Diisonitrile Natural Product SF2768 Functions As a Chalkophore That Mediates Copper Acquisition in Streptomyces thioluteus

Abstract

A nonribosomal peptide synthetase (NRPS) gene cluster (sfa) was identified in Streptomyces thioluteus to direct the biosynthesis of the diisonitrile antibiotic SF2768. Its biosynthetic pathway was reasonably proposed based on bioinformatics analysis, metabolic profiles of mutants, and the elucidation of the intermediate and shunt product structures. Bioinformatics-based alignment found a putative ATP-binding cassette (ABC) transporter related to iron import within the biosynthetic gene cluster, which implied that the product might be a siderophore. However, characterization of the metal-binding properties by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), metal-ligand titration, thin-layer chromatography (TLC), and chrome azurol S (CAS) assays revealed that the final product SF2768 and its diisonitrile derivatives specifically bind copper, rather than iron, to form stable complexes. Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that the intracellular cupric content of S. thioluteus significantly increased upon incubation with the copper-SF2768 complex, direct evidence for the copper acquisition function of SF2768. Further in vivo functional characterization of the transport elements for the copper-SF2768 complexes not only confirmed the chalkophore identity of the compound but also gave initial clues into the copper uptake mechanism of this nonmethanotrophic microorganism.

A Histidine Residue and a Tetranuclear Cuprous‐thiolate Cluster Dominate the Copper Loading Landscape of a Copper Storage Protein from Streptomyces lividans

“…[15a] Interestingly,d iisonitrile derivativesw ith nitrogen and/or oxygen as Cu I donor,with 1:2(Cu I :ligand) stoichiometry, have been discovered in Streptomycest hioluteus and identified to play ar ole in Cu uptake mechanisms. [16] This poses the question of whether similar compounds serve as donors to Csp3s, as opposed to thiol donors such as CopZ Cu chaperones. Thus, our kineticstudies of Cu I transfer to SlCsp3 utilizing an itrogen Cu I donor in the guise of the [Cu(BCA) 2 ] 3À complex and the discoveryo farole for aH is residuei nf acilitating the Cu I transfer bear significance on potential biological events.…”

Chalkophores

“…Like siderophore and chalkophore operons, the SF2768 operon contains exporters [members of the major facilitator superfamily (MFS)] as well as importers (an ABC transporter annotated as an iron chelate importer). SF2768 binds Cu(I) and Cu(II), and like Mbn, it appears to be able to bind Cu(II) reductively, followed by reinternalization after copper binding (184). Similar operons were identified in other gram-positive species, and characterization of the resulting compounds in Mycobacterium species indicated that they form diisonitrile lipopeptides with a possible role in zinc homeostasis (185).…”

Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

“…ICH is one of only two enzymes characterized to date that degrades organic isocyanides, and homologs are present in many microbes that inhabit soil and water. Some naturally occurring isocyanides are potent antimicrobials (27) and others have recently been shown to be chalkophores, facilitating copper uptake by certain bacteria (28). ICH homologs appear to be important in both of these aspects of isocyanide biochemistry (29)(30)(31) as well as for generating biologically active formamides derived from isocyanide precursors (30).…”