Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis

Jenner, Matthew; Hai, Yang; Nguyen, Hong H.; Passmore, Munro; Skyrud, Will; Kim, Junyong; Garg, Neil K.; Zhang, Wenjun; Ogorzalek Loo, Rachel R.; Tang, Yi

doi:10.1038/s41467-023-38484-8

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Published: 17 May 2023

Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis

Nature Communications volume 14, Article number: 2832 (2023) Cite this article

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Abstract

Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) enzymes remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC reveals its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometry uncovers several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds.

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Introduction

Iron is an indispensable cofactor for all microbial life. The ability to coordinate and activate molecular oxygen, in addition to optimal redox properties for electron transport, places it central to numerous cellular processes^1,2. Equally, high intra-cellular iron concentrations give rise to Fenton and Haber–Weiss reactions, producing reactive oxygen species capable of cell damage³. It is therefore vital that iron homoeostasis is carefully managed. Although iron has a high natural abundance, it exists predominantly as Fe³⁺ in aerobic environments and tends to form insoluble ferric hydroxides rendering it inaccessible to microorganisms⁴. As a result, organisms have evolved complex strategies for iron acquisition and storage. Whilst several mechanisms are known, a common approach employed by bacteria and fungi is the production of low-molecular-weight compounds known as siderophores, which serve as high-affinity iron chelators^5,6.

In fungi, the majority of siderophore compounds produced belong to the hydroxamate class. This functionality originates from l-ornithine, which is N^δ-hydroxylated and subsequently N^δ-acylated to yield either N^δ-acetyl-N^δ-hydroxy-l-ornithine (l-AHO) or N^δ-anhydromevalonyl-N^δ-hydroxy-l-ornithine (AMHO)⁷. Typically, siderophores possess three hydroxamate units, producing a hexadentate ligand which promotes formation of a polyhedral Fe³⁺ complex with binding constants in the 10²²–10³² range⁸. The hydroxamate-containing units, l-AHO and cis-/trans-AMHO, are enzymatically incorporated into chemical scaffolds and define two separate families of hydroxamate siderophores. These include the depsipeptides, typified by fusarinine C (FSC) (1)⁹ and coprogen^10,11, which utilise either cis- or trans-AMHO as monomeric units and are excreted primarily to capture ferric iron (Fig. 1a)¹². In contrast, members of the ferrichrome family, such as ferricrocin (2) and ferrichrome (3), are generally considered to be intracellular and can incorporate l-AHO or cis-/trans-AMHO, in combination with other amino acids, and are principally used for iron storage, although not exclusively (Fig. 1b, Supplementary Fig. 1)^13,14. Both extra- and intra-cellular siderophores are essential for the survival and virulence of many problematic fungal species, including the opportunistic pathogen Aspergillus fumigatus and the rice blast fungus Magnaporthe oryzae^15,16.

**Fig. 1: Hydroxamate-containing siderophores produced by fungi and the non-linear NRPSs responsible for their biosynthesis.**

Whilst the physiological function of hydroxamate siderophores in fungi is well established, in some cases, the molecular details underpinning their biosynthesis remain poorly understood. Genes encoding for large non-ribosomal peptide synthetase (NRPS) enzymes are known to be responsible for the assembly of peptidyl siderophores^10,17,18. These modular multi-domain enzymes are typically comprised of three domain types: condensation (C), adenylation (A) and thiolation (T). During the biosynthetic process, the peptidyl intermediates are covalently tethered to the T domains via a thioester linkage, afforded by a 4′-phosphopantetheine (Ppant) moiety post-translationally appended to each T domain¹⁹. Within a module, the A domain specifically selects and loads an amino acid starter unit (module 1 only) or extender units onto the Ppant thiol of the T domains. This allows the C domain to catalyse amide bond formation between the growing peptidyl intermediate appended to the T domain of the upstream module, and the amino acid extender unit primed on the T domain^20,21. Once all cycles of chain elongation are complete, the nascent peptidyl chain is cleaved from the NRPS by either a thioesterase (TE) domain, or more commonly in fungal NRPSs, a C_T domain, which catalyse chain-length-specific intramolecular cyclisation to release the final product^22,23.

In bacterial NRPSs, the colinear relationship between the domain organisation and the final product allows rational assignment of biosynthetic pathways and even prediction of products from sequence data alone²¹. In contrast, fungal NRPSs typically exhibit highly aberrant domain organisations, making understanding their biosynthetic pathways challenging^18,24. A recurring non-canonical feature of fungal NRPSs is modules lacking a functional A domain, implying that the T domain must be loaded by an A domain from a different module. This can be observed in the SidD and Nps6 NRPSs, where module 2 (M2) possesses a truncated A domain (dA) that lacks ~290 amino acids from the N-terminus rendering it catalytically inactive (Fig. 1a)²⁵. Our previous work has demonstrated that the SidD A₁ domain loads cis-AMHO onto both the T₁ and T₂ domains, thus circumventing the requirement for an A domain in module 2²⁶. Another distinctive trait is that the length and sequence of the peptide product does not correlate to the number/order of domains in the NRPS, indicative of nonlinear behaviour. This is also exemplified by the SidD NRPS, which incorporates three cis-AMHO units yet harbours only 2 modules. Our characterisation of SidD revealed that the NRPS acts in an iterative manner, loading another cis-AMHO unit onto the T₁ domain after formation of the di-cis-AMHO species on T₂, allowing a third cis-AMHO residue to be condensed and thereby yielding the fusarinine C (1) product (Fig. 1a)²⁶. Whilst currently limited to the SidD NRPS, these examples of nonlinear behaviour begin to highlight the intricate and highly unusual molecular programming of fungal NRPSs.

The biosynthesis of siderophores belonging to the ferrichrome family have been linked to an evolutionarily-related group of NRPSs which exhibit unusual nonlinear domain organisations. An example of one such systems is the SidC NRPS from Aspergillus nidulans, which is known to produce ferricrocin (2) (Fig. 1b)²⁷. Variations in the domain architecture of the NRPS exist, some of which give rise to the structurally related ferrichrome (3), such as Sib1 from Schizosaccharomyces pombe and Sid2 from Ustilago maydis (Supplementary Fig. 1a)^18,28,29. The majority of ferrichrome family siderophores are cyclic hexapeptides with the exceptions of the cyclic heptapeptide, tetraglycylferrichrome, and cyclic octapeptide, epichloenin A (Supplementary Fig. 1b and c)^30,31. Structurally, they are comprised of three N^δ-acetyl-N^δ-hydroxy-L-ornithine (AHO) residues for Fe³⁺ chelation, and three amino acids forming a variable backbone, of which two residues are either alanine, serine or glycine, and the third a glycine residue⁷. Similar to SidD, the domain organisation of the SidC NRPS suggests a high degree of nonlinear behaviour. The SidC NRPS possesses only three A domains, yet the siderophore product requires incorporation of six amino acids. Using a linear biosynthetic logic, modules 4 and 5 both lack integrated A domains required for priming the T₄ and T₅ domains, presumably with AHO units. However, based on our previous observations of the SidD A₁ domain, we hypothesise that the SidC A₃ domain is likely to load the T₄ and T₅ domains with AHO units in an inter-modular manner (Fig. 1b). Assuming the T₄ and T₅ domains are indeed primed by the A₃ domain, under a linear paradigm this would only permit synthesis of a five-residue peptide product, suggesting further aberrant behaviour to allow incorporation of an additional Gly residue into the backbone of both ferricrocin (2) and ferrichrome (3) (Fig. 1b). It is worth noting that this level of nonlinearity has not been observed before and the exact molecular mechanism cannot be explained by any previous model.

To understand the enigmatic programming rules of this particular class of siderophore-producing NRPSs, we employ a combination of in vitro biochemical assays and intact protein mass spectrometry (MS) to interrogate SidC with respect to its ability to produce peptidyl siderophore products. Our results uncover several non-canonical events during peptidyl siderophore biosynthesis, which add previously unobserved capabilities to NRPS assembly-line enzymology and sets the stage for efforts towards reprogramming the SidC NRPS towards novel chemical scaffolds.

Results

Reconstitution of the SidC NRPS and determination of adenylation domain specificity

In the first instance, we elected to examine SidC activity in vivo using Saccharomyces cerevisiae as a heterologous host. This was conducted to allow production of the associated siderophore(s) and to ascertain whether S. cerevisiae would be an appropriate host for recombinant overproduction of SidC for subsequent purification and in vitro analysis. To achieve this, the sidC NRPS gene from A. nidulans FSGC A1145 (Supplementary Data 1), in addition to sidA and sidL (required for production of the l-AHO precursor), were cloned into vectors with distinct selection markers, and transformed into S. cerevisiae BJ5464-npgA (a strain with the fungal Ppant transferase, NpgA, integrated into its chromosome to ensure phosphopantetheinylation of the resulting proteins) for siderophore production³². Analysis of the small molecule extract from a 3-day culture indicated that ferricrocin (2) was produced (Fig. 2a, trace i), and large scale cultures allowed purification and isolation of ferricrocin (2) for structural elucidation, which was in agreement with previous reports (see Supplementary Fig. 2–4). Having established that an active form of SidC can be produced in S. cerevisiae, recombinant SidC protein was overproduced in S. cerevisiae JHY686 as a polyhistidine-tagged fusion protein, and was purified to near-homogeneity using immobilised metal-ion affinity chromatography (IMAC) (Supplementary Fig. 5), thereby allowing controlled exposure to substrates/cofactors³³. To ensure protein samples were completely in the holo-form prior to assays, purified SidC was enzymatically phosphopantetheinylated using the fungal phosphopantetheinyl transferase, NpgA (A. nidulans), as described previously³⁴. Following addition of ATP and Mg²⁺ cofactors to recombinant SidC, incubation with l-AHO alone (synthesised according to literature protocols^35,36, Supplementary Fig. 6–8), or a combination of l-AHO + l-Ser, yielded no detectable products (Fig. 2a, trace ii and iii). However, the combination of l-AHO + Gly + l-Ser resulted in the production of ferricrocin (2) (Fig. 2a, trace v). Interestingly, incubation with l-AHO + Gly resulted in formation of a species consistent with ferrichrome (3) (Fig. 2a, trace iv), which was confirmed by comparison to a chemical standard (Fig. 2a, trace vii). These observations suggest that the SidC NRPS is capable of producing both ferricrocin (2) and ferrichrome (3) depending upon the availability of amino acid substrates, yet appears to produce exclusively ferricrocin (2) in the native host, probably due to the abundance of l-Ser.

**Fig. 2: Reconstitution of SidC NRPS and inter-modular loading of l-AHO residues by the A₃ domain.**

Our initial biosynthetic model hypothesised that each of the three A domains within SidC are responsible for activation and loading of l-Ser, Gly and l-AHO (Fig. 1b). In bacteria, bioinformatic analysis allows accurate prediction of the substrate specificity for A domains, primarily based on highly conserved amino acid motifs within the enzyme active site. However, this approach is not possible for fungal A domains, largely due to limited sequence and structural information. Unable to predict the substrate specificity of the SidC A₁, A₂ and A₃ domains using bioinformatics, excised constructs of SidC A₁, A₂ and A₃ were cloned, overproduced in E. coli and purified to homogeneity for determination of substrate specificity. Using an ATP/PPi exchange assay to measure the reverse reaction of the adenylation step, the SidC A₁ domain showed activation of l-Ala and l-Ser, with a clear preference towards the latter (Supplementary Fig. 9, top). Interestingly, the SidC A₂ domain exhibited no detectable activity when subjected to the ATP/PPi exchange assay, suggesting that PPi may not formed during the activation step. However, activity was observed for SidC A₂ using the hydroxylamine release assay, which indicates formation of the corresponding aminoacyl-adenylate, and allowed a substrate specificity profile to be obtained revealing Gly as the preferred substrate (Supplementary Fig. 9, middle). The discrepancy between these two assays indicates that the A₂-catalysed adenylation reaction is not reversible^37,38. Activity for the SidC A₃ domain was obtained using the ATP/PPi exchange assay and displayed a clear preference towards l-AHO (Supplementary Fig. 9, bottom). The SidC A₁ and A₂ assays were also reconstituted with their cognate T₁ and T₂ domains to examine the aminoacyl transfer step. Intact protein mass spectrometry (MS) analysis showed mass shifts corresponding to loading of l-Ser and Gly onto the respective T domains (Supplementary Fig. 10).

The SidC A₃ domain catalyses intra- and inter-modular loading of l-AHO

The biosynthesis of ferricrocin (2) and ferrichrome (3) both require installation of three l-AHO residues, yet the SidC NRPS possesses only a single A domain capable of activating l-AHO; the A₃ domain situated in module 3. Furthermore, modules 4 and 5 lack integrated A domains for loading amino acid units to their cognate T domains (Fig. 1b). Based on our previous observations in the SidD NRPS, we postulated that the SidC A₃ domain may be capable of loading l-AHO units onto the T₄ and T₅ domains, in addition to its cognate T₃ domain. This would result in the T₃, T₄ and T₅ domains being charged with l-AHO units, which can then be condensed together by the sequential activity of the C₄ and C₅ domains to yield the tri- l-AHO motif. An alternative model would involve iterative activity of module 3 to generate tri-l-AHO appended to the T₃ domain; however, this would render the C₄T₄C₅T₅ region redundant for the biosynthesis and seemed less likely.

To investigate this aspect, a SidC C₃A₃T₃ tri-domain construct was cloned, overproduced and purified to examine the covalently tethered intermediates loaded onto the T₃ domain. Following conversion to its holo form and subsequent incubation with ATP, Mg²⁺ and l-AHO (15 min), intact protein MS analysis of C₃A₃T₃ revealed loading of a single l-AHO unit, indicated by a + 172 Da mass shift relative to the mass of holo-C₃A₃T₃ (Supplementary Fig. 11). This highlighted that the standalone SidC C₃A₃T₃ tri-domain is only capable of loading a single l-AHO unit onto its cognate T₃ domain and ruled out the possibility of iterative loading. We next generated a SidC C₃A₃T₃C₄T₄ penta-domain construct to examine the ability of the A₃ domain to catalyse loading of l-AHO onto the T₄ domain. Under the same conditions, two sequential +172 Da mass shifts were observed in the mass spectrum, congruent with loading of two l-AHO units (Fig. 2b, top). The measured masses were consistent with either two l-AHO units loaded in an uncondensed form onto the T₃ and T₄ domains, or in a condensed di-l-AHO species on the T₄ domain. To validate these observations, a mutant of the SidC C₃A₃T₃C₄T₄ construct was produced, where the Ser residue that serves as the Ppant group attachment site was mutated to Ala (S3140A, designated as T₃⁰), allowing only the T₄ domain to be converted to its holo form. When subjected to the loading assay, the SidC C₃A₃T₃⁰C₄T₄ protein was able to activate and transfer an l-AHO unit onto the T₄ domain, indicated by a single +172 Da mass shift in the intact protein mass spectrum (Fig. 2b, bottom).

Inter-modular loading of T₄ by the A₃ domain in the SidC NRPS is reminiscent of behaviour observed for the SidD NRPS, where the A₁ domain is able to prime T₂, situated in the downstream module. In both of these instances, the A domain is interacting with a T domain situated one module downstream. However, in the SidC NRPS, we hypothesised that the A₃ domain is capable of loading the T₅ domain, situated two modules downstream. To probe this, we conducted a bimolecular assay between SidC C₃A₃T₃ and SidC C₅T₅C_T in the presence of ATP, Mg²⁺ and l-AHO. This resulted in ~35% of l-AHO loading onto the T₅ domain after a 60 min incubation (Fig. 2c, top), and indicated that the A₃ domain is capable of loading the T₅ domain, situated two modules downstream. An equivalent experiment using a C₄T₄ construct yielded comparable levels of l-AHO loading, serving as a control measurement, and also highlighting the reduced efficiency when domains are not covalently tethered in megasynth(et)ases (Supplementary Fig. 12). Interestingly, spectra obtained from this experiment also gave rise to a small peak congruent with a di- l-AHO species. The relatively small amount of this condensed species relative to the mono- l-AHO suggests that the C₃ domain does not preferentially condense l-AHO units together, indicating that the species in Fig. 2b (top) is likely two uncondensed l-AHO units. Analogous assays using the SidC T₅C_T didomain and SidC T₄ domain (i.e. without the N-terminal C domain), resulted in no detectable l-AHO loading (Fig. 2c, bottom and Supplementary Fig. 12), implying that the N-terminal C domains facilitate the loading reaction. These results suggest two architectural models for non-linear l-AHO loading by the A₃ domain. One possibility is that intra-chenar loading of l-AHO is promoted by a 3-dimensional arrangement of the SidC NRPS that enables proximity of the A₃ domain to the T₄ and T₅ domains (Supplementary Fig. 13a). Here, the presence of the C₄ and C₅ domains may be essential to provide an interaction ‘platform’ for the T₄ and T₅ domains to access the A₃ domain. A second possibility involves inter-chenar communication between two SidC proteins, allowing the A₃ domain to load l-AHO onto the T₄ and T₅ domain in trans, whilst loading the T₃ domain conventionally (Supplementary Fig. 13b). Interestingly, modelling of the A₃T₃C₄T₄C₅T₅ region using AlphaFold³⁹ suggests that both C₄ and C₅ domains form interfaces with the A₃ domain, conceivably providing a platform for their respective T domains to access the A₃ domain active site, adding credence to an intra-chenar loading model (Supplementary Figs. 13c, 14 and 15).

SidC C₂A₂T₂ tri-domain catalyses non-canonical loading of Gly residues

We next turned our attention to the biosynthetic steps required for the formation of the tripeptide backbone. This region is the sole structural difference between ferricrocin (2) and ferrichrome (3), possessing [Gly]-[l-Ser]-[Gly] and [Gly]-[Gly]-[Gly] motifs, respectively. The SidC A domain specificity assays determined that the A₁ domain preferentially activates l-Ser, and that the A₂ domain only activates Gly (Supplementary Fig. 9, 10). Based on these observations, linear assembly of the amino acid units would yield a T₂-[Gly]-[l-Ser]-NH₂ species, requiring a second Gly to be non-canonically condensed onto the amine of l-Ser to yield the T₂-[Gly]-[l-Ser]-[Gly]-NH₂ tripeptide intermediate necessary for ferricrocin (2) production. However, for ferrichrome (3), two scenarios seemed plausible: i). the A₁ domain would instead load Gly onto the T₁ domain (note, some activity towards Gly observed in specificity assays (Supplementary Fig. 9)), allowing a T₂-[Gly]-[Gly]-NH₂ species to be formed, followed by non-canonical condensation of a third Gly to yield the T₂-[Gly]-[Gly]-[Gly]-NH₂ intermediate. ii). the A₁ and T₁ domains are not utilised, leaving the A₂ domain to generate a T₂-[Gly]-NH₂ species, which must undergo two sequential non-canonical condensation events to yield the T₂-[Gly]-[Gly]-[Gly]-NH₂ intermediate.

In order to unpick these biosynthetic steps, we first produced a SidC(ΔA₁T₁) construct to examine whether the domains of module 1 are essential for siderophore production. Using S. cerevisiae as a heterologous host, SidC (ΔA₁T₁) was observed to produce ferrichrome (3) exclusively, with no ferricrocin (2) detected (Fig. 3a, trace i), which was the product of full-length SidC under the same culture conditions (Fig. 2a). Purification of the recombinant SidC(ΔA₁T₁) protein allowed controlled exposure to amino acid substrates. Here, SidC(ΔA₁T₁) produced ferrichrome (3) exclusively, provided that both Gly and l-AHO were present (Fig. 3a, traces ii–iii). The inclusion of l-Ser did not promote ferricrocin (2) production (Fig. 3a, trace ii), and omission of l-AHO resulted in no detectable products (Fig. 3a, trace iv). These data indicated that the A₁T₁ domains are essential for ferricrocin (2) production, but are not required for ferrichrome (3) formation. Furthermore, these data indicated that during ferrichrome (3) biosynthesis, neither A₁ or T₁ domain participate in the recruitment of the additional Gly residue. This left the intriguing possibility that module 2 alone (i.e. C₂A₂T₂) could be responsible for constructing the T₂-[Gly]-[Gly]-[Gly]-NH₂ intermediate required for ferrichrome (3) biosynthesis.

**Fig. 3: Iterative loading of Gly residues by the A₂ domain and chain-length control by the C₃ domain.**

To explore this prospect, we cloned an MBP-SidC C₂A₂T₂ fusion construct, which was overexpressed in E. coli as a soluble protein and purified to homogeneity for in vitro studies. Using our established intact protein MS approach, incubation of SidC C₂A₂T₂ with excess Gly, ATP and Mg²⁺ for 10 mins led to emergence of two new peaks which were + 171 Da and + 285 Da relative to the holo-C₂A₂T₂ species, mass shifts which correspond to condensed tri- and penta-Gly chains, respectively, attached to the T₂ domain (Fig. 3b, spectra (i–ii). Upon extension of the incubation period to 60 min, the relative abundance of the penta-Gly species increased as the tri-Gly decreased, suggesting that the tri-Gly intermediate is a precursor to the penta-Gly chain (Fig. 3b, spectrum iii). In the presence of excess Gly and ATP conversion to the tri-Gly and penta-Gly intermediates was fast, preventing observation of early intermediates of the poly-Gly chain. Therefore, assays were repeated with reduced relative concentrations of 4:1 and 2:1 (Gly/ATP:protein) to capture early-stage intermediates. At a 4:1 ratio, a peak at + 57 Da relative to the holo-C₂A₂T₂ species was observed, which correlates to a single Gly unit loaded onto the T₂ domain, in addition to the previously observed tri-Gly species (Fig. 3b, spectrum iv). Switching to a 2:1 ratio yielded predominantly the mono-Gly species, with low levels of di- and tri-Gly intermediates observable in the spectrum (Fig. 3b, spectrum v).

These observations indicated that the domains of module 2 alone are capable of constructing Ppant-bound poly-Gly chains, appearing to favour the formation of tri- and penta-Gly species. While loading of a single Gly unit onto the T₂ domain is likely catalysed by the A₂ domain in a canonical fashion, the mechanism by which the remaining two Gly units are condensed onto the free amine remained elusive. The use of an isolated C₂A₂T₂ construct in our experiments, meant it was possible that inter-chenar communication between individual C₂A₂T₂ proteins could allow the C₂ domain to catalyse condensation reactions between Gly chains, assuming that the T₂ domain is able to act as an aminoacyl donor and acceptor (i.e. C₂A₂T₂-[Gly]-NH₂ + C₂A₂T₂-[Gly]-NH₂ → C₂A₂T₂-SH + C₂A₂T₂-[Gly]-[Gly]-NH₂). To test this possibility, the catalytically essential active site His₅₆₉ residue of the C₂ domain was mutated to Ala to produce a catalytically inactive C₂ domain, denoted as C₂⁰. Upon incubation with Gly and co-factors, the SidC C₂⁰A₂T₂ protein was able to generate poly-Gly chains in a near-identical manner to that of the wild-type construct (Supplementary Fig. 16). We attempted to produce a SidC A₂T₂ construct, however, in the absence of the N-terminal C₂ domain the resulting protein was highly unstable and degraded quickly, suggesting that the C domain plays an important structural role within the module.

Taken together, our data strongly indicate that the SidC A₂ domain is responsible for catalysing both canonical loading of a single Gly unit onto the Ppant thiol of the T₂ domain, and subsequent amide bond formation steps to generate the tri-Gly intermediate required for ferrichrome (3) biosynthesis. Whilst two non-canonical amide bond-forming steps are required for ferrichrome (3) biosynthesis, this is only required once for ferricrocin (2) biosynthesis, adding a Gly to the free amine of the T₂-[Gly]-[l-Ser]-NH₂ species. Poly-amide bond formation catalysed by an A domain is unprecedented within the context of a multi-modular NRPS, making this system particularly interesting. However, similar activity has been observed for a standalone A domain during the biosynthesis of streptothricin. Here, following canonical loading of a l-β-lysine residue onto a T domain, a separately encoded adenylation domain, ORF19, generates poly-l-β-lysine chains via amide bond formation with the free ε-NH₂ group (Supplementary Fig. 17)⁴⁰. Interestingly, the SidC A₂ domain differs from ORF19, in that the amide bond is formed using the α-NH₂ group and the domain is found integrated into the NRPS. It is worth noting that stand-alone adenylation domains have also been observed to catalyse amide bond formation in the biosynthesis of pacidiamycin (PacU), coumermycin A₁ (CouL) and novobiocic acid (NovL) (Supplementary Fig. 17)^41,42,43. However, these examples involve single condensation events, not formation of poly-amino acid chains as observed for the ORF19 and SidC A₂ domains.

The SidC C₃ domain is a chain-length gatekeeper

Our biochemical investigations of SidC C₂A₂T₂ demonstrated its ability to generate poly-Gly chains of up five residues in length (Fig. 3b). However, the biosynthetic products of SidC, ferricrocin (2) and ferrichrome (3), both require the T₂-tethered intermediate to be three residues in length: T₂-[Gly]-[l-Ser]-[Gly]-NH₂ and T₂-[Gly]-[Gly]-[Gly]-NH₂, respectively. Therefore, in order to maintain biosynthetic fidelity, we postulated that the SidC C₃ domain imposes a selective requirement for three-residue chains appended to the T₂ domain in order to catalyse condensation with the first l-AHO unit, effectively acting as a gatekeeper. To explore this hypothesis, we incubated holo-SidC C₂A₂T₂ with holo-SidC C₃A₃T₃ in the presence of Gly, l-AHO, ATP and Mg²⁺, and monitored the SidC C₃A₃T₃ protein using intact protein MS at several time points. After 10 min, a new peak at + 171 Da from the l-AHO-C₃A₃T₃ species had emerged, indicating condensation of a tri-Gly unit onto the l-AHO (Fig. 3c, spectra (i) and (ii)), with the intensity of this species increasing over a 60 min period (Fig. 3c, spectrum (iii)). The absence of signals corresponding to mono- (+ 57 Da) or di-Gly (+ 114 Da) species condensed with l-AHO-C₃A₃T₃ during the time-course strongly indicated that the entire T₂-[Gly]-[Gly]-[Gly] intermediate is condensed with l-AHO, rather than sequential addition of Gly residues.

To examine whether the SidC C₃ domain can discriminate between tri-, tetra- and penta-Gly intermediates, we pre-incubated holo-SidC C₂A₂T₂ with Gly, ATP and Mg²⁺ for 60 min to generate a mixture of poly-Gly chain lengths, represented by Fig. 3c, spectrum iii. The remaining Gly in the reaction was then removed by multiple cycles of ultrafiltration, before addition to holo-SidC C₃A₃T₃ in the presence of l-AHO, ATP and Mg²⁺ for 60 min. Subsequent intact protein MS analysis revealed only the tri-Gly species condensed with T₃-tethered l-AHO (Fig. 3c, spectrum iv), suggesting that the SidC C₃ domain possesses strict selectivity for poly-amino acid chain lengths where n = 3, thereby acting as a critical checkpoint during the biosynthesis.

A biosynthetic model for siderophore production by the SidC NRPS

Our data allows proposal of a rational biosynthetic model for the construction of ferricrocin (2) and ferrichrome (3) by the SidC NRPS (Fig. 4). In ferricrocin (2) biosynthesis, the process is initiated by the A₁ domain loading a l-Ser residue onto the T₁ domain, which is subsequently condensed with a Gly residue tethered to the downstream T₂ domain by the C₂ domain, as a result of A₂ domain loading, yielding the T₂-[Gly]-[l-Ser]-NH₂ intermediate (Fig. 4a). This initial step is not required for ferrichrome (3) biosynthesis, which commences with A₂ domain-catalysed loading of a single Gly residue onto the T₂ domain, and is then condensed with a second Gly residue, catalysed by the amide bond-forming capabilities of the A₂ domain, yielding a T₂-[Gly]-[Gly]-NH₂ intermediate (Fig. 4b). Both T₂-tethered dipeptide intermediates during ferricrocin (2) and ferrichrome (3) biosynthesis then undergo addition of a Gly residue to the free NH₂ group, catalysed by the A₂ domain, producing T₂-[Gly]-[l-Ser]-[Gly]-NH₂ and T₂-[Gly]-[Gly]-[Gly]-NH₂ intermediates. Whilst the A₂ domain is capable of adding further Gly residues to extend the peptidyl chain over time (Fig. 3b), the nascent tripeptide intermediates are rapidly and selectively condensed with the T₃-tethered l-AHO species by the C₃ domain.

**Fig. 4: Proposed biosynthetic models for SidC-catalysed formation of ferricrocin and ferrichrome.**

Discussion

Ferrichrome NRPSs are found in the vast majority of Ascomycetes, providing the biosynthetic machinery for siderophore production. Despite producing near-identical products, ferricrocin (2) and ferrichrome (3), substantial differences in the NRPS domain architecture exist within the NRPS family (Types I–V, Fig. 5). Phylogenetic work has suggested that ferrichrome NRPSs originate from an ancestral colinear hexamodule NRPS, created by adjacent duplication of complete NRPS modules resulting in two lineages: NSP2 and NSP1/SidC. The recently reported Sid1 NRPS responsible for AS2488059 biosynthesis, a related ferrichrome siderophore, might be considered as a contender for this ancestral gene (Supplementary Fig. 18)^44,45,46. Here, dedicated A domains are employed to load each of the three l-AHO residues, in addition to the three backbone residues (Asn, Val and Phe), totalling six A domains in the NRPS. However, despite the similarities, phylogenetic analyses suggest that the Sid1 NRPS is of a different evolutionary origin to siderophores of the ferrichrome family⁴⁶. All combinations of the ferrichrome family of NRPSs give rise to unusual non-linear domain organisations, which cannot be reconciled with standard biosynthetic logic of NRPSs. Plausible biosynthetic proposals linking the domain organisation to the peptidyl product require inter-modular loading of amino acid substrates by A domains up to n + 2 modules downstream (blue arrows), and/or A domains capable of creating (poly)amide chains on the same T domain (red arrows). Our study of the SidC NRPS highlights that both activities are possible in NRPSs and allow evidence-based biosynthetic proposals for all variations of the ferrichrome NRPS (Fig. 5).

**Fig. 5: Biosynthetic schemes for the six modular architectures of ferrichrome class of NRPSs.**

Members of the NPS2 lineage (Types III, IV and V) all possess the correct number of C domains required for the number of amide bonds formed in the peptidyl product. However, loss or degeneration of A domains requires inter-module loading of T domains with amino acid substrates. This is observed in the tri-Orn region, as characterised for SidC, and in the tripeptide region for Type III and IV. In contrast, the Type I and II members of the NPS1/SidC lineage both require an amide bond-forming A domain to compensate for the lack of C domains in the NRPS, in addition to inter-module loading capabilities in the tri-Orn region. The CsNPS2 and Cpf1 NRPSs (Type VI) are the most truncated variation and appears to have lost much of the N-terminus, leaving just the tri-Orn region that requires inter-module loading capabilities. Recent assignments of the CsNPS2 and Cpf1 products as basidioferrin (4) and coprinoferrin (5) revealed a structure comprised of three condensed l-AHO units for basidioferrin (4)⁴⁷, and three l-hydroxyhexanoyl ornithine (l-hhOrn) units for coprinoferrin (5)⁴⁸. In the latter, this suggests that the Cpf1 A₁ domain (equivalent to A₃ in SidC and Sid2) has evolved specificity towards a larger hydroxamate substrate, whilst retaining the ability to conduct inter-module loading (Fig. 5).

Taken together, our observations highlight the impressive evolutionary changes employed by fungal NRPSs to improve atom economy and increase structural diversity in their biosynthetic assembly-lines. Our improved understanding of the biosynthetic rules has set the stage for manipulating and recombining these pathways towards novel hydroxamate-containing scaffolds.

Methods

Molecular cloning and site directed mutagenesis

Yeast Expression Constructs: the SidC (Genbank: XM_653119 [https://www.ncbi.nlm.nih.gov/nuccore/XM_653119.2]), SidA (Genbank: XM_658335 [https://www.ncbi.nlm.nih.gov/nuccore/XM_658335.1]), and SidL (Genbank: XM_652967 [https://www.ncbi.nlm.nih.gov/nuccore/XM_652967]) gene exon fragments were cloned from the cDNA library prepared from the mRNA extract of A. nidulans FSGC A1145 strain⁴⁹ cultured on Czapek-Dox (CD) agar. The corresponding yeast expression plasmids were assembled through yeast homologous recombination using a Frozen-EZ Yeast Transformation II Kit (Zymo research). Gene fragments were integrated into a 2µ-based yeast expression vector with auxotrophic markers and ADH2 promoter and terminator regions. All proteins were cloned in-frame with an N-terminal pHis₈ tag to facilitate purification. E. coli Expression Constructs: target regions of SidC were subcloned into either pHis_6-MBP-pET28a or pHis₆-pET28a vectors. All proteins were cloned in-frame with an N-terminal TEV-cleavable tag (either MBP or pHis₆), allowing removal post-purification. Primers used for the cloning of SidC constructs and mutagenic primers to generate point-mutations/truncations are detailed in Supplementary Table 1.

Protein overproduction and purification

Yeast expression constructs

The full-length proteins were expressed in S. cerevisiae JHY686³³ strain cultured in YPD medium. Briefly, single colonies of yeast cells harbouring expression plasmids were inoculated into SDCt uracil drop-out culture and left growing at 28 °C for 2 days. The seed culture was then inoculated into YPD culture (20 ml to 1000 mL) and left growing at 28 °C for another 2 days. Cells were harvested by centrifugation and washed once with cell lysis buffer (50 mM K₂HPO₄ (pH 7.5), 10 mM imidazole, 300 mM NaCl, 5% glycerol). Cells were flash frozen in liquid nitrogen and lysed by using a stainless-steel Waring blender. The cell lysate was cleared by centrifugation at 26,000g for 60 min at 4 °C and the supernatant was filtered through a 0.22 µm filter (Millipore). The filtrate was incubated with Ni²⁺-NTA resin for 30 min at 4 °C and then the slurry was loaded onto a gravity column. The resin was washed and eluted with increasing concentrations of imidazole in cell lysis buffer. The fractions were examined by SDS-PAGE gels. Pure fractions were concentrated to ~20 mg/mL by Amicon concentrators (Millipore), supplemented with 10% glycerol and stored at −80 °C. Protein concentrations were determined by Bradford assay. Typically, 2 L cell culture could yield 1 − 10 mg of protein depending on the nature of the protein construct.

E. coli expression constructs

A single colony of E. coli BL21 (DE3) that had been transformed with the appropriate expression vector was picked and used to inoculate LB medium (5 or 10 mL) containing kanamycin (50 µg/mL). The resulting culture was incubated overnight at 37 °C and 180 rpm then used to inoculate LB medium (0.5 or 1 L) containing kanamycin (50 µg/mL). The resulting culture was incubated at 37˚C and 180 rpm until the optical density of the culture at 595 nm reached 0.6, then IPTG (1 mM) was added and growth was continued overnight at 15 °C and 180 rpm. The cells were harvested by centrifugation (4000g, 15 min, 4 ˚C) and re-suspended in buffer (20 mM Tris-HCl, 100 mM NaCl, 20 mM Imidazole, pH 7.4) at 10 mL/L of growth medium then lysed using a Constant Systems cell disruptor. The lysate was centrifuged (37,000g, 30 min, 4 °C) and the resulting supernatant was loaded onto a HiTrap FF Chelating Column (GE Healthcare), which had been pre-loaded with 100 mM NiSO₄ and equilibrated in re-suspension buffer (20 mM Tris-HCl, 100 mM NaCl, 20 mM Imidazole, pH 7.4). Proteins were eluted in a stepwise manner using re-suspension buffer containing increasing concentrations of imidazole—50 mM (5 mL), 100 mM (3 mL), 200 mM (3 mL) and 300 mM (3 mL). The presence of the protein of interest in fractions was confirmed by SDS-PAGE, and an additional gel filtration step (Superdex 75/200, GE Healthcare) was used to further purify proteins where necessary. Fractions containing the protein of interest were pooled and concentrated to 250–400 µM using a Viva-Spin centrifugal concentrator (Sartorius) at an appropriate MWCO. Samples were snap-frozen in liquid N₂ and stored at −80 °C.

Siderophore isolation/preparation

Desferriferricrocin and ferricrocin were obtained through coexpression of sidC, sidA, and sidL genes in S. cerevisiae BJ5464-npgA strain⁵⁰. Briefly, competent yeast cells were transformed with plasmids XW55-SidC, XW06-SidL and XW02-SidA and the colonies harbouring these three plasmids were selected using minimal medium dropping out uracil, tryptophan, and leucine. The colony was inoculated into the corresponding liquid minimal medium, and the cell culture was grown at 28 °C for 2 days. To induce production, the starting culture was inoculated to YPD medium and left growing at 28 °C for 3 days. The cell pellet was harvested through centrifugation and the produced siderophore compound was extracted using acetone. The organic extract was dried using rotary evaporation and the residue was dissolved in methanol and subjected to LC-MS analysis on a Shimadzu 2020 LC-MS (Phenomenex Kinetex, 1.7 µm, 2.0 × 100 mm, C18 column) using positive and negative mode electrospray ionisation with a linear gradient of 5–95% MeCN - H₂O supplemented with 0.1% (v/v) formic acid in 15 min followed by 95% MeCN for 3 min with a flow rate of 0.3 mL/min. To convert desferriferricrocin into ferricrocin, FeCl₃ (final concentration at 1 mM) was added into the organic extract. To purify the fermentation product for structural analysis, similar extraction procedure was performed on 4 L cell culture pellet. The organic extract was dried and dissolved in H₂O and fractionated with Amberlite XAD-16 (Sigma-Aldrich) resin. The desferriferricrocin and ferricrocin were eluted from a gradient from 20% MeOH to 70% MeOH. The eluent was combined and purified by semipreparative HPLC using a reverse-phase column (Phenomenex Kinetics, C18, 5 µm, 100 Å, 250 × 4.6 mm). The identity of desferri-ferricrocin was confirmed by HR-MS and NMR analysis. The NMR spectra data are consistent with the literature data⁵¹. ¹H-NMR (500 MHz, CD₃OD): δ 8.36 (s,), 4.46–4.30 (m, overlap, 3H), 4.24 (d, J = 17.1 Hz, 1H, Gly C_αH₂), 4.08, (m, overlap, 1H, Ser C_αH), 4.07 (d, J = 16.0 Hz, 1H, Gly C_αH₂), 3.86 (ddd, J = 56.5, 11.1, 5.4 Hz, 2H Ser C_βH₂), 3.69 (d, J = 15.7 Hz, 1H, Gly C_αH₂), 3.62 (d, J = 17.0 Hz, 1H, Gly C_αH₂), 3.76-3.53 (m, overlap, 6H), 2.11 (s, 3H, hydroxamic CH₃), 2.105 (s, 3H, hydroxamic CH₃), 2.102 (s, 3H, hydroxamic CH₃), 2.00-1.90 (m, 1H, Orn C_βH₂), 1.90-1.85 (m, overlap, 1H, Orn C_βH₂), 1.85-1.80 (m, overlap, 1H, Orn C_βH₂),1.80-1.75 (m, overlap, 1H, Orn C_βH₂), 1.78-1.73 (m, overlap, 1H, Orn C_βH₂), 1.75-1.70 (m, overlap, 1H, Orn C_βH₂), 1.74-1.60 (m, overlap, 6H, Orn C_βH₂). ¹³C-NMR (125 MHz, CD₃OD): δ 174.6 C, 174.4, 174.2, 173.8 (overlap), 173.7, 172.5, 171.90, 171.88, 62.3 (Ser C_β), 56.7(Orn_α), 56.2 (Ser C_α), 54.9 (Orn C_α), 54.5 (Orn C_α), 48.41 (Orn C_δ), 48.39 (Orn C_δ), 48.3 (Orn C_δ), 44.4 (Gly C_α), 43.7(Gly C_α), 30.3 (Orn C_β), 30.2 (Orn C_β), 28.3 (Orn C_β), 24.4 (Orn C_γ), 24.3(Orn C_γ), 24.0(Orn C_γ), 20.31 (hydroxamic CH₃), 20.29 (hydroxamic CH₃), 20.22(hydroxamic CH₃). HRMS: calc. for [M + H]⁺ C₂₈H₄₈N₉O₁₃⁺, 718.3367, found 718.3365.

Synthesis of l-AHO amino acid substrate

The amino acid N⁵-acetyl-N⁵-hydroxy-l-ornithine (l-AHO) is synthesised from N²-Cbz-N²-Boc-l-ornithine according to the literature^35,36. ¹H-NMR (500 MHz, CD₃OD): δ 3.65 (t, J = 6.0 Hz, 1H, C_αH), 3.56 (t, J = 6.7 Hz, 2H, C_δH₂, cis-trans isomers not resolved), 2.04 (s, 3H, acetyl CH₃, with a small shoulder at 2.01 due to cis-trans isomerization), 1.83-1.56 (m, overlap,.4H, C_βH₂, C_γH₂). ¹³C-NMR (125 MHz, CD₃OD): δ 174.3 (carboxylate), 173.7 (amide carbonyl), 169.3 (amide carbonyl, minor isomer), 54.3 (C_α), 50.9 (C_δ), minor isomer), 47.2 (C_δ, major isomer), 27.5 (C_β), 27.3 (C_β, minor isomer) 22.1 (C_γ), minor isomer), 19.7 (acetyl CH₃, minor isomer), 19.4 (acetyl CH₃). HRMS: calc. for [M + H]⁺ C₇H₁₅N₂O₄⁺, 191.1027; found 191.1097.

Biochemical characterisation of SidC in vitro

Purified SidC and associated variants/mutants were converted to their holo- form by incubation in 20 mM Tris HCl, 100 mM NaCl, 2 µM of NpgA, 0.1 mM CoA and 10 mM MgCl₂ in a total volume of 50 µL for 1 h at 25 °C. Reactions were initiated by addition of ATP (5 mM) and either all or various combinations of the following: l-AHO (1 mM)/l-Ser (1 mM)/Gly (1 mM) in a final volume of 50 µL, and the reaction was allowed to proceed at 25 °C. At different time points, the reaction was quenched by mixing with an equal volume of methanol. The reaction products were analysed on an UHPLC-MS on a Shimadzu 2020 EVLC–MS controlled using Shimadzu LabSolutions (Phenomenex kinetex, 1.7 µm, 2.0 × 100 mm, C₁₈ column) using positive and negative mode electrospray S5 ionisation with a linear gradient of 5−95% MeCN−H₂O supplemented with 0.1% (v/v) formic acid in 15 min followed by 95% MeCN for 5 min with a flow rate of 0.3 mL/min. All data were analysed using Shimadzu LabSolutions.

Biochemical assays to determine adenylation domain specificity

ATP-PPi exchange assays

The amino acid substrate specificity profiles for the SidC A₁ and SidC C₃A₃ constructs were conducted using ATP-PPi exchange assays. Assays performed in 100 μL of reaction buffer (50 mM Tris-HCl pH 8, 2 mM MgCl₂) containing 1 mM TCEP, 5 mM ATP, 1 mM tetrasodium pyrophosphate (Na₄PPi), 5 mM substrate, and 5 μM enzyme. Before the addition of enzyme, Na₄[³²P]-PPi was added to a final intensity of ∼2.5 × 106 cpm/mL. Reactions were allowed to proceed for 2 h at 25 °C and then quenched by the addition of 500 μL of charcoal (3.6% w/v activated charcoal, 150 mM Na₄PPi, 5% HClO₄). Samples were centrifuged, and supernatant was discarded. To remove residual free [³²P]PPi, the pellet was washed twice with wash solution (0.1 M Na₄PPi, 5% HClO4). The pellet was resuspended in 500 μL of water and added to scintillation fluid at a final volume of 5 mL. Radioactivity was measured using a Beckman LS 6500 scintillation counter.

Hydroxylamine Release Assays

The hydroxylamine-trapping assay for detecting adenylation activity was conducted for the SidC C₂A₂ construct, and performed according to a reported protocol³⁸. Briefly, the reaction was initiated by mixing 150 µL of substrate mixture [50 mM Tris, (pH 8.0), 30 mM MgCl₂, 300 mM hydroxylamine (pH 8.0), 10 mM carboxylic acid substrate] with an equal volume of enzyme mixture [100 mM Tris (pH 8.0), 20 mM ATP, 20 µM enzyme]. For some hydrophobic substrates, 2–5% (v/v) DMSO was included to facilitate dissolving the substrate. The reaction mixture was then incubated at 30 o C for 16 h. The reaction was stopped by mixing with 300 µL of stopping solution [10% (w/v) FeCl₃•6H₂O and S5 3.3% TCA dissolved in 0.7 M HCl]. The precipitated enzymes were removed by centrifugation at 17,000g for 5 min, and 200 µL of the supernatant were transferred to a 96-well plate and the absorbance of the ferric-hydroxamate complex at 540 nm was measured by using a Tecan M200 plate reader.

Biochemical characterisation of intra-molecular l-AHO loading by SidC A₃ domain

Purified SidC C₃A₃T₃/C₃A₃T₃C₄T₄ and C₃A₃T₃⁰C₄T₄ proteins were converted to their holo- form by incubation in 20 mM Tris, 100 mM NaCl, 2 µM Sfp PPtase, 1 mM CoA and 10 mM MgCl₂ in a total volume of 50 µL for 1 h at 25 °C. Loading of l-AHO was initiated by addition of ATP (5 mM) and l-AHO (1 mM) in a final volume of 50 µL, and the loading reaction was allowed to proceed for 1 h at 25 °C before intact protein analysis by UHPLC-ESI-Q-TOF-MS.

Biochemical characterisation of inter-molecular l-AHO loading by SidC A₃ domain

Purified SidC C₃A₃T₃/C₄T₄/T₄/C₅T₅C_T and T₅C_T proteins were converted to their holo- form by incubation in 20 mM Tris, 100 mM NaCl, 2 µM Sfp PPtase, 1 mM CoA and 10 mM MgCl₂ in a total volume of 50 µL for 1 h at 25 °C. Loading of l-AHO was initiated by the addition of ATP (5 mM) and l-AHO (1 mM) to a solution of holo-C₃A₃T₃ (100 μM) and one of holo-C₄T₄/T₄/C₅T₅C_T and T₅C_T (100 μM). The loading reaction was allowed to proceed for 1 h at 25 °C before intact protein analysis by UHPLC-ESI-Q-TOF-MS.

Biochemical characterisation of intra-molecular Gly loading by SidC C₂A₂T₂

Purified SidC C₂A₂T₂ was converted to its holo- form by incubation in 20 mM Tris, 100 mM NaCl, 2 µM Sfp PPtase, 1 mM CoA and 10 mM MgCl₂ in a total volume of 50 µL for 1 h at 25 °C. To a solution of SidC C₂A₂T₂ (100 μM), loading of Gly was initiated by the addition of ATP (5 mM, or limited to a 2:1, 4:1 ratio with protein) and Gly (1 mM, or limited to a 2:1, 4:1 ratio with protein) in a total volume of 50 µL at 25 °C. Loading reactions were allowed to proceed for various time intervals before intact protein analysis by UHPLC-ESI-Q-TOF-MS.

Biochemical characterisation of inter-molecular condensation reaction between SidC C₂A₂T₂-Gly₃ and SidC C₃A₃T₃-l-AHO

Purified SidC C₂A₂T₂ and SidC C₃A₃T₃ proteins were converted to their holo- form by incubation in 20 mM Tris, 100 mM NaCl, 2 µM Sfp PPtase, 1 mM CoA and 10 mM MgCl₂ in a total volume of 50 µL for 1 h at 25 °C. Reactions were initiated by addition of ATP (5 mM), l-AHO (1 mM), Gly (1 mM) to a solution containing holo-C₂A₂T₂ (50 μM) and holo-C₃A₃T₃ (100 μM) in a total volume of 50 µL at 25 °C. Reactions were allowed to proceed for either 10 min or 60 min before intact protein analysis by UHPLC-ESI-Q-TOF-MS. A variation of this reaction was conducted which allowed Gly₃ and Gly₅ to be formed in situ on SidC C₂A₂T₂ (following procedure for intra-molecular Gly loading by SidC C₂A₂T₂) before its addition (at a final concentration of 50 μM) to a solution containing holo-C₃A₃T₃ (100 μM), ATP (5 mM), l-AHO (1 mM).

UHPLC-ESI-Q-TOF-MS analysis of intact proteins

Biochemical assays were analysed on a Bruker MaXis II ESI-Q-TOF-MS connected to a Dionex 3000 RS UHPLC fitted with an ACE C₄−300 RP column (100 × 2.1 mm, 5 μm, 30 °C), controlled using Bruker Otof control 4.0. The column was eluted with a linear gradient of 5–100% MeCN containing 0.1% formic acid over 30 min. The mass spectrometer was operated in positive ion mode with a scan range of 200–3000 m/z. Source conditions were: end plate offset at −500 V; capillary at −4500 V; nebuliser gas (N₂) at 1.8 bar; dry gas (N₂) at 9.0 L min⁻¹; dry temperature at 200 °C. Ion transfer conditions were: ion funnel RF at 400 Vpp; multiple RF at 200 Vpp; quadrupole low mass at 200 m/z; collision RF at 2000 Vpp; transfer time at 110.0 µs; pre-pulse storage time at 10.0 µs. All spectra were analysed using Bruker DataAnalysis 4.4. Measured masses for all species are displayed in Table S2 and S3.

AlphaFold modelling of SidC fragments

Structural models of the A₃T₃C₄T₄ and C₄T₄C₅T₅ regions of SidC were constructed using AlphaFold³⁹. The full amino acid sequences of the excised tri-/tetra-domain regions were submitted to the AlphaFold Colab notebook (v1.5.2)⁵², which uses a slightly simplified version of AlphaFold v2.3.1, with the run_relax parameter enabled. Structures were assessed for their reliability via inspection of PAE and pLDDT plots. The resulting structures were then aligned to each other via the C₄ domain present in both structures using PyMOL v1.3, yielding the final model of SidC A₃T₃C₄T₄C₅T₅. Structure co-ordinate files for the SidC A₃T₃C₄T₄C₅T₅ region and associated fragments used to assemble the model are available for download from Mendeley Data https://doi.org/10.17632/c3ymyp3yx4.1.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The minimum dataset necessary to interpret, verify and extend the work is provided in the manuscript and supplementary information. The raw data for Figs. 2 and 3, and Supplementary Figs. 11, 12 and 16, which were processed via standard deconvolution, are available upon written request to the corresponding authors. GenBank accessions have been provided for SidC (XM_653119, [https://www.ncbi.nlm.nih.gov/nuccore/XM_658335.1]), SidA (XM_658335, [https://www.ncbi.nlm.nih.gov/nuccore/XM_658335.1] and SidL (XM_652967, [https://www.ncbi.nlm.nih.gov/nuccore/XM_652967]) in the methods section. The DNA and amino acid sequence of the full-length SidC construct used in this study is reported in the supplementary information. Co-ordinate files for structural models of SidC have been deposited in Mendeley Data https://doi.org/10.17632/c3ymyp3yx4.1. A reporting summary for this Article is available as a supplementary information file.

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Acknowledgements

This work was supported by an NIH 1R35GM118056 grant to Y.T. Y.H. was supported by a Life Sciences Research Foundation Fellowship sponsored by the Mark Foundation for Cancer Research, and also a general start-up fund supported by UCSB. M.J. is the recipient of a BBSRC Discovery Fellowship (BB/R012121/1) and a UKRI Future Leaders Fellowship (MR/W011247/1), and M.P. is supported by a Midlands Integrative Bioscience Doctoral Training Partnership studentship (BB/M01116X/1). The Bruker MaXis II instrument used in this study was funded by the BBSRC (BB/M017982/1). R.R.O.L. and H.H.N. acknowledge funding from the U.S. Department of Energy (DOE) Office of Science (BER) contract DE-FC-02-661 02ER63421 (PI Yeates; UCLA/DOE Institute for Genomics and Proteomics). W.Z. and W.S. acknowledge funding from NIH DP2AT009148 grant. N.K.G. acknowledges shared instrumentation grants from the NSF (CHE-1048804) and the National Center for Research Resources (S10RR025631).

Author information

Yang Hai
Present address: Department of Chemistry and Biochemistry, University of California, Santa Barbara, USA
Hong H. Nguyen
Present address: Transmed Co., Ltd., Ho Chi Minh City, Vietnam
Will Skyrud
Present address: Arzeda, 3421 Thorndyke Ave W, Seattle, WA 98119, USA
These authors contributed equally: Matthew Jenner, Yang Hai.

Authors and Affiliations

Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK
Matthew Jenner & Munro Passmore
Warwick Integrative Synthetic Biology Centre (WISB), University of Warwick, Coventry, CV4 7AL, UK
Matthew Jenner
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, USA
Yang Hai & Yi Tang
Department of Chemistry and Biochemistry, University of California, Los Angeles, USA
Hong H. Nguyen, Junyong Kim, Neil K. Garg & Rachel R. Ogorzalek Loo
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, USA
Will Skyrud & Wenjun Zhang

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Matthew Jenner
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Yi Tang
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Contributions

Y.H., M.J. and Y.T. designed the study. Y.H. generated expression constructs and performed heterologous production of siderophores. Y.H., M.J. and M.P. overexpressed and purified proteins, constructed mutant plasmids and performed biochemical assays. M.J., R.R.O.L. and H.H.N. conducted mass spectrometry analysis of intact proteins. J.K. and N.K.G. synthesised l-AHO. W.S. and W.Z. conducted adenylation domain activity assays. M.P. and M.J. constructed and analysed AlphaFold models. M.J. and Y.H. wrote the manuscript with input from all authors.

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Correspondence to Matthew Jenner or Yang Hai.

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Jenner, M., Hai, Y., Nguyen, H.H. et al. Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis. Nat Commun 14, 2832 (2023). https://doi.org/10.1038/s41467-023-38484-8

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Received: 18 October 2022
Accepted: 25 April 2023
Published: 17 May 2023
DOI: https://doi.org/10.1038/s41467-023-38484-8

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