Photooxidation of the Antimicrobial, Nonribosomal Peptide Bacitracin

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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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Environmental Science & Technology

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

ACS Paragon Plus Environment

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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

27

past six decades, very few studies have addressed the environmental fate of bacitracin

28

and zinc-bacitracin complexes. In this study, the photochemical transformation of

29

bacitracin components (i.e., cyclic dodecapeptides) in the aquatic environment was

30

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)

36

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

38

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

59

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

140

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

142

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

144

aquatic DOM.

145

All solutions were prepared in 18 MΩ•cm water (MQ; Barnstead Nanopure Diamond

146

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

150

or 5.8). Stock solutions of model 1O2 sensitizers, either Rose Bengal (RB) or lumichrome,

151

were prepared in MQ water and stored at 4 °C. SRNOM solutions were prepared in MQ

152

water as described in Chu et al.39 (see SI). Furfuryl alcohol (FFA) was distilled and stock

153

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

155

H2016), containing 0.5 mg of five peptides, into one mL of MQ water and stored at -

156

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

161

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

171

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

173

separate HPLC vials for 1) bacitracin peptide analysis by LC-HRMS or 2) FFA analysis

174

(vide infra). Aliquots for bacitracin LC-HRMS analysis were immediately spiked with

175

the mixture of five peptide standards, lightly vortexed, and kept at 4 °C until same day

176

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)

183

at constant SRNOM concentration (11.4 mgC/L). The photolysis solutions contained the

184

following: FFA (40 µM), SRNOM (0-17.1 mgC/L), bacitracin A (60 µM), and buffer (5

185

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

187

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

189

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

191

of bacitracin components, solutions were prepared as described above but without

192

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

194

processed as described above.

195

Bacitracin Analysis by nanoUPLC-HRMS. Aliquots from the bacitracin photolysis

196

experiments were analyzed by nanoUPLC coupled to an Orbitrap HRMS (Thermo

197

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

200

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

204

EDTA and effectively divert residual EDTA and Zn-EDTA complexes to waste prior to

205

nanoUPLC-ESI-HRMS analysis of bacitracin components (see Section S2). The Orbitrap

206

HRMS was tuned and calibrated according to the manufacturer’s recommendations prior

207

to data acquisition. All analyses were carried out in positive mode at a spray voltage of

208

3.8 kV and capillary temperature of 275 °C. High energy collisional dissociation (HCD)

209

fragmentation was conducted simultaneously, switching scan modes between full MS and

210

HCD. Ultra-high purity argon was used as HCD collision gas and collision energy was

211

set at 30 eV. Mass spectra of eluting peptides were obtained at high resolving power with

212

a scan range of 100-1600 m/z in full MS mode and 80-1600 m/z in HCD. Data acquisition

213

and processing was done through Thermo Xcalibur software.

214

Determination of the Steady-State Concentration of 1O2.

FFA was used as a

215

chemical probe to determine the steady-state concentration of 1O2, [1O2]ss, in the bulk

216

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

219

(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

224

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,

228

many of the bacitracin congeners have not been structurally characterized, in large part

229

due to the challenges of determining cyclic peptide fragmentation patterns.6,8,12,41

230

To reduce the complexity of the analyses, we used an isolated bacitracin A analytical

231

standard (see Experimental Section). While bacitracin A was found to be the most

232

abundant component in the analytical standard (Figures S1-S2), it also contained traces of

233

bacitracin F, consistent with it being a common byproduct and perhaps indicative of

234

conversion of bacitracin A to bacitracin F. For bacitracin A, we observed a well-resolved

235

chromatographic peak corresponding to three observable charge states in the full MS

236

spectrum (observed [M+nH]n+; where n=1, 1422.7536; n=2, 711.8813; n=3, 474.9228;

237

Table S1). Bacitracin F had a longer retention time and two charged ions observed at

238

high signal-to-noise ratios (observed [M+H]+ = 1419.7064; [M+2H]2+ = 710.3579; Table

239

S1).

240

Several key peptide fragment ions were observed at high mass resolution in the HCD

241

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

243

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

245

between bacitracin A and bacitracin F (Figure 1). For bacitracin A, we observed an

246

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

248

corresponding to cleavage of the N-terminus at the L-Leu residue and subsequent

249

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

252

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

255

peptides are complex, not well characterized and accurate assignment may require

256

multiple stages of collision-induced dissociation.8,42

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In the following sections, we discuss the photochemical transformations and kinetics

258

of the bacitracin A analytical standard under varying experimental conditions. For the

259

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

261

transformations of bacitracin F, a non-interfering, trace component in the standard.

262

Evidence for 1O2 involvement in SRNOM-sensitized photolysis of bacitracin A.

263

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

265

standard (monitoring only bacitracin A) in the presence of increasing SRNOM

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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

268

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

271

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

279

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

291

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

294

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

296

photolyses. The strong agreement between the measured and the predicted kobs values

297

supports the view that 1O2 is the dominant contributor to the degradation of bacitracin A

298

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.

301

pH-dependence on the photooxidation of bacitracin A.

302

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

313

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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335

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


16

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358

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


17


Page 18 of 36

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


18

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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


19


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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


20

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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


21


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470

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


22

Page 23 of 36


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


23


Page 24 of 36

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


24

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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)


25


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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)


26

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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


27


Page 28 of 36

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)


28

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573

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)


29


588 589 590 591 592 593 594 595 596 597 598 599

Page 30 of 36

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)


30

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Table of Contents (TOC) Art:

601

602 603 604 605 606

(Actual Size 8.47 cm x 4.76 cm)


31


607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651

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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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Environmental Science & Technology Page 36 of 36

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

Photooxidation of the Antimicrobial, Nonribosomal Peptide Bacitracin

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