The ancient roots of nicotianamine: diversity, role, regulation and evolution of nicotianamine-like metallophores

International audienceNicotianamine(NA)isametabolitesynthesized by allplantsinwhichitisinvolved inthehomeostasisofdifferentmicronutrientssuchasiron,nickelorzinc.Insomeplantsitalsoserves as a precursor of phytosiderophores that are used for extracellular ironscavenging.PreviousworkshavealsoestablishedthepresenceofNA infilamentousfungiandsomemosseswhereasananalogueofNAwasinferredinanarchaea.Morerecently,opine-typemetallophoreswithhomologytoNAwereuncoveredinbacteria, especiallyinhuman pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa or Yersiniapestis, synthesizing respectively staphylopine, pseudopalineand yersinopine.Here, wereviewthecurrentstateofknowledgeregardingthediscovery,biosynthesis,functionandregulationofthesemetallophores.WealsodiscussthegenomicenvironmentofthecntLgene,whichishomologous totheplantNAsynthase(NAS)gene,andplaysacentralroleinthesynthesisofNA-like metallophores.Thisreveals alargediversityofbiosynthetic,export and import pathways.Using sequence similarity networks, we uncovered thatthese metallophores are widespread in numerous bacteria thriving in very differentenvironments,suchasthose livingatthehost-pathogeninterfacebutalsointhe soil. Weadditionally establishedaphylogenyoftheNAS/cntLgeneand,asaresult,weproposethatthisgene isanancientgeneandNA, oritsderivatives, isan ancientmetallophore thatplayedaprominentroleinmetalacquisition ormetalresistance.Indeed,ourphylogeneticanalysis suggests an evolutionary model where the possibility to synthesize thismetallophore waspresentearlyintheapparitionoflife, althoughit waslaterlostbymostliving microorganisms, unless facing metal starvation such as at the host-pathogeninterface or in some soils. According to our model, NA then re-emerged as a centralmetabolitefor metalhomeostasis in fungi,mossesandallknownhigherplants

Simple rules govern the diversity of bacterial nicotianamine-like metallophores

International audienceIn metal-scarce environments, some pathogenic bacteria produce opine-type metallophores mainly to face the host's nutritional immunity. This is the case of staphylopine, pseudopaline and yersinopine, identified in $Staphylococcus\ aureus$, $Pseudomonas\ aeruginosa$ and $Yersinia\ pestis$ respectively. Depending on the species, these metallophores are synthesized by two (CntLM) or three enzymes (CntKLM), CntM catalyzing the last step of biosynthesis using diverse substrates (pyruvate or $\alpha$-ketoglutarate), pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH). Here, we explored substrate specificity of CntM by combining bioinformatics and structural analysis with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity was mainly due to the amino acid at position 33 ($S.\ aureus$ numbering) which ensures a preferential binding to NADPH when it is an arginine. Moreover, whereas CntM from $P.\ aeruginosa$ preferentially uses yNA over xNA, the staphylococcal enzyme is not stereospecific. Most importantly, selectivity towards $\alpha$- ketoacids is largely governed by a single residue at position 150 of CntM ($S.\ aureus$ numbering): an aspartate at this position ensures selectivity towards pyruvate whereas an alanine leads to the consumption of both pyruvate and $\alpha$-ketoglutarate. Modifying this residue in $P.\ aeruginosa$ led to a complete reversal of selectivity. Thus, opine-type metallophore diversity is governed by the absence/presence of a $cntK$ gene encoding a histidine racemase, and the amino acid residue at position 150 of CntM. These two simple rules predict the production of a fourth metallophore by $Paenibacillus\ mucilaginosus$, which was confirmed $in\ vitro$ and called bacillopaline

Involvement of the Pseudomonas aeruginosa MexAB–OprM efflux pump in the secretion of the metallophore pseudopaline

International audienceTo overcome the metal restriction imposed by the host’s nutritional immunity, pathogenic bacteria use high metal affinity molecules called metallophores. Metallophore-mediated metal uptake pathways necessitate complex cycles of synthesis, secretion, and recovery of the metallophore across the bacterial envelope. We recently discovered staphylopine and pseudopaline, two members of a new family of broad-spectrum metallophores important for bacterial survival during infections. Here, we are expending the molecular understanding of the pseudopaline transport cycle across the diderm envelope of the Gram-negative bacterium Pseudomonas aeruginosa. We first explored pseudopaline secretion by performing in vivo quantifications in various genetic backgrounds and revealed the specific involvement of the MexAB–OprM efflux pump in pseudopaline transport across the outer membrane. We then addressed the recovery part of the cycle by investigating the fate of the recaptured metal-loaded pseudopaline. To do so, we combined in vitro reconstitution experiments and in vivo phenotyping in absence of pseudopaline transporters to reveal the existence of a pseudopaline modification mechanism, possibly involved in the metal release following pseudopaline recovery. Overall, our data allowed us to provide an improved molecular model of secretion, recovery, and fate of this important metallophore by P. aeruginos

Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis

International audienceEnzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a pi-pi interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level