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To survive in the metal-scarce environment within the host, pathogens synthesize various small molecular metallophores to facilitate the acquisition of transition metals. The cobalt and nickel transporter (Cnt) system synthesizes and transports staphylopine, a nicotianamine-like metallophore, and serves as a primary transition metal uptake system in Gram-positive bacteria including the human pathogen Staphylococcus aureus. In this study, we report the design of the first inhibitor of the Cnt system by targeting the key aminobutanoyltransferase CntL which is involved in the biosynthesis of staphylopine. Through structure-guided fragment linking and optimization, a class of acceptor-adenosine dual-site inhibitors against S. aureus CntL (SaCntL) were designed and synthesized. The most potent inhibitor, compound 9, demonstrated a ΔT m value of 9.4 °C, a K d value of 0.021 ± 0.004 μM, and an IC 50 value of 0.06 μM against SaCntL. The detailed mechanism by which compound 9 inhibits SaCntL has been elucidated through a high-resolution co-crystal structure. Treatment with compound 9 resulted in a moderate downregulation of intracellular concentrations of iron, nickel, and cobalt ions in the S. aureus cells cultured in the metal-scarce medium, providing the first chemical validation of the important role of Cnt system in bacterial metal acquisition. Our findings pave the way for the development of CntL-based antibacterial agents in future., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Staphylopine (StP) and other nicotianamine-like metallophores are crucial for many pathogens to acquire the transition metals from hosts during invasion. CntL from Staphylococcus aureus (SaCntL) catalyzes the condensation of the 2-aminobutyrate (Ab) moiety of S-adenosylmethionine (SAM) with D-histidine in the biosynthesis of StP. Here, we report the crystal structures of SaCntL in complex with either SAM or two products. The structure of SaCntL consists of an N-terminal four-helix bundle (holding catalytic residue E84) and a C-terminal Rossmann fold (binding the substrates). The sequence connecting the N- and C-terminal domains (N-C linker) in SaCntL was found to undergo conformational alternation between open and closed states. Our structural and biochemical analyses suggested that this intrinsically dynamic interdomain linker forms an additional structural module that plays essential roles in ligand diffusion, recognition, and catalysis. We confirmed that SaCntL stereoselectively carries out the catalysis of D-His but not its enantiomer, L-His, and we found that the N-C linker and active site of SaCntL could accommodate both enantiomers. SaCntL is likely able to bind L-His without catalysis, and as a result, L-His could show inhibitory effects toward SaCntL. These findings provide critical structural and mechanistic insights into CntL, which facilitates a better understanding of the biosynthesis of nicotianamine-like metallophores and the discovery of inhibitors of this process., (© 2021 Federation of American Societies for Experimental Biology.)

Pseudopaline and staphylopine are opine metallophores biosynthesized by Pseudomonas aeruginosa and Staphylococcus aureus , respectively. The final step in opine metallophore biosynthesis is the condensation of the product of a nicotianamine (NA) synthase reaction ( i.e. l-HisNA for pseudopaline and d-HisNA for staphylopine) with an α-keto acid (α-ketoglutarate for pseudopaline and pyruvate for staphylopine), which is performed by an opine dehydrogenase. We hypothesized that the opine dehydrogenase reaction would be reversible only for the opine metallophore product with ( R )-stereochemistry at carbon C2 of the α-keto acid (prochiral prior to catalysis). A kinetic analysis using stopped-flow spectrometry with ( R )- or ( S )-staphylopine and kinetic and structural analysis with ( R )- and ( S )-pseudopaline confirmed catalysis in the reverse direction for only ( R )-staphylopine and ( R )-pseudopaline, verifying the stereochemistry of these two opine metallophores. Structural analysis at 1.57-1.85 Å resolution captured the hydrolysis of ( R )-pseudopaline and allowed identification of a binding pocket for the l-histidine moiety of pseudopaline formed through a repositioning of Phe-340 and Tyr-289 during the catalytic cycle. Transient-state kinetic analysis revealed an ordered release of NADP + followed by staphylopine, with staphylopine release being the rate-limiting step in catalysis. Knowledge of the stereochemistry for opine metallophores has implications for future studies involving kinetic analysis, as well as opine metallophore transport, metal coordination, and the generation of chiral amines for pharmaceutical development., (© 2019 McFarlane et al.)

Siderophores, iron-scavenging small molecules, are fundamental to bacterial nutrient metal acquisition and enable pathogens to overcome challenges imposed by nutritional immunity. Multimodal imaging mass spectrometry allows visualization of host-pathogen iron competition, by mapping siderophores within infected tissue. We have observed heterogeneous distributions of Staphylococcus aureus siderophores across infectious foci, challenging the paradigm that the vertebrate host is a uniformly iron-depleted environment to invading microbes., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Opine dehydrogenases (ODHs) from the bacterial pathogens Staphylococcus aureus , Pseudomonas aeruginosa , and Yersinia pestis perform the final enzymatic step in the biosynthesis of a new class of opine metallophores, which includes staphylopine, pseudopaline, and yersinopine, respectively. Growing evidence indicates an important role for this pathway in metal acquisition and virulence, including in lung and burn-wound infections ( P. aeruginosa ) and in blood and heart infections ( S. aureus ). Here, we present kinetic and structural characterizations of these three opine dehydrogenases. A steady-state kinetic analysis revealed that the three enzymes differ in α-keto acid and NAD(P)H substrate specificity and nicotianamine-like substrate stereoselectivity. The structural basis for these differences was determined from five ODH X-ray crystal structures, ranging in resolution from 1.9 to 2.5 Å, with or without NADP + bound. Variation in hydrogen bonding with NADPH suggested an explanation for the differential recognition of this substrate by these three enzymes. Our analysis further revealed candidate residues in the active sites required for binding of the α-keto acid and nicotianamine-like substrates and for catalysis. This work reports the first structural kinetic analyses of enzymes involved in opine metallophore biosynthesis in three important bacterial pathogens of humans., (© 2018 McFarlane et al.)