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Acinetobacter baumannii is an opportunistic human pathogen which can use host-derived L-carnitine as sole carbon and energy source. Recently, an L-carnitine transporter (Aci1347) and a specific monooxygense (CntA/CntB) for the intracellular cleavage of L-carnitine have been characterized. Subsequent conversion of the resulting malic semialdehyde into the central metabolite L-malate was hypothesized. Alternatively, L-carnitine degradation via D-malate with subsequent oxidation into pyruvate was proposed. Here we describe the in vitro and in vivo reconstitution of the entire pathway, starting from the as yet uncharacterized gene products of the carnitine degradation gene operon. Using recombinantly purified enzymes, enantiomer-specific formation of D-malate by the NAD(P)+-dependent malic semialdehyde dehydrogenase (MSA-DH) is demonstrated. The solved X-ray crystal structure of tetrameric MSA-DH reveals the key catalytic residues Cys290 and Glu256, accessible through opposing substrate and cofactor funnels. Specific substrate binding is enabled by Arg166, Arg284 and Ser447 while dual cofactor specificity for NAD+ and NADP+ is mediated by Asn184. The subsequent conversion of the unusual D-malate reaction product by an uncharacterized NAD+-dependent malate dehydrogenase (MDH) is shown. Tetrameric MDH is a ß-decarboxylating dehydrogenase that synthesizes pyruvate. MDH experiments with alternative substrates showed a high degree of substrate specificity. Finally, the entire A. baumannni pathway was heterologously reconstituted, allowing E. coli to grow on L-carnitine as a carbon and energy source. Overall, the metabolic conversion of L-carnitine via malic semialdehyde and D-malate into pyruvate, CO2 and trimethylamine was demonstrated. Trimethylamine is also an important gut microbiota-dependent metabolite that is associated with an increased risk of cardiovascular disease. The pathway reconstitution experiments allowed us to assess the TMA forming capacity of gut microbes which is related to human cardiovascular health. [ABSTRACT FROM AUTHOR]

Legionella pneumophila, the causative agent of a life‐threatening pneumonia, intracellularly replicates in a specialized compartment in lung macrophages, the Legionella‐containing vacuole (LCV). Secreted proteins of the pathogen govern important steps in the intracellular life cycle including bacterial egress. Among these is the type II secreted PlaA which, together with PlaC and PlaD, belongs to the GDSL phospholipase family found in L. pneumophila. PlaA shows lysophospholipase A (LPLA) activity which increases after secretion and subsequent processing by the zinc metalloproteinase ProA within a disulfide loop. Activity of PlaA contributes to the destabilization of the LCV in the absence of the type IVB‐secreted effector SdhA. We here present the 3D structure of PlaA which shows a typical α/β‐hydrolase fold and reveals that the uncleaved disulfide loop forms a lid structure covering the catalytic triad S30/D278/H282. This leads to reduction of substrate access before activation; however, the catalytic site gets more accessible when the disulfide loop is processed. After structural modeling, a similar activation process is suggested for the GDSL hydrolase PlaC, but not for PlaD. Furthermore, the size of the PlaA substrate‐binding site indicated preference toward phospholipids comprising ~16 carbon fatty acid residues which was verified by lipid hydrolysis, suggesting a molecular ruler mechanism. Indeed, mutational analysis changed the substrate profile with respect to fatty acid chain length. In conclusion, our analysis revealed the structural basis for the regulated activation and substrate preference of PlaA. [ABSTRACT FROM AUTHOR]

The non‐canonical terpene cyclase AsR6 is responsible for the formation of 2E,6E,9E‐humulene during the biosynthesis of the tropolone sesquiterpenoid (TS) xenovulene A. The structures of unliganded AsR6 and of AsR6 in complex with an in crystallo cyclized reaction product and thiolodiphosphate reveal a new farnesyl diphosphate binding motif that comprises a unique binuclear Mg2+‐cluster and an essential K289 residue that is conserved in all humulene synthases involved in TS formation. Structure‐based site‐directed mutagenesis of AsR6 and its homologue EupR3 identify a single residue, L285/M261, that controls the production of either 2E,6E,9E‐ or 2Z,6E,9E‐humulene. A possible mechanism for the observed stereoselectivity was investigated using different isoprenoid precursors and results demonstrate that M261 has gatekeeping control over product formation. [ABSTRACT FROM AUTHOR]

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In the search for new antitubercular compounds, we leveraged target-directed dynamic combinatorial chemistry (tdDCC) as an efficient hit-identification method. In tdDCC, the target selects its own binders from a dynamic library generated in situ , reducing the number of compounds that require synthesis and evaluation. We combined a total of 12 hydrazides and six aldehydes to generate 72 structurally diverse N -acylhydrazones. To amplify the best binders, we employed anti-infective target 4-diphosphocytidyl-2 C -methyl-d-erythritol kinase (IspE) from Mycobacterium tuberculosis ( Mtb ). We successfully validated the use of tdDCC as hit-identification method for IspE and optimized the analysis of tdDCC hit determination. From the 72 possible N -acylhydrazones, we synthesized 12 of them, revealing several new starting points for the development of IspE inhibitors as antibacterial agents., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa, which is on the WHO list of antibiotic resistant "priority pathogens". In this study the isomerase PhzF, a key bacterial enzyme of the pyocyanin biosynthetic pathway, was investigated as a pathoblocker target. The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it, thus preventing the rapid development of resistance. Based on crystal structures of PhzF, derivatives of the inhibitor 3-hydroxyanthranilic acid were designed. Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF in the closed conformation. In contrast, ligands aligned to the open conformation of PhzF provided more room for structural modifications. The intrinsic fluorescence of small 3-hydroxyanthranilic acid derivatives enabled direct affinity determinations using FRET assays. The analysis of structure-activity relationships showed that the carboxylic acid moiety is essential for binding to the target enzyme. The results of this study provide fundamental structural insights that will be useful for the design of PhzF-inhibitors., (© 2024 Wiley‐VCH GmbH.)

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile . D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridial toxins TcdA and TcdB. D-proline reduction is catalyzed by the proline reductase Prd, which belongs to the pyruvoyl-dependent enzymes. These enzymes are translated as inactive proenzymes and require subsequent processing to install the covalently bound pyruvate. Whereas pyruvoyl formation by intramolecular serinolysis has been studied in unrelated enzymes, details about pyruvoyl generation by cysteinolysis as in Prd are lacking. Here, we show that Prd maturation requires a small dimeric protein that we have named PrdH. PrdH (CD630_32430) is co-encoded with the PrdA and PrdB subunits of Prd and also found in species producing similar reductases. By producing stable variants of PrdA and PrdB, we demonstrate that PrdH-mediated cleavage and pyruvoyl formation in the PrdA subunit requires PrdB, which can be harnessed to produce active recombinant Prd for subsequent analyses. We further created PrdA- and PrdH-mutants to get insight into the interaction of the components and into the processing reaction itself. Finally, we show that deletion of prdH renders C. difficile insensitive to proline concentrations in culture media, suggesting that this processing factor is essential for proline utilization. Due to the link between Stickland fermentation and pathogenesis, we suggest PrdH may be an attractive target for drug development., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

The resolution of 3D electron diffraction (ED) data of small-molecule crystals is often relatively poor, due to either electron-beam radiation damage during data collection or poor crystallinity of the material. Direct methods, used as standard for crystal structure determination, are not applicable when the data resolution falls below the commonly accepted limit of 1.2 Å. Therefore an evaluation was carried out of the performance of molecular replacement (MR) procedures, regularly used for protein structure determination, for structure analysis of small-molecule crystal structures from 3D ED data. In the course of this study, two crystal structures of Bi-3812, a highly potent inhibitor of the oncogenic transcription factor BCL6, were determined: the structure of α-Bi-3812 was determined from single-crystal X-ray data, the structure of β-Bi-3812 from 3D ED data, using direct methods in both cases. These data were subsequently used for MR with different data types, varying the data resolution limit (1, 1.5 and 2 Å) and by using search models consisting of connected or disconnected fragments of BI-3812. MR was successful with 3D ED data at 2 Å resolution using a search model that represented 74% of the complete molecule., (open access.)

The non-canonical terpene cyclase AsR6 is responsible for the formation of 2E,6E,9E-humulene during the biosynthesis of the tropolone sesquiterpenoid (TS) xenovulene A. The structures of unliganded AsR6 and of AsR6 in complex with an in crystallo cyclized reaction product and thiolodiphosphate reveal a new farnesyl diphosphate binding motif that comprises a unique binuclear Mg 2+ -cluster and an essential K289 residue that is conserved in all humulene synthases involved in TS formation. Structure-based site-directed mutagenesis of AsR6 and its homologue EupR3 identify a single residue, L285/M261, that controls the production of either 2E,6E,9E- or 2Z,6E,9E-humulene. A possible mechanism for the observed stereoselectivity was investigated using different isoprenoid precursors and results demonstrate that M261 has gatekeeping control over product formation., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)