between drug binding of AdeB and Escherichia coli AcrB is made via activity analysis of 20 AdeB variants, selected on basis of side chain interactions with antibiotics observed in the AcrB periplasmic domain X-ray co-structures with fusidic acid (2.3 Å), doxycycline (2.1 Å) and levofloxacin (2.7 Å). AdeABC, compared to AcrAB-TolC, confers higher resistance to E. coli towards polyaromatic compounds and lower resistance towards antibiotic compounds. Resistance-nodulation-cell division (RND)-type tripartite efflux pumps confer multidrug resistance to Gram-negative bacteria. Here, structural and functional analyses of AdeB from Acinetobacter baumannii and AcrB from Escherichia coli provide insight into their different drug-binding and conformational drug transport states.

The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens.

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 Å resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interlocked in a tautomeric equilibrium. Formation of the keto tautomer is associated with a loss of aromaticity of the cofactor. The alternate confinement of electrons to neighboring atoms rather than π -conjugation seems to be of importance for the enzyme-catalyzed, redox-coupled acyl transfer to phosphate, which requires a dramatic inversion of polarity of the reacting substrate carbon in two subsequent catalytic steps. The ability to oscillate between a nucleophilic (carbanion) and an electrophilic (ketone) substrate center highlights a hitherto unrecognized versatility of the thiamin cofactor. It remains to be studied whether formation of the keto tautomer is a general feature of all thiamin enzymes, as it could provide for stable storage of the carbanion state, or whether this feature represents a specific trait of thiamin oxidases. In addition, the protonation state of the two-electron reduced flavin cofactor can be fully assigned, demonstrating the power of high-resolution cryocrystallography for elucidation of enzymatic mechanisms.

X-ray crystallography and EPR spectroscopy are used to characterize a soluble, oxygen-tolerant reductive dehalogenase from Nitratireductor pacificus pht-3B; the data suggest that the cobalt in the cobalamin cofactor ligates the halogen atom of the substrate, directly abstracting the halogen atom via an oxidative addition. Mechanism of action of a reductive dehalogenase Reductive dehalogenases are cobalamin-dependent enzymes that catalyse the removal of a halogen atom from organohalides, organic molecules that contain one or more halogen atoms. A large proportion of environmental pollutants are organohalides and reductive dehalogenases are responsible for the biological dehalogenation of these compounds, including polychlorinated biphenyls. In this manuscript, the authors used X-ray crystallography and EPR spectroscopy to characterize a soluble, oxygen-tolerant reductive dehalogenase, pht-3B, from Nitratireductor pacificus . Their data suggest that the cobalt in the cobalamin cofactor forms a covalent bond with the halogen atom of the substrate, directly abstracting the halogen atom via an oxidative addition. This mechanism is fundamentally different from the mechanisms of other cobalamin-containing enzymes. These findings will be of relevance to future use of reductive dehalogenases in bioremediation and biocatalysis. Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides 1 . Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle 2 , 3 . Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria 4 , 5 , with substrates including polychlorinated biphenyls or dioxins 6 , 7 . Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies 8 , 9 , 10 , 11 , 12 . Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon–cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies 13 , we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen–cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 Å resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interlocked in a tautomeric equilibrium. Formation of the keto tautomer is associated with a loss of aromaticity of the cofactor. The alternate confinement of electrons to neighboring atoms rather than π-conjugation seems to be of importance for the enzyme-catalyzed, redox-coupled acyl transfer to phosphate, which requires a dramatic inversion of polarity of the reacting substrate carbon in two subsequent catalytic steps. The ability to oscillate between a nucleophilic (carbanion) and an electrophilic (ketone) substrate center highlights a hitherto unrecognized versatility of the thiamin cofactor. It remains to be studied whether formation of the keto tautomer is a general feature of all thiamin enzymes, as it could provide for stable storage of the carbanion state, or whether this feature represents a specific trait of thiamin oxidases. In addition, the protonation state of the two-electron reduced flavin cofactor can be fully assigned, demonstrating the power of high-resolution cryocrystallography for elucidation of enzymatic mechanisms.

The evolution of microorganisms over millions of years has led to an impressive adaptability regarding the utilisation of different environmental conditions. The identification of bacterial species with the fascinating features to use cobalamin-dependent metalloenzymes to (i) extract energy from halogenated organic compounds (organohalides) and (ii) transfer methyl groups from lignin breakdowns into central carbon pathways, are examples for this adaptability. The biochemical study of these two cobalamin-dependent enyzmes is the topic of this PhD project.For the extraction of growth energy, organohalides serve as terminal electron acceptorsand are reductively dehalogenated in a respiratory manner termed organohalide respiration. Reductive dehalogenases, the key enzymes in organohalide respiration, catalyse the chemical cleavage between the halogen substituent and the carbon moiety. They use cobalamin and two Fe-S clusters as cofactors and constitute a new and distinct class of cobalamin-dependent enzymes. Their three-dimensional structure and the mechanism of catalysis are unknown, because their hydrophobicity and oxygen sensitivity have hampered their biochemical investigation. Here, a novel purification technology in Escherichia coli for the reductive dehalogenase PceA from Dehalobacter restrictus has been developed, accompanied by methods that allow the in vitro reconstitution of PceA with both cofactors, cobalamin and Fe-S clusters. It has been demonstrated that the soluble expression of PceA is dependent on the covalent fusion of the enzyme to a trigger factor chaperone. Based on these findings, the PceA specific trigger factor PceT has been studied biochemically, resulting in its successful crystallisation. The established protocols for PceA and PceT are transferable to other members of their respective families, which will therefore allow detailed studies of reductive dehalogenases and their associated chaperones in the future.In addition to reductive dehalogenases, organohalide respiring bacteria contain anothercobalamin-dependent enzyme system, termed O-demethylase, which is involved in the carbon metabolism of different anaerobic bacteria. O-demethylases are three-component enzyme systems that transfer methyl groups from aromatic methyl ethers totetrahydrofolate via methylcobalamin intermediates. The different cofactors (substrate,cobalamin and tetrahydrofolate), bind to either of the three individual proteins involvedin O-demethylation. It has been speculated that the same or similar halogenated aromatic molecules are substrates for both organohalide respiration and O-demethylation in the same bacteria. In order to test this proposal, a O-demethylase from Desulfitobacterium hafniense DCB-2 has been studied using X-ray crystallography and biochemistry. As a result, the first crystal structures of the cobalamin-binding protein in complex with cobalamin, and of the methyl acceptor protein in complex with substrate (tetrahydrofolate) and product (methyltetrahydrofolate) from a O-demethylase have been solved toresolutions of 1.5 A, 1.8 A and 1.6 A, respectively. The crystal structures, in combinationwith spectroscopic and biophysical analyses, have led to a proposed mechanism forthe catalysed methyl transfer reaction from methylcobalamin to tetrahydrofolate.