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Photooxidation of the antimicrobial, nonribosomal peptide bacitracin A by singlet oxygen under environmentally relevant conditions Rachel A. Lundeen, Chiheng Chu, Michael Sander, and Kristopher McNeill Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01131 • Publication Date (Web): 29 Apr 2016 Downloaded from http://pubs.acs.org on May 2, 2016
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Manuscript
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Photooxidation of the antimicrobial, nonribosomal peptide bacitracin A by singlet
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oxygen under environmentally relevant conditions
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Rachel A. Lundeen,|| Chiheng Chu, Michael Sander and Kristopher McNeill* Institute of Biogeochemistry and Pollutant Dynamics, Department of Environmental Systems Science, ETH Zurich, 8092 Zurich, Switzerland *Corresponding author Tel. +41 (0)44 632 47 55; Fax. +41 (0)44 632 14 38 Email:
[email protected] ||
R.A.L. Present address: School of Oceanography, University of Washington, Seattle, WA 98195, United States
Number of pages: 35 Number of figures: 5 Number of tables: 0 Number of words: 7,379
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Abstract
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Bacitracin is a mixture of nonribosomal peptides (NRPs) that is extensively used as an
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antibiotic in both human and veterinary medicine. Despite its widespread use over the
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past six decades, very few studies have addressed the environmental fate of bacitracin
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and zinc-bacitracin complexes. In this study, the photochemical transformation of
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bacitracin components (i.e., cyclic dodecapeptides) in the aquatic environment was
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investigated. A high-resolution mass spectrometry (HRMS)-based approach enabled
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monitoring of the photochemical degradation kinetics of individual bacitracin
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components, investigation of the relative contribution of reactive oxygen species (e.g.,
33
singlet oxygen,
34
identification of oxidative modifications in bacitracin photoproducts. The results of this
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study support the hypothesis that indirect photochemical oxidation of the histidine (His)
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residue by 1O2 is a major degradation pathway for bacitracin A, the most potent congener
37
of the mixture. Furthermore, the photooxidation rate of bacitracin A with 1O2 decreased
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upon bacitracin A coordination with Zn2+, demonstrating that the photochemistry of
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metal-bound His is different from metal-free His. Overall, these results provide insight
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into the fate of bacitracin components in the aquatic environment and highlight the
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potential of utilizing this HRMS-based methodology to study transformations of other
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environmentally relevant NRPs.
1
O2) in dissolved organic matter-sensitized photoreactions, and
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Introduction
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Nonribosomal peptides (NRPs)1 are a group of biomolecules that have potent
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bioactivities and complex molecular structures. Many strains of bacteria and fungi are
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known to synthesize NRPs, which are often exploited for their pharmacological
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properties due to their pronounced antimicrobial (e.g., bacitracin or vancomycin),
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immunosuppressive (e.g., cyclosporine), and antitumor (e.g., bleomycin) activities.2,3
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Other examples of bioactive NRPs include siderophores (e.g., pyoverdin or enterobactin),
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toxins (e.g., microcystins),4,5 or surfactants (e.g., surfactin). Compared to ribosomally
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derived peptides, which are linear in structure, NRPs often have complex structural
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motifs and can be branched, cyclic or polycyclic. Since they are produced independent of
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mRNA, NRPs are not limited to the 20-proteinogenic amino acids and may incorporate
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non-proteinogenic amino acid monomers (e.g., D-stereoisomers) or unique NRP
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monomer building blocks (e.g., thiazoline, N-formyl or halogenated groups).6
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Furthermore, NRP biosynthesis1,2,7 often results in the production of mixtures of closely
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related peptide congeners, or peptide variants that have identical structures except for
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monomers located in various positions.
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Bacitracin is an antimicrobial NRP synthesized by Bacillus subtilis and Bacillus
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licheniformis as a complex mixture of cyclic dodecapeptides. While more than 30
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components have been resolved in bacitracin mixtures,8-12 bacitracin A is a major
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component and the most potent antimicrobial congener of the mixture. As shown in
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Figure 1a, the bacitracin structural motif consists of seven amino acid residues in a ring
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and five residues in a side-chain. Bacitracin contains both L- and D-amino acids,
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including D-ornithine (D-Orn), and known photooxidizable amino acids, including a
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histidine (His) residue and a cysteine (Cys)-derived heterocyclic residue. The N-terminus
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of bacitracin A contains an isoleucine residue condensed with Cys to form an inter-
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residue 2-thiazoline ring (Ile’-Cys’, respectively; Figure 1a). The other components of the
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bacitracin mixture vary in their structural arrangement of monomers or differ in one
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specific residue; for instance, an oxidative degradation product of bacitracin A is
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bacitracin F, where the N-terminal inter-residue has been further oxidized to a 2-thiazole
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ring (Ile”-Cys”, Figure 1a).
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Since its discovery in 1945 as a potent antibiotic effective against Gram-positive
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bacteria (e.g., Staphylococcus aureus), bacitracin has been extensively used as a
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commercial antimicrobial in both human and veterinary medicine. Coordination of a
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divalent metal ion with bacitracin, particularly zinc, has been found to increase its
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antimicrobial potency;10,13-15 thus, Zn2+-bacitracin is widely used in antibiotic
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formulations. The bacitracin zinc complex is marketed as an over-the-counter topical
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antibiotic and it is a main ingredient in triple antibiotic ointments (e.g., Neosporin®).
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This zinc complex was also one of the first antimicrobial agents exploited in large-scale
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animal feed operations to promote feed efficiency and growth.16 The overuse of zinc
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bacitracin as an animal feed additive for over 70 years led the European Union to ban its
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use in 1999 on the basis of the ‘precautionary principle’, the justification that bacterial
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resistance to antibiotics might be transferred to humans.17 Currently, there is little
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evidence for bacitracin resistance in gram-positive bacteria or evidence that resistance has
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prevailed or increased over time;18 however, little is known about the mechanism of
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bacterial resistance to bacitracin.19
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Despite its widespread use today and over the past six decades, there exist very few
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studies addressing the fate of bacitracin components in the environment. This is
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surprising since a recent United States Environmental Protection Agency water quality
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report listed bacitracin as one of the top antimicrobials of concern and one of the only
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polypeptide antimicrobials currently approved in the U.S. for use in humans, livestock
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and poultry.20 One study has assessed the fate and transport of antimicrobials and
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antimicrobial resistance genes (ARGs) in soil and agricultural runoff and estimated
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bacitracin concentrations to be at the same order of magnitude as chlortetracycline and
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tylosin.21 A recent study by Li et al.22 examined the co-occurrence of environmental
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ARGs and uncovered that bacitracin ARGs are amongst the most abundant ARGs in
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complex environmental samples, including river water and wastewater from livestock
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farms. Overall, the scarcity of studies on the fate of bacitracin components in the
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environment is alarming and perhaps may be due to fact that the intrinsic complexities of
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NRPs present significant analytical challenges in terms of identifying components and
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following their transformations.
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The goal of this study was to establish whether direct and/or indirect photochemical
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transformations play a role in the degradation of bacitracin components in natural waters.
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From a photochemical standpoint, bacitracin components are both interesting and
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complex as they contain not only a normal target of photooxidation (i.e., His) but also a
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Cys-derived heterocyclic residue with unknown photoreactivity.23-31 We hypothesized
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that the His residue in bacitracin A, the primary focus of this study, would react
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selectively with photochemically produced singlet oxygen (1O2) and that the rate of His
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photooxidation would be modulated by its accessibility in the bacitracin protein
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structure.32 In addition, given that the Zn2+ atoms are known to be coordinated by the His
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and Cys’ residues of bacitracin A,13 we further hypothesize that Zn2+ ligation affects the
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rate and mechanism of photochemical degradation of bacitracin A.33,34
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To enable the study of bacitracin photochemical transformations, we first developed
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a liquid chromatography high-resolution mass spectrometry (LC-HRMS)-based approach
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to identify bacitracin peptides and quantify individual component degradation. An
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analogous methodological approach was successfully used in prior studies by our group
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to monitor the photochemical transformations of His-containing oligopeptides and
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peptides generated from proteolytic digestion of intact proteins.32,35 Herein, peptide
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sequencing techniques were used to characterize bacitracin components (see bacitracin A
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in Figure 1b) and to allow further elucidation of photochemical transformation products.
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We sought to monitor bacitracin photochemical degradation kinetics under
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environmentally relevant conditions, including various light and pH regimes,
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concentrations of Zn2+, and concentrations of dissolved organic matter (DOM). In natural
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waters, chromophoric DOM (CDOM) acts as both a major sensitizer of photochemically
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produced reactive intermediates (PPRIs), including 1O2, hydroxyl radical and triplet-
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excited CDOM, and a sorbent for amino acid-based biomolecules.36 From these studies,
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we assessed the contribution of PPRI-mediated degradation of bacitracin A in CDOM-
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sensitized reactions and investigated site-specific photooxidative modifications to
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individual amino acid residues. We also examined whether sorption of bacitracin A to
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DOM is an important fate process. It has been shown that association of compounds to
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CDOM macromolecules exposes them to much higher concentrations of PPRI than in the
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bulk solution,37,38 resulting in enhanced rates of indirect photochemical transformation.
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Both free His monomers and His-containing peptides are known to be susceptible to
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sorption-enhanced phototransformation processes in solutions containing CDOM.35,39
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Experimental Section
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Chemicals and Sample Preparation. All chemicals were received from commercial
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sources and used as received unless otherwise indicated (see Supporting Information, SI).
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Bacitracin A (VETRANALTM) was purchased from Fluka and a technical mixture of
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bacitracin from Sigma (B0125). Suwannee River Natural Organic Matter (SRNOM) was
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obtained from the International Humic Substances Society and used as a model for
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aquatic DOM.
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All solutions were prepared in 18 MΩ•cm water (MQ; Barnstead Nanopure Diamond
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Water Purification System). Bacitracin A stock solutions (2 mM) were prepared daily in a
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LoBind Eppendorf tube by dissolving bacitracin A VETRANAL in MQ water. The
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following buffers were used based on the pH regime required for the experiment:
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tris(hydroxymethyl)aminomethane (Tris; pH 7.0 or 8.0) and ammonium acetate (pH 5.0
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or 5.8). Stock solutions of model 1O2 sensitizers, either Rose Bengal (RB) or lumichrome,
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were prepared in MQ water and stored at 4 °C. SRNOM solutions were prepared in MQ
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water as described in Chu et al.39 (see SI). Furfuryl alcohol (FFA) was distilled and stock
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solutions were prepared daily in MQ water. An internal standard stock solution was
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prepared by dissolving the contents of the HPLC peptide standard mixture vial (Fluka
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H2016), containing 0.5 mg of five peptides, into one mL of MQ water and stored at -
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20°C. A dilution of the peptide internal standard was prepared daily by diluting 1:10 in
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MQ water.
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Photolysis of Bacitracin A with Model 1O2 Sensitizers. All photolysis solutions
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contained the following: FFA (100 µM), RB or lumichrome (5 or 10 µM, respectively),
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bacitracin A (60 µM), buffer (5 mM; varying pH 5-8), and zinc acetate (Zn(OAc)2; 0-200
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µM). To assess direct photolysis, solutions were prepared as above but without sensitizer.
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RB-sensitized experiments were conducted in cork-stoppered borosilicate test tubes with
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a Xenon lamp (300 W) equipped with a 455 nm long-pass filter. Xenon lamp intensities
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were measured using an externally calibrated fiber optic probe (Jaz, Ocean Optics) to
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ensure stable, reproducible intensities during and across photolyses.
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photolysis, the solutions were lightly stirred. Bacitracin A photolysis experiments in the
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presence of Zn(OAc)2 where conducted only using the filtered Xenon lamp (> 455 nm).
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Lumichrome-sensitized photolysis experiments were conducted in open borosilicate test
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tubes on a turntable apparatus inside a Rayonet photochemical reactor equipped with 2 ×
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365-nm bulbs (Southern New England Ultraviolet Co., RPR-3500 Å), hereafter referred
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to as low intensity UV-A light.
During the
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Aliquots were taken at designated time points for kinetic analysis and split into two
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separate HPLC vials for 1) bacitracin peptide analysis by LC-HRMS or 2) FFA analysis
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(vide infra). Aliquots for bacitracin LC-HRMS analysis were immediately spiked with
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the mixture of five peptide standards, lightly vortexed, and kept at 4 °C until same day
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analysis. Aliquots for LC-HRMS analysis that contained Zn(OAc)2 were first spiked with
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EDTA (2-fold molar excess), vortexed, allowed to sit ten minutes, and then spiked with
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the peptide internal standards. Over the course of all photolysis experiments, the pH of
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the bacitracin solutions increased by at most 0.2 pH units.
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Photolysis of Bacitracin A with SRNOM. Two series of SRNOM-sensitized
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bacitracin A photolysis experiments were conducted: 1) under varied concentrations of
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SRNOM (0-17.1 mgC/L) at constant pH (pH 8) and 2) under varied solution pH (pH 5-8)
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at constant SRNOM concentration (11.4 mgC/L). The photolysis solutions contained the
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following: FFA (40 µM), SRNOM (0-17.1 mgC/L), bacitracin A (60 µM), and buffer (5
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mM). Quenching of 1O2 experiments (pH 7.9; 11.4 mgC/L SRNOM) were conducted in
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the presence of either sodium azide (NaN3; 1 mM) or isopropanol (0.2% v/v, 25 mM). All
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solutions were mixed and allowed to sit overnight at 4 °C to equilibrate. The SRNOM-
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sensitized photolysis experiments were conducted in open borosilicate test tubes on a
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turntable apparatus inside a Rayonet photochemical reactor equipped with 14 × 365-nm
190
bulbs, hereafter referred to as high intensity UV-A light. To monitor any direct photolysis
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of bacitracin components, solutions were prepared as described above but without
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SRNOM. Aliquots of each photolysis were taken at designated time points for kinetic
193
analysis and split for 1) bacitracin peptide analysis by LC-HRMS or 2) FFA analysis, and
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processed as described above.
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Bacitracin Analysis by nanoUPLC-HRMS. Aliquots from the bacitracin photolysis
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experiments were analyzed by nanoUPLC coupled to an Orbitrap HRMS (Thermo
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Exactive) equipped with an electrospray ionization (ESI) source. The bacitracin-internal
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standard peptide mixture (5 µL) was injected onto the column of a Waters
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nanoACQUITY UPLC. Details regarding UPLC column conditions and gradients are
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provided in the SI. Additionally, previous studies that analyzed Zn-peptide complexes by
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MS observed unfavorable zinc deposition within the ESI source,40 which could
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potentially complicate quantitation of Zn2+-bacitracin complexes. We, therefore,
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conducted a series of control studies with Zn2+-bacitracin complexes to chelate Zn2+ with
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EDTA and effectively divert residual EDTA and Zn-EDTA complexes to waste prior to
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nanoUPLC-ESI-HRMS analysis of bacitracin components (see Section S2). The Orbitrap
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HRMS was tuned and calibrated according to the manufacturer’s recommendations prior
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to data acquisition. All analyses were carried out in positive mode at a spray voltage of
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3.8 kV and capillary temperature of 275 °C. High energy collisional dissociation (HCD)
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fragmentation was conducted simultaneously, switching scan modes between full MS and
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HCD. Ultra-high purity argon was used as HCD collision gas and collision energy was
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set at 30 eV. Mass spectra of eluting peptides were obtained at high resolving power with
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a scan range of 100-1600 m/z in full MS mode and 80-1600 m/z in HCD. Data acquisition
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and processing was done through Thermo Xcalibur software.
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Determination of the Steady-State Concentration of 1O2.
FFA was used as a
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chemical probe to determine the steady-state concentration of 1O2, [1O2]ss, in the bulk
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aqueous phase. [1O2]ss was calculated by dividing the observed FFA degradation rate
217
constant by the FFA reaction rate constant with 1O2, 8.3 × 107 M-1s-1. FFA analysis was
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conducted using a Waters ACQUITY UPLC equipped with a photodiode array detector
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(Section S1 for details).
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Results and Discussion
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Identification of bacitracin components.
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Bacitracin is a complex mixture of up to 30 cyclic oligopeptide congeners. A
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nanoUPLC-ESI-HRMS analysis of a technical bacitracin mixture illustrated the analytical
225
complications of attempting to identify individual bacitracin components within a
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complex mixture (Figure S4). Although the high resolving power of an Orbitrap HRMS
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is in principal helpful in identifying individual peptide congeners by their exact masses,
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many of the bacitracin congeners have not been structurally characterized, in large part
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due to the challenges of determining cyclic peptide fragmentation patterns.6,8,12,41
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To reduce the complexity of the analyses, we used an isolated bacitracin A analytical
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standard (see Experimental Section). While bacitracin A was found to be the most
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abundant component in the analytical standard (Figures S1-S2), it also contained traces of
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bacitracin F, consistent with it being a common byproduct and perhaps indicative of
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conversion of bacitracin A to bacitracin F. For bacitracin A, we observed a well-resolved
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chromatographic peak corresponding to three observable charge states in the full MS
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spectrum (observed [M+nH]n+; where n=1, 1422.7536; n=2, 711.8813; n=3, 474.9228;
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Table S1). Bacitracin F had a longer retention time and two charged ions observed at
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high signal-to-noise ratios (observed [M+H]+ = 1419.7064; [M+2H]2+ = 710.3579; Table
239
S1).
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Several key peptide fragment ions were observed at high mass resolution in the HCD
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fragmentation spectra of bacitracin A and bacitracin F. The fragment ions found were
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consistent with a previous low resolution study by Govaerts et al.8 (Table S2). Several
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fragment ions were derived from the intact KOIFHDN(I) ring, which is conserved
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between bacitracin A and bacitracin F, and the N-terminal side-chain, which differ
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between bacitracin A and bacitracin F (Figure 1). For bacitracin A, we observed an
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abundant N-terminal fragment ion from the inter-residue thiazoline ring (observed m/z =
247
199.0898; Figure 1b). Analogously, for bacitracin F, we observed a fragment ion
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corresponding to cleavage of the N-terminus at the L-Leu residue and subsequent
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decarboxylation (observed m/z = 281.1317; Figure S2). The presence of His could easily
250
be identified in these HCD spectra due to the highly abundant His-derived immonium
251
fragment ion (expected m/z = 110.0718; Figures S1-S2). It should be noted that the other
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fragment ions that appear in the HCD spectra of bacitracin A and bacitracin F could be
253
indicative of the fragment ions formed upon ring opening; however, in this study, we
254
omitted these fragments from our analysis because the fragmentation patterns of cyclic
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peptides are complex, not well characterized and accurate assignment may require
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multiple stages of collision-induced dissociation.8,42
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In the following sections, we discuss the photochemical transformations and kinetics
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of the bacitracin A analytical standard under varying experimental conditions. For the
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majority of these experiments, we present results that monitor only the transformations of
260
bacitracin A, the major component of the standard. Later discussions address
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transformations of bacitracin F, a non-interfering, trace component in the standard.
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Evidence for 1O2 involvement in SRNOM-sensitized photolysis of bacitracin A.
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We conducted the photolysis of the bacitracin A standard in a SRNOM-containing
264
solution. Figure 2a shows the SRNOM-sensitized degradation of the bacitracin A
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standard (monitoring only bacitracin A) in the presence of increasing SRNOM
266
concentrations (i.e., 0 – 17.1 mgC/L SRNOM) and under high intensity UVA light. The
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loss of bacitracin A in the presence of SRNOM (at pH 8.0) followed first-order
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degradation kinetics and the observed degradation rate constant (kobs, s-1) increased with
269
increasing concentration of SRNOM (Table S4). Bacitracin A did not degrade under
270
direct photolysis (at 0 mgC/L SRNOM; Figure 2a) or in dark incubations at all SRNOM
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concentrations (Figure S5). The data also show that addition of NaN3, a 1O2 quencher,
272
drastically slowed the rate of bacitracin A degradation in the presence of SRNOM (11.4
273
mgC/L SRNOM, pH 8.0; Figure 2a, Table S4) and the magnitude of quenching was
274
consistent with the concentration of azide (1 mM) and its rate constant (see SI Section
275
S4). Typically, experiments with both quenchers (e.g., N3- and isopropanol) and
276
enhancers (e.g., D2O) are conducted to assess fully the contribution of 1O2; however,
277
enhancement experiments in D2O resulted in partial H/D exchange in bacitracin A and, as
278
a consequence, largely complicated the HRMS data analysis. Overall, these results
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support 1O2-mediated degradation of bacitracin A.
280
In order to assess the relative contribution of 1O2 in the SRNOM-sensitized
281
photodegradation of bacitracin A, we conducted the photolysis of the bacitracin A
282
standard at pH 8.0 using two different 1O2 sensitizers: RB and lumichrome. With both
283
sensitizers, bacitracin A followed first-order degradation kinetics. No bacitracin A losses
284
were observed during photolysis in the absence of sensitizer (Figures S6-S7). Bacitracin
285
A 1O2 reaction rate constants (krxn, M-1 s-1) for either RB- or lumichrome-sensitized
286
photolysis were determined using their respective kobs values and the measured [1O2]ss.
287
The krxn values for bacitracin A where similar to one another, with lumichrome-sensitized
288
krxn slightly higher than RB-sensitized krxn values (c.f., krxn,lumichrome = 6.2 ± 0.4 × 107 M-1
289
s-1 and krxn,RB = 5.2 ± 0.2 × 107 M-1 s-1, Tables S5-S6).
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Next, we plotted kobs values of bacitracin A obtained at different SRNOM
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concentrations versus the measured [1O2]ss in the bulk solution (Figure 2b). We observed
292
a linear correlation between bacitracin A observed degradation rate constants and [1O2]ss
293
from SRNOM-sensitized photolyses. The solid lines correspond to the predicted kobs
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values, which were calculated using the 1O2 krxn value of bacitracin A with lumichrome or
295
RB under the same pH conditions and the measured [1O2]ss from SRNOM-sensitized
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photolyses. The strong agreement between the measured and the predicted kobs values
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supports the view that 1O2 is the dominant contributor to the degradation of bacitracin A
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in SRNOM-sensitized photolyses. Additionally, the linearity of the data in Figure 2b and
299
its agreement with predicted kobs values suggests no enhancement of the degradation of
300
bacitracin A due to sorption with SRNOM at pH 8.0.
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pH-dependence on the photooxidation of bacitracin A.
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The data from the SRNOM-sensitized experiments point to a predominantly 1O2-
303
mediated degradation pathway for bacitracin A; however, the site of 1O2 reaction remains
304
unknown. Although the N-terminal thiazoline residue of bacitracin A has unknown
305
reactivity with 1O2, it is known that free His reacts predominantly with 1O2 in natural
306
waters43 and the rate of His residue 1O2 photooxidation is modulated by its accessibility
307
within the protein structure.32 In addition, the photooxidization of free His is largely pH
308
dependent with lower His 1O2 reactivity at pH values below the imidiazole pKa (approx.
309
6.0).24,39 Thus, if the His residue was the primary site of 1O2 reactivity, the rates of
310
bacitracin A 1O2 photooxidation would also be pH dependent and may be modulated by
311
its location in the KOIFHDN ring.
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The pH-dependent krxn values of bacitracin A with
1
O2 were independently
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determined for both RB- and lumichrome-sensitized photolyses. The observed
314
photochemical degradation of bacitracin A followed first-order kinetics at all investigated
315
pH values (Table S5-S6). No direct photolysis of bacitracin A was observed under both
316
light conditions and across all experimental pH. The krxn values of bacitracin A increased
317
from pH 5.0 to 8.0 (Figure 3a, data shown only for RB-sensitized photolyses; refer
318
further to Section S5), which is consistent with our hypothesis that His is the primary site
319
of 1O2 photooxidation and previous results on the pH-dependent 1O2 reactivities of both
320
free His24,39 and His-containing oligopeptides.35 The solid line represents the calculated
321
pH-dependent krxn of bacitracin A with 1O2 (krxn,calc), which was determined using the
322
respective 1O2 krxn values of His residues in either the neutral or protonated form (
323
and ) and the respective fraction of each of these species to total His ( and
324
; see equation 1).35,39
325
+
, =
(1)
326
The good agreement between observed and the calculated 1O2 krxn values from RB-
327
sensitized photolyses in Figure 3a strongly supports that the His residue was involved in
328
the 1O2-mediated degradation of bacitracin A. The apparent His pKa value in bacitracin A
329
around 6.5, as indicated by the inflection in Figure 3a, was in very good agreement with
330
the pKa values of His residues in short oligopeptides (e.g., AAAHAAA His pKa at 6.6),35
331
supporting His as the 1O2 reactive site. This recent study by Chu et al.35 demonstrated that
332
the shifts in the pKa values and hence the variation 1O2 krxn of His residues in short,
333
unstructured oligopeptides as compared to free His were modulated through electrostatic
334
effects originating from ionizable residues neighboring the His residue.
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The magnitude of the krxn values of bacitracin A indicate that the reactivity of the His
336
residue with 1O2 may be modulated by the peptide structure. For instance, the observed
337
krxn values for bacitracin A (krxn,RB = 5.2 ± 0.2 × 107 M-1 s-1, pH 8.0) were slightly lower
338
than the krxn values of both free His (krxn = 7.0 ± 0.1 × 107 M-1s-1, pH 7.8)32,39 and
339
unstructured, His-containing oligopeptides (e.g., AAAHAAA krxn = 7.2 ± 0.2 × 107 M-1s-
340
1
341
environment resulting from neighboring D-Phe and D-Asp residues to L-His in the
342
KOIFHDN ring or resulting from bacitracin A secondary protein structure. We have
343
previously shown that the 1O2 reactivity of His residues in proteins is dependent on the
344
1
345
relationship developed
346
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the rate constant for reaction of
347
1
348
His side-chain being 75% exposed relative to free His (1O2-ASA =160 Å2).
, pH 8.0).35 This difference in photooxidation kinetics could be due to the local steric
O2-accessible surface area (1O2-ASA) of the respective residues.32 Using the same from
Lundeen and
McNeill32 for the intact protein
O2 with the L-His residue in bacitracin A is consistent with a 1O2-ASA of 120 Å2 or the
349
Sorption-enhanced phototransformation of bacitracin A in SRNOM-sensitized
350
photolysis. The phototransformation kinetics of bacitracin A with 1O2 in SRNOM
351
solutions (at 11.4 mgC/L) followed first-order degradation kinetics at all pH values (Table
352
S7). In addition to showing the calculated fit of bacitracin A krxn values from RB-
353
sensitized photolyses, Figure 3a also shows the pH-dependence on krxn values from
354
SRNOM photolyses with bacitracin A. In Figure 3b, the ratio of bacitracin A krxn to the
355
krxn,calc from these SRNOM experiments was plotted as a function of pH; where the
356
SRNOM krxn,calc values were determined from equation 1 using the calculated fit from
357
RB-sensitized rate constants and the pH during SRNOM experiments (see Section S5). At
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solution pH values greater than 6.5, there was good agreement between experimentally
359
measured and calculated rate constants, where SRNOM krxn/krxn,calc ratios were near unity.
360
At pH values below 6.5, the measured krxn values were higher than the calculated rate
361
constants (apparent in Figure 3a), where SRNOM krxn/krxn,calc ratios were much greater
362
than one (Figure 3b). We ascribe this increase in the krxn/krxn,calc ratios at low solution pH
363
to association of positively charged residues in bacitracin A with SRNOM, which
364
enhances indirect phototransformation. This has been previously demonstrated by Chu et
365
al.,35,39 which established that sorption of His and His-containing oligopeptides to
366
SRNOM resulted in enhanced rates of 1O2 phototransformation. For instance, krxn/krxn,calc
367
ratios for AAAHAAA (replotted for comparison in Figure 3b) and of RRRHRRR were
368
13 and 34 at solution pH 5.0, respectively. These enhanced phototransformation rates
369
reflected sorption of these oligopeptides to SRNOM driven by the electrostatic attraction
370
between the positively charged imidazolium moiety in AAAHAAA and also guanidium
371
moieties in RRRHRRR with the negatively charged carboxylate moieties in SRNOM.
372
Analogous to AAAHAAA, a similar enhancement was observed for bacitracin A
373
(krxn/krxn,calc = 11 at pH 5.1, Table S7) indicating that protonation of the His residue
374
resulted in sorption-enhanced phototransformation.
375
Phototransformation products of bacitracin components.
376
Site-specific photooxidative modification to His in bacitracin A. Previous sections that
377
reported on the 1O2 reaction kinetics of bacitracin A provided evidence that the His
378
residue was the dominant photooxidizable residue. Yet, no evidence was provided to
379
exclude the thiazoline moiety as a potential additional target of bacitracin A 1O2
380
photooxidation. Below we give evidence that oxidation of bacitracin A by 1O2 selectively
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381
attacks the His residue in the KOIFHDN ring. The proposed pathway of 1O2-mediated
382
bacitracin component photooxidation is presented in Figure 4 along with the results of
383
bacitracin product peptide sequencing by HCD-HRMS.
384
Mass spectrometric monitoring of bacitracin A during both lumichrome- and RB-
385
sensitized photolyses and SRNOM-sensitized photolysis revealed the production of
386
several identical products, where photooxidative modifications were directed entirely at
387
His residues. The exact masses of the products corresponded to the 2-oxo-histidine
388
(His+14) and imidazolone (His+32) oxidation of the His residue in bacitracin A (Figures 4,
389
Table S9).25,31 The extracted ion chromatograms resulted in four distinct chromatographic
390
peaks for the product bacitracin A–His+14 (Figure S12) and one chromatographic peak for
391
bacitracin A–His+32. In the fragmentation spectrum of each bacitracin A–His+14 peak, we
392
observed the loss of fragment ions corresponding to intact KOIFHDN(I) and the presence
393
of several key fragments corresponding to KOIFH+14DN(I) (Table S10; Figure 4). We
394
also observed the disappearance of the His-derived immonium ion, which was highly
395
abundant in the parent bacitracin A HCD spectrum and an important indicator of the
396
presence of the His residue. Furthermore, the HCD spectrum of bacitracin A–His+14
397
contains identical fragment ions corresponding to the N-terminal thiazoline ring of
398
bacitracin A (observed m/z = 199.0899; Figure 4). In the fragmentation spectrum for
399
bacitracin A– His+32, we observed the KOIFH+32DN fragment ion (observed m/z =
400
901.4512, Table S10); yet, simultaneously, water loss during fragmentation yielded
401
identical ions as KOIFH+14DN. Overall, the agreement between the expected m/z and
402
HCD fragmentation spectrum provide strong evidence for the His+14 oxidation of
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403
bacitracin A; however, the reason for the observed splitting into four chromatographic
404
peaks currently remains unclear.
405
Site-specific N-terminal residue dark oxidation of bacitracin A by H2O2. Figure 4
406
also displays the observed oxidation pathway of the bacitracin A standard with hydrogen
407
peroxide (H2O2). It is known that free thiols (e.g., Cys) are irreversibly oxidized to the
408
sulfonic acid via the sulfenic acid and sulfinic acid under strong oxidizing conditions
409
(Figure S14a).44 Thus, we hypothesized that reaction of bacitracin A with H2O2 would
410
react selectively with the N-terminal thiazoline residue. Following a dark, overnight
411
incubation of the bacitracin A standard with H2O2 (approx. 1 M), we were able to identify
412
transformation products indicative of the complete conversion of bacitracin A to
413
bacitracin F and their corresponding sulfonic acids (labeled as SO3H, Figure 4; refer to
414
Section S9). This reaction provided further support that 1O2 selectively attacked His
415
moieties whereas H2O2 primarily reacted with the N-terminal Cys-containing residues.
416
Though each step of sulfur oxidation along the pathway to sulfonic acid was not observed
417
in this study, it is apparent this HCD-HRMS methodology could be used in further
418
mechanistic studies, for instance, aimed at detecting oxidative intermediates (e.g.,
419
sulfenic acids).44
420
Preliminary evidence for the direct photochemical isomerization of bacitracin F. An
421
additional photoproduct was observed that resulted from the direct phototransformation
422
of bacitracin F under high intensity UVA light, which was likely attributed to a small
423
spectral overlap in the UV-B region (near 300 nm). Based on its exact mass and
424
fragmentation pattern, the photoproduct is an isomer of bacitracin F. We speculate that
425
the N-terminal thiazole in bacitracin F may undergo direct photochemical isomerization
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426
to an isothiazole (scheme 1, proposed structure of Ile”-iso-Cys”) during UV-B
427
irradiation. This hypothesis is supported by studies that observe the photochemical
428
isomerization of thiazoles and other thiazole-containing NRPs.45,46 Further studies
429
addressing bacitracin F direct photochemistry are ongoing.
430
431 432
Scheme 1. Proposed isomerization of N-terminal thiazole in bacitracin F.
433 434
Effect of Zn-ligation on 1O2 reactivity of bacitracin A.
435
In commercial antibiotic formulations, bacitracin is most often administered with
436
complexed Zn2+; thus, Zn-bacitracin complexes may be the dominant, anthropogenic
437
metal-bacitracin species in aquatic environments. Figure 5a shows the absorbance spectra
438
of the bacitracin A analytical standard upon the addition of Zn(OAc)2. Coordination of
439
Zn2+ to the bacitracin A standard was evident from the increase of its molar extinction
440
coefficient at 252 nm (at pH 8.0, 25°C).14 The inset in Figure 5a shows the binding
441
isotherm of bacitracin with Zn2+ (based on bacitracin absorbance at 252 nm) and the
442
calculated model fit of the dissociation constant (Kd (Zn2+-bacitracin) = 14 µM). It is
443
noteworthy that this binding isotherm cannot differentiate between Zn2+ ligation with
444
bacitracin A or bacitracin F, which is present as a byproduct in the standard. For
445
bacitracin A, it has been previously shown that the Zn atom coordinates with L-His, D-
446
Glu and the N-terminal thiazoline ring (Figure 5);13 yet, studies have not been conducted
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447
with pure bacitracin F standards and it is neither known if Zn2+ complexes form with
448
bacitracin F nor if the coordination involves the His and thiazole moieties.
449
The reactivity of Zn2+-bacitracin A with 1O2 was tested and compared with metal-free
450
bacitracin A (vide supra). We conducted visible light RB-sensitized photolyses of the
451
bacitracin A standard in the presence of increasing concentrations of Zn2+. We chose
452
visible light sensitized photolysis to help elucidate the 1O2 reactivity of Zn2+-bacitracin A
453
without evoking other mechanisms of oxidation, including metal-catalyzed oxidation
454
reactions.47 Control bacitracin A visible light photolysis experiments at all Zn(OAc)2
455
concentrations (without RB) showed that the Zn2+-bacitracin A did not undergo direct
456
photolysis under these conditions (Figure S9).
457
The 1O2-mediated photodegradation of bacitracin A followed first-order degradation
458
kinetics at all Zn2+ concentrations (pH 8.0). The data in Figure 5b show the kobs values
459
over increasing Zn2+ concentrations. The kobs values of bacitracin A without Zn2+ were in
460
agreement with previous RB-sensitized results (kobs values from Figure 3 at pH 8.0 are
461
shown as thresholds in Figure 5b) and also provided evidence that the addition of EDTA
462
to the aliquots following photolysis did not introduce sample loss (refer to Experimental
463
Section). Upon addition of Zn2+, the kobs values for bacitracin A decreased significantly
464
from 0 to 40 µM Zn2+ and then remained constant up to 200 µM Zn2+ (kobs,RB = 2.3 ± 0.09
465
× 10-3 s-1 at 40 µM Zn2+; Table S8). This observed decrease in kobs indicates that ligation
466
of Zn2+ by the His residue inhibited the rate of His photooxidation with 1O2. The data
467
support the idea that the photochemistry of Zn2+-His complexes may be different from
468
free His regarding reaction pathways and kinetics. These results are consistent with a
469
recent study by Lebrun et al.,33 which highlighted that binding of Zn2+ inhibits the
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photooxidation of His residues but not Cys residues in the zinc finger tetrahedral complex
471
Zn(Cys)2(His)2.
472
Environmental Implications.
473
Due to their large-scale exploitation in human and agricultural settings, bacitracin
474
and other NRPs of its class are compounds of environmental concern alongside more
475
traditional antibiotic (micro-) pollutants. Just as with other pollutants, a mechanistic
476
understanding of the environmental fate of NRPs is essential in order to adequately
477
predict their activity and persistence in the environment. For instance, the intrinsic
478
biological activity of NRPs as well as their unusual structural features may have evolved
479
in order to protect these peptides from degradation by proteases or inhibit biological
480
transformation. The study presented here provides evidence that the photochemical
481
transformations of bacitracin under environmentally relevant conditions may be an
482
important fate process and further highlights the need to include both abiotic and biotic
483
transformation rates in our assessment of the cycling of amino acid-based (pollutant-)
484
biomolecules in sunlit surface waters.
485
This work helps contribute to a more comprehensive and accurate evaluation of the
486
abiotic transformations governing proteinogenic and non-proteinogenic amino acid-
487
containing biomolecules in the aquatic environment. Past and ongoing work in our group
488
has uncovered that photochemical transformation of amino acid-containing biomolecules,
489
including photochemically generated 1O2 with free His (and other photooxidizable amino
490
acids) and His residues in oligopeptides and proteins, is important fate processes in
491
natural waters.32,35,39,43,48 However, despite our growing understanding on the
492
photooxidation of amino acid residues, studies with Zn2+-bacitracin indicate that the
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493
photooxidation rates of metal-bound photooxidizable residues are likely to differ
494
significantly from their metal-free counterparts. This calls for more mechanistic studies
495
examining the effect of ligated metals on the photooxidation kinetics of amino acid
496
residues in metal-containing oligopeptides, NRPs or metalloenzymes.
497
photochemical investigations of non-proteinogenic amino acid monomers are largely
498
missing from the literature and detailed studies are needed.
Furthermore,
499
This study presented new lines of evidence that (photo)oxidative transformations of
500
bacitracin components generates a suite of oxidation products. These findings contradict
501
previous assumptions that bacitracin F was the final oxidation product of bacitracin A,
502
the most potent bacitracin congener.49 Overall, this raises potential concern regarding the
503
antimicrobial activity of oxidized bacitracin component by-products and isomers in the
504
environment. For instance, higher concentrations of mixtures of residual parent and
505
oxidized bacitracin components could exert selective pressure on microbial communities
506
and induce the emergence of diverse ARGs across environmental compartments.22
507
Lastly, this study showed that the developed nanoUPLC-HRMS approach is a
508
powerful tool (and the only tool thus far) to study environmental transformations of large
509
and structurally complex NRPs. This method provides a foundation for studying the fate
510
of other NRPs of interest, for instance, cyanobacteria microcystins,4,5 or ribosomally
511
synthesized and post-translationally modified peptides50 in the environment or engineered
512
systems.
513
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514 515
Supporting Information Available
516
Supporting figures, tables, detailed experimental methods and additional experiments
517
described within the manuscript are provided. This information is available free of charge
518
via the Internet at http://pubs.acs.org.
519 520
Acknowledgements
521
This study was supported by funding from the Swiss National Science Foundation
522
through
523
(postdoctoral fellowship for R.A.L). We thank Prof. Tamar Kohn (EPFL), Prof. P. Lee
524
Ferguson (Duke University), and Dr. Elisabeth M.-L. Janssen (ETH Zurich) for helpful
525
discussions.
project
grants
200021_138008,
200020_159809
and
P2EZP2_155522
526
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527 528 529 530 531 532 533 534 535 536 537
Figure 1. a. Structural motif of the nonribosomal peptide bacitracin. Bacitracin A, the most potent congener, and bacitracin F differ only in their N-terminal R-group, with either a thiazoline (Cys’) or thiazole (Cys’’) residue, respectively. b. Bacitracin A peptide-sequencing and key fragment ion assignments from HCD-HRMS. Observed and expected m/z values of bacitracin A parent ion [M+2H]2+ as well as fragment ions (blue, solid arrows) containing the intact KOIFHDN(I) ring or the N-terminus are labeled. (Small: Actual Size 17.78 cm × 8.38 cm)
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538 539 540 541 542 543 544 545 546 547 548 549 550
Figure 2. Photolysis of bacitracin A in the presence of Suwannee River natural organic matter (SRNOM) at pH 8.0. a. Kinetic traces for the direct and indirect photochemical degradation of only bacitracin A under high intensity UV-A light at SRNOM concentrations 0-17.1 mgC/L (triangles). Red circles indicate an analogous photodegradation at 11.4 mgC/L SRNOM but in the presence of a 1O2 quencher, sodium azide (NaN3). Error bars represent error in the averaged normalized MS peak areas across multiple charge states. When error bars are not visible, they are contained within the symbols. b. Observed rate constants for bacitracin A during SRNOM-sensitized photolysis (0-17.1 mgC/L) versus the measured steady-state 1O2 concentration. Predicted lines calculated from lumichrome (green) and Rose Bengal (RB, pink) 1O2 reaction rate constants (krxn, lumichrome (pH 7.9) = 6.15 ± 0.42 × 107 M-1s-1 or krxn,RB (pH 8.0) = 5.17 ± 0.16 × 107 M-1s-1; see Tables S5-S6).
551 552 553
(Small: Actual Size 8.46 cm × 14.22 cm)
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554 555 556 557 558 559 560 561 562 563 564 565 566 567
Figure 3. pH dependency on bacitracin A reactivity with singlet oxygen (1O2). Reaction rate constants with 1O2 (krxn; 107 M-1s-1) of a. bacitracin A as a function of solution pH from the following experiments: Rose Bengal (RB)-sensitized visible light photolysis (pink, filled squares) and SRNOM-sensitized photolysis under high intensity UVA light (brown, open squares). The solid line represents the calculated fit of reaction rates (krxn,calc) from equation 1, according to Chu et al.35,39 Error bars signify propagated error from observed rate constant and measured [1O2]ss. b. Ratios of observed bacitracin A 1O2 krxn to krxn,calc (brown, open squares) from SRNOM-sensitized photolysis, where krxn,calc values were calculated from the RB predicted fit (Figure 3a, refer also to Section S5). For comparison, previously published values from Chu et al.35 for 1O2 mediated krxn of the peptide AAAHAAA in SRNOM-sensitized experiments over the krxn,calc (from AAAHAAA lumichrome-sensitized experiments) as a function of pH (brown, open circles). Error bars represent the ratio of the standard deviation of experimental krxn
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568 569 570
divided by the error in the predicted fit. The dashed line at krxn/krxn,calc = 1 is the ratio expected for RB-sensitized systems. When error bars are not visible, they are contained within the symbols.
571 572
(Large: Actual Size 8.46 cm × 15.8 cm)
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574 575 576 577 578 579 580 581 582 583 584 585 586 587
Figure 4. Predicted photooxidative and oxidative degradation pathways for bacitracin components addressed in this study. Sites of oxidative modification are displayed and illustrated by peptide-sequencing (c.f., Figure 1b) and key fragment ion assignments from HCD-HRMS. Observed m/z values of fragment ions of the intact KOIFHDN(I) ring (His+14 or His+32; red, dashed lines; Section S8) or the N-terminus are labeled as well as photoproduct parent ion observed and expected monoisotopic masses. The oxidative degradation of bacitracin A with hydrogen peroxide (H2O2) is also provided with proposed structures for N-terminal sulfonic acids (Cys-SO3H) (refer to Section S9, for more detailed fragmentation). Abbreviation N.R. indicates no reaction. (Large: Actual Size 17.8 cm × 14.3 cm)
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Figure 5. a. Binding of Zn2+ to bacitracin A VETRANAL standard (60 µM, pH 8.0 Tris, 25°C). Traces represent the UV-absorption spectra of bacitracin upon addition of zinc acetate (at pH 8.0). The color dependent-traces illustrate the formation of Zn2+-bacitracin complexes (from grey to dark blue) and an increase in bacitracin component molar extinction coefficient at 252 nm.14 Inset displays only absorbance units at 252 nm against log of Zn2+ concentrations with predicted fit of Zn2+-bacitracin dissociation constant (Kd). b. Observed rate constants of bacitracin A from RB-sensitized visible light photolysis under increasing concentrations of zinc acetate (Zn2+). Solid line displays the kobs value from RB-sensitized photolysis of bacitracin A without Zn2+ from Figure 3 at pH 8.0 only and pink shading depicts error in kobs value. (Small: Actual Size 8.46 cm × 17.4 cm)
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602 603 604 605 606
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References 1. Cane, D. E.; Walsh, C. T.; Khosla, C. Harnessing the biosynthetic code: combinations, permutations, and mutations. Science 1998, 282 (5386), 63-68. 2. Sieber, S. A.; Marahiel, M. A. Molecular mechanisms underlying nonribosomal peptide synthesis: Approaches to new antibiotics. Chem. Rev. 2005, 105 (2), 715-738. 3. Walsh, C. T. Antibiotics: Actions, Origins, Resistance. ASM Press: Washington, DC, 2003. 4. Schwarzenbach, R. P.; Escher, B. I.; Fenner, K.; Hofstetter, T. B.; Johnson, C. A.; von Gunten, U.; Wehrli, B. The challenge of micropollutants in aquatic systems. Science 2006, 313 (5790), 1072-1077. 5. Svrcek, C.; Smith, D. W. Cyanobacteria toxins and the current state of knowledge on water treatment options: a review. J. Environ. Eng. Sci. 2004, 3 (3), 155-185. 6. Caboche, S.; Pupin, M.; Leclere, V.; Fontaine, A.; Jacques, P.; Kucherov, G. NORINE: a database of nonribosomal peptides. Nucleic Acids Res. 2008, 36 (Database issue), D326-331. 7. Challis, G. L.; Naismith, J. H. Structural aspects of non-ribosomal peptide biosynthesis. Curr Opin Struct Biol 2004, 14 (6), 748-756. 8. Govaerts, C.; Li, C.; Orwa, J.; Van Schepdael, A.; Adams, E.; Roets, E.; Hoogmartens, J. Sequencing of bacitracin A and related minor components by liquid chromatography/electrospray ionization ion trap tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17 (12), 1366-1379. 9. Ikai, Y.; Oka, H.; Hayakawa, J.; Matsumoto, M.; Saito, M.; Harada, K.; Mayumi, Y.; Suzuki, M. Total structures and antimicrobial activity of bacitracin minor components. J. Antibiot. 1995, 48 (3), 233-242. 10. Ming, L.-J.; Epperson, J. D. Metal binding and structure–activity relationship of the metalloantibiotic peptide bacitracin. Journal of Inorganic Biochemistry 2002, 91 (1), 4658. 11. Morris, M. Primary structural confirmation of components of the bacitracin complex. Biomed. Environ. Mass Spectrom. 1994, 23 (2), 61-70. 12. Siegel, M. M.; Huang, J.; Lin, B.; Tsao, R.; Edmonds, C. G. Structures of bacitracin A and isolated congeners: Sequencing of cyclic peptides with blocked linear side chains by electrospray ionization mass spectrometry. Biomed. Environ. Mass Spectrom. 1994, 23 (4), 186-204. 13. Economou, N. J.; Cocklin, S.; Loll, P. J. High-resolution crystal structure reveals molecular details of target recognition by bacitracin. Proc. Natl. Acad. Sci. USA 2013, 110 (35), 14207-14212. 14. Scogin, D. A.; Mosberg, H. I.; Storm, D. R.; Gennis, R. B. Binding of nickel and zinc ions to bacitracin A. Biochemistry 1980, 19, 3348-3352. 15. Stone, K. J.; Strominger, J. L. Mechanism of action of bacitracin: Complexation with metal ion and C55-isoprenyl pyrophosphate. Proc. Natl. Acad. Sci. USA 1971, 68 (12), 3223-3227. 16. Sarmah, A. K.; Meyer, M. T.; Boxall, A. B. A. A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 2006, 65 (5), 725-759.
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O
bacitracin A
O H 2N
NH O
O S H 2N
N
H N O
OH
HN
H N
OH O N H
N
HN
Ph
O
O
HN
O
ln (Bacitracin At / Bacitracin A0)
anthropogenic inputs
direct photolysis
0
+ DOM + 1O2 quencher
-0.5 -1
+ Dissolved
-1.5
Organic Matter ACS Paragon Plus Environment O
N H
H N
O
O
N H
H N
O
HN
O NH 2
(DOM)
-2 0
6000
12000
18000
Time (sec)
24000