Fungal Isocyanide Synthases and Xanthocillin Biosynthesis in Aspergillus fumigatus

Lim, Fang Yun; Won, Tae Hyung; Raffa, Nicholas; Baccile, Joshua A.; Wisecaver, Jennifer H.; Rokas, Antonis; Schroeder, Frank C.; Keller, Nancy P.

doi:10.1128/mbio.00785-18

Cited by 47 publications

(69 citation statements)

References 57 publications

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“…Isocyanides also inhibited pigmentation in P. aeruginosa 9 (PAO1), which we hypothesize is due to an interference with the copper-pyocyanin complex (36). Furthermore, we have confirmed the roles of most Xan proteins in the biosynthesis of the xan BGC metabolites, solidifying the proposed biosynthetic pathway presented in a previous study (25). Two xan proteins that did not affect biosynthesis under the tested conditions were XanD and XanA.…”

Section: Discussionsupporting

confidence: 86%

Dual-purpose isocyanides produced byAspergillus fumigatuscontribute to cellular copper sufficiency and exhibit antimicrobial activity

Raffa

Won

Sukowaty

et al. 2020

Preprint

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The maintenance of sufficient but non-toxic pools of metal micronutrients is accomplished through diverse homeostasis mechanisms in fungi. Siderophores play a well-established role for iron homeostasis; however, no copper-binding analogs have been found in fungi. Here we demonstrate that in Aspergillus fumigatus isocyanides derived from the xan biosynthetic gene cluster (BGC) bind copper, impact cellular copper content, and have significant metal-dependent antimicrobial properties. xan BGC-derived isocyanides are secreted and bind copper as visualized by a chrome azurol S (CAS) assay and inductively coupled plasma-mass spectrometry (ICP-MS) analysis of A. fumigatus intracellular copper pools demonstrated a role for xan cluster metabolites in the accumulation of copper. A. fumigatus coculture with A. nidulans, Candida albicans and a variety of pathogenic bacteria establish copper-dependent antimicrobial properties of xan BGC metabolites including inhibition of laccase activity. Similarly, inhibition of Pseudomonas aeruginosa by low concentrations of the xan isocyanide xanthocillin was copper-dependent. Other metals also reduced xanthocillin’s antimicrobial properties, but less efficiently than copper. As variations of the xan BGC exist in other filamentous fungi, we suggest that xanthocillin-like natural products represent a first example for fungal small molecules that serve to maintain copper sufficiency and mediate interactions with competing microbes.SignificanceMetal homeostasis is an integral part of metabolism for any organism. A vast array of small molecules are already known to mediate metal homeostasis in fungi and bacteria; however, unlike their bacterial counterparts, to-date there are no known fungal small molecules that function to maintain copper homeostasis. Discovery of copper binding small molecules produced by A. fumigatus gives insight into mechanisms other than the extensively studied copper transporters or metalloproteins for how fungi can regulate copper. This has important ecological implications as securing scarce nutrients is central for fitness and survival. Additionally, studying this mechanism in A. fumigatus provides a basis for investigation of copper regulation pathways in other fungi.

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Section: Discussionsupporting

confidence: 86%

Dual-purpose isocyanides produced byAspergillus fumigatuscontribute to cellular copper sufficiency and exhibit antimicrobial activity

Raffa

Won

Sukowaty

et al. 2020

Preprint

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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

Dasgupta

Budday

Oliveira

et al. 2019

Preprint

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Post-translational modification of cysteine residues can regulate protein function and is essential for catalysis by cysteine-dependent enzymes. Covalent modifications neutralize charge on the reactive cysteine thiolate anion and thus alter the active site electrostatic environment. Although a vast number of enzymes rely on cysteine modification for function, precisely how altered structural and electrostatic states of cysteine affect protein dynamics remains poorly understood.Here we use X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in isocyanide hydratase (ICH) affects the protein conformational ensemble. ICH exhibits a concerted helical displacement upon cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained residue Ile152. The mobile helix samples alternative conformations in crystals exposed to synchrotron X-ray radiation due to the X-rayinduced formation of a cysteine-sulfenic acid at the catalytic nucleophile (Cys101-SOH). This oxidized cysteine residue resembles the proposed thioimidate intermediate in ICH catalysis. Neither cysteine modification nor helical disorder were observed in X-ray free electron laser (XFEL) diffraction data. Computer simulations confirm cysteine modification-gated helical motion and show how structural changes allosterically propagate through the ICH dimer. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce the ICH catalytic rate and alter its presteady state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. Our results suggest that cysteine modification may be a common and likely underreported means for regulating protein conformational dynamics. Figure 2: Cys101 oxidation leads to a weakening of the Ile152-Cys101 H-bond. Electrostatic Poisson-Boltzmann surfaces (red negative, blue positive charge) calculated from the Cys101-Ile152 (A) and Cys-SOH-Ile152 (B) environments. Cysteine photooxidation neutralizes the negative charge of the sulfur atom, weakening the N-H … S hydrogen bond. (C-E) Schematic showing covalent modification of Cys101 weakens the hydrogen bond (red dotted/dashed line) to Ile152 and allows relaxation of backbone strain (curved arrow). (F) The catalytic thioimidate covalent intermediate resembles photooxidized Cys101-SOH. Both modifications neutralize negative charge on Cys Sγ.

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“…‐ Fungal isocyanide synthase . In microorganisms of fungal origin, especially species involved in the biosynthesis of xanthocillin ( 1 ) and analogs, isocyanide synthase gene clusters were identified . Protein XanB (standing for xanthocillin) has been proposed for the two‐step conversion of l ‐tyrosine to intermediate 2 in Aspergillus fumigatus and is also localized in other genera of fungi.…”

Section: Vinyl Isocyanides Biosynthesis: An Early Incorporation Fomentioning

confidence: 99%

“…Protein XanB (standing for xanthocillin) has been proposed for the two‐step conversion of l ‐tyrosine to intermediate 2 in Aspergillus fumigatus and is also localized in other genera of fungi. Further dimerization of 2 is necessary for the biosynthesis of xanthocillin ( 1 ) and involves a cytochrome P450 named XanG …”

Section: Vinyl Isocyanides Biosynthesis: An Early Incorporation Fomentioning

confidence: 99%

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Biosynthetic Routes to Natural Isocyanides

2020

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As intriguing as it may appear, natural products bearing an isocyanide function are well known in different kingdoms of living organisms. An important question in terms of biochemistry is, of course, the origin of such a functional group and the mechanisms that have evolved in biosynthetic pathways to afford the formation of isocyanides. The latest studies dedicated to such endeavours are analysed therein in view of the producing organism metabolisms (e.g. micro‐organisms, marine invertebrates). The in‐situ reactivity of natural isocyanides is also discussed as well as their biological activities.

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Fungal Isocyanide Synthases and Xanthocillin Biosynthesis in Aspergillus fumigatus

Cited by 47 publications

References 57 publications

Dual-purpose isocyanides produced byAspergillus fumigatuscontribute to cellular copper sufficiency and exhibit antimicrobial activity

Dual-purpose isocyanides produced byAspergillus fumigatuscontribute to cellular copper sufficiency and exhibit antimicrobial activity

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

Biosynthetic Routes to Natural Isocyanides

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