Simple rules govern the diversity of bacterial nicotianamine-like metallophores

Abstract

In 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 α-ketoglutarate), pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH). Here, we explored the substrate specificity of CntM by combining bioinformatic and structural analysis with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity is 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 toward α-ketoacids is largely governed by a single residue at position 150 of CntM (S. aureus numbering): an aspartate at this position ensures selectivity toward pyruvate, whereas an alanine leads to the consumption of both pyruvate and α-ketoglutarate. Modifying this residue in P. aeruginosa led to a complete reversal of selectivity. Thus, the diversity of opine-type metallophore 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.

Intracellular Accumulation of Staphylopine Can Sensitize Staphylococcus aureus to Host-Imposed Zinc Starvation by Chelation-Independent Toxicity

“…A wide variety of Gram-positive and Gram-negative microbes possess the ability to make StP-like molecules. Analysis of the synthesis machinery revealed that the metallophores produced from these loci by Yersinia pestis and Paenibacillus mucilaginosus differ from StP and pseudopaline only by the utilization of D-or L-histidine and pyruvate or alpha-ketoglutarate (56,57). Combined with recently proposed rules that govern the alpha-keto acid incorporated into the zincophore (56), it seems likely that many of the uncharacterized loci produce molecules similar to StP and pseudopaline.…”

“…Analysis of the synthesis machinery revealed that the metallophores produced from these loci by Yersinia pestis and Paenibacillus mucilaginosus differ from StP and pseudopaline only by the utilization of D-or L-histidine and pyruvate or alpha-ketoglutarate (56,57). Combined with recently proposed rules that govern the alpha-keto acid incorporated into the zincophore (56), it seems likely that many of the uncharacterized loci produce molecules similar to StP and pseudopaline. These data, combined with the conserved colocalization of an efflux pump with zincophore synthesis loci (12), suggest that StP-like molecules may be toxic, independent of their metal-chelating ability.…”

“…Notably, in S. aureus, the StP system, not the Adc system, is the primary importer that enables successful competition with CP for Zn (12). Loci encoding for analogous zincophore-based importers are present in a wide array of Gram-positive and Gram-negative pathogens, suggesting that zincophores are a common strategy used to obtain Zn in a metal-limited environment, such as the host (10)(11)(12)56).…”

“…In S. aureus, D-histidine and pyruvate are used, while in P. aeruginosa, L-histidine and alphaketoglutarate are used (9)(10)(11). Yersinia pestis and Paenibacillus mucilaginosus also produce putative zincophores that are chemically similar to but distinct from StP and pseudopaline (56,57). In S. aureus, all of the machinery necessary for synthesis (cntKLM), import (cntABCDF), and export (cntE) of StP is present at a single locus (9).…”

Bioinformatic Mapping of Opine-Like Zincophore Biosynthesis in Bacteria

“…While only a few bacterial opine-like zincophores have been characterized to date, two general "rules" have been described for predicting the type of zincophore that will be produced based on a biosynthetic gene cluster (BGC) (27). The first of these, the presence or absence of a histidine racemase, simply determines which stereoisomer of histidine will be utilized by NAS synthase, thereby changing the stereochemistry of the histidine moiety in the zincophore.…”