Investigation of Amide Bond Formation during Dehydrophos Biosynthesis

Article abstract of DOI:10.1021/acschembio.7b00949

Dehydrophos is a tripeptide phosphonate antibiotic produced by Streptomyces luridus. Its biosynthetic pathway involves the use of aminoacyl-tRNA (aa-tRNA) for amide bond formation. The first amide bond during biosynthesis is formed by DhpH-C, a peptidyltransferase that utilizes Leu-tRNA^Leu. DhpH-C is a member of a burgeoning family of natural product biosynthetic enzymes that make use of aa-tRNA outside of canonical translation activities in the cell. Here, we used site-directed mutagenesis of both DhpH-C and tRNA^Leu to investigate the enzyme mechanism and substrate specificity, respectively, and analyzed the substrate scope for the production of a set of dipeptides. DhpH-C appears to recognize both the amino acyl group on the tRNA and the tRNA acceptor stem, and the enzyme can accept other hydrophobic residues, in addition to leucine. These results contribute to a better understanding of enzyme-aa-tRNA interactions and the growing exploration of aa-tRNA usage beyond translation.

Full text of DOI:10.1021/acschembio.7b00949

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Letter
Investigation of Amide Bond Formation during Dehydrophos Biosynthesis
Emily C. Ulrich, Despina J. Bougioukou, and Wilfred A. van der Donk
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00949 • Publication Date (Web): 05 Jan 2018
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Investigation of Amide Bond Formation during Dehydrophos Biosynthesis
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Emily C. Ulrich1,2, Despina J. Bougioukou2,†, and Wilfred A. van der Donk1,2,3*
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1Department of Chemistry, 2Carl R. Woese Institute for Genomic Biology, 3Howard Hughes
Medical Institute, University of Illinois at UrbanaꢀChampaign, Urbana, Illinois 61801, United
States; †Present address: E. I. du Pont de Nemours and Company, Wilmington, Delaware 19803,
United States
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*Correspondence to: Eꢀmail: vddonk@illinois.edu
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Abstract
Dehydrophos is a tripeptide phosphonate antibiotic produced by Streptomyces luridus. Its
biosynthetic pathway involves the use of aminoacylꢀtRNA (aaꢀtRNA) for amide bond formation.
The first amide bond during biosynthesis is formed by DhpHꢀC, a peptidyltransferase that
utilizes LeuꢀtRNALeu. DhpHꢀC is a member of a burgeoning family of natural product
biosynthetic enzymes that make use of aaꢀtRNA outside of canonical translation activities in the
cell. Here we used siteꢀdirected mutagenesis of both DhpHꢀC and tRNALeu to investigate the
enzyme mechanism and substrate specificity, respectively, and analyzed the substrate scope for
the production of a set of dipeptides. DhpHꢀC appears to recognize both the amino acyl group on
the tRNA and the tRNA acceptor stem, and the enzyme can accept other hydrophobic residues in
addition to leucine. These results contribute to a better understanding of enzymeꢀaaꢀtRNA
interactions and the growing exploration of aaꢀtRNA usage beyond translation.
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Several recent studies have uncovered a role for aminoacylꢀtRNA (aaꢀtRNA) in antibiotic
biosynthesis.1ꢀ5 Biosynthetic enzymes that make use of aaꢀtRNA originate from at least three
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different protein folds to perform amide or ester bond ligations.6,
Examples include
cyclodipeptide synthases (CDPSs) that are structural homologs of aaꢀtRNA synthetases, amide
bondꢀforming ligases that are homologs of FemX peptidyltransferases from cell wall
biosynthesis, and ribosomally synthesized and posttranslationally modified peptide dehydratases
with a new protein fold.2ꢀ4
Enzymes known to interact with aaꢀtRNA share several common features, despite their
strong variations in sequence. Studies of FemX and MprF (multiple peptide resistance factor),
which transfer amino acids from aaꢀtRNA to UDPꢀMurNAc pentapeptide and
phosphatidylglycerol, respectively, have revealed a PheꢀLys motif potentially important for the
binding of the amino acyl group on the tRNA and the tRNA acceptor stem.8, 9 In fact, this Lys
residue appears to be conserved across many of the biochemically validated aaꢀtRNAꢀdependent
biosynthetic enzymes.6 Additionally, several studies have highlighted the importance of the
tRNA acceptor stem for substrate recognition.10 For example, the activities of two unrelated
enzymes, the CDPS AlbC and the lantibiotic dehydratase MibB, are both negatively affected if
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nucleotides at the top of the acceptor stems of their aaꢀtRNA substrates are changed.11, 12 In this
study, we investigated whether these features are important for the function of an aaꢀtRNAꢀ
dependent enzyme involved in phosphonate natural product biosynthesis.
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Currently, the biosyntheses of two characterized phosphonopeptides with antibacterial or
antifungal properties are known to involve aaꢀtRNAꢀdependent enzymes.13, 14 This discovery was
made through a comparison of enzymes encoded in the dehydrophos and fosfazinomycin
biosynthetic gene clusters with FemX peptidyltransferases. We were intrigued by the use of aaꢀ
tRNA as a requirement for amide bond formation, given that previously investigated
phosphonopeptides are produced by other amide bondꢀforming mechanisms. For instance, the
biosynthetic pathway to the herbicide phosphinothricin tripeptide includes two nonꢀribosomal
peptide synthetases, which activate amino acids by adenylation.15ꢀ17 In contrast, ATPꢀgrasp
enzymes activate amino acids by phosphorylation during the production of the antifungal
rhizocticins.18, 19 In dehydrophos biosynthesis, a new mode of amino acid activation was
uncovered for phosphonopeptides with the determination that leucine and glycine are transferred
to the phosphonate warhead using aaꢀtRNA as a substrate (Figure 1a). The Cꢀterminal domain of
DhpH (DhpHꢀC) catalyzes the formation of an amide bond between (R)ꢀ(−)ꢀ1ꢀ
aminoethylphosphonate (LꢀAlaP) and Leu using LeuꢀtRNALeu as substrate (Figure 1a).
Dehydrophos is a ‘Trojan horse’ antibiotic in which the proteinogenic amino acids Gly and Leu
help deliver the phosphonate warhead via peptide permeases.20, 21 Once the tripeptide enters the
cell, internal peptidases release 1ꢀaminovinylphosphonate, which spontaneously converts to
methyl acetylphosphonate, a potent inhibitor of pyruvate dehydrogenase. Thus, the function of
DhpHꢀC is linked to the antibacterial potential of dehydrophos. Here, we investigated the
features of DhpHꢀC and aaꢀtRNA that are necessary for dipeptide formation. We performed siteꢀ
directed mutagenesis on the putative active site of DhpHꢀC to determine residues important for
activity, investigated the ability of DhpHꢀC to recognize other amino acids in addition to Leu,
and analyzed the effects of mutating tRNALeu acceptor stems on product formation. These
findings contribute to the growing understanding of aaꢀtRNA usage by secondary metabolite
enzymes.
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We first investigated which residues of DhpHꢀC may play a role in enzyme activity. A
recent structure of FemX from Weissella viridicens8 coꢀcrystallized with the last three
nucleotides of the tRNAAla acceptor stem and an IꢀTASSER22 model of DhpHꢀC facilitated
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analysis of the putative DhpHꢀC active site (Figure 1bꢀc). Furthermore, previous work on AlbC,
a cyclodipeptide synthase involved in the production of albonoursin, revealed an Argꢀrich basic
patch suitable for interacting with the negatively charged phosphate groups of aaꢀtRNA.11 Thus,
Arg residues inside the potential aaꢀtRNA binding pocket of DhpHꢀC were identified, and siteꢀ
directed mutagenesis to Ala was performed to determine the contribution of each residue to
product formation (Figure 1c). Each Ala variant was then tested in a coupled enzyme assay based
on a previously described method13 using E. coli leucylꢀtRNA synthetase (LeuRS) to generate
aaꢀtRNA in situ from E. coli total tRNA, LꢀLeu, and ATP. Product formation was monitored
over the course of 6 h by 31P NMR spectroscopy (Figure 1d and S1). All Ala mutations
negatively impacted product formation, with the most pronounced decline resulting from
mutated residues Arg233 and Arg290 located in a helix and loop region lining the bottom of the
putative active site, respectively (Figure 1cꢀd and Figure S2). These results imply that the use of
Arg residues to bind aaꢀtRNA may be a shared feature of aaꢀtRNAꢀdependent enzymes
regardless of protein structural fold, considering that the CDPS AlbC is not related by sequence
or structure to DhpHꢀC and other FemXꢀlike enzymes.
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Next, we examined the consequences of mutating three residues of DhpHꢀC that
structurally align with three residues important for FemX activity. In the IꢀTASSER model,
Arg233 is structurally in a similar location as Arg205 in the crystal structure of FemX, which is
proposed to be important for hydrogenꢀbonding to the phosphate group of the final nucleotide at
the 3’ end of the tRNA acceptor stem.8 As discussed above, the R233A mutant had a pronounced
negative effect on product formation over time. Residues Phe328 and Lys329 are structurally
similar to Phe304 and Lys305 of FemX, possibly forming the PheꢀLys motif for tRNA
interaction also seen in MprF.9 The DhpHꢀCꢀF328L mutant was still functional, however product
formation was drastically lowered compared to wild type (Figure 1e). We also removed positive
charge with a K329M mutant, or displaced the positive charge with a K329R mutant. Both
DhpHꢀC K329M and K329R showed no observable activity after 12 h, indicating that the
placement of the charge on Lys329 is critical for enzyme function (Figure S3). Finally, we chose
to investigate the effects of mutating a set of residues (Trp284, Asn325, and Glu326) in the
vicinity of the critical Lys329 residue (Figure 1c). The most striking effect on product formation
came from the W284L and N325A mutants, which provides more evidence that this region of
DhpHꢀC is critical for function (Figure 1e). These results lend support to the proposed pocket of
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DhpHꢀC for substrate interaction, and suggest that the importance of a conserved Lys residue in
other aaꢀtRNAꢀdependent enzymes extends to DhpꢀC.
Our next goal was to determine how DhpHꢀC activity with LeuꢀtRNALeu compares with
other hydrophobic aaꢀtRNA cognate pairs carrying Val, Ile, and Phe. Each amino acid and its
corresponding aminoacylꢀtRNA synthetase (aaRS) were used to substitute LꢀLeu and LeuRS in
the coupled enzyme assay described above for DhpHꢀC activity. When the concentration of each
aaRS was doubled, the reaction rate did not increase, indicating that the production of aaꢀtRNA
was not the rateꢀlimiting step under the assay conditions (Figure S4). Furthermore, the amount of
total E. coli tRNA used, which has different amounts of the tRNAs for various amino acids, was
normalized to produce equal amounts of each aaꢀtRNA substrate at the initiation of each
experiment (Figures S5 and S6). Under these conditions, production of the dipeptides was
observed (Figure S7) and was most efficient with LeuꢀtRNALeu as the substrate (Figure 2 and
S8). When IleꢀtRNAIle, ValꢀtRNAVal, or PheꢀtRNAPhe was used, both the rate and extent of
product formation decreased. Interestingly, similar rates and yields were observed for each of
these alternative substrates. Because of the experimental setꢀup that regenerates the aaꢀtRNA, we
cannot identify if the observed change is an effect on kcat or KM, but the overall strong decrease
of activity is clear. To distinguish whether the effect on product formation was a result of the
change in amino acid or the change in the tRNA sequence delivering the amino acid, we set up
an assay with IleꢀtRNALeu as a substrate. In other words, using the tRNA for the cognate amino
acid, but with the nonꢀcognate amino acid Ile attached. The mutant enzyme LeuRSꢀ
Y330A/D342A/D345A (LeuRS*m) has been shown previously to misacylate tRNALeu with Ile.23
When IleꢀtRNALeu was the substrate for DhpHꢀC, similar levels of product formation were
observed to the IleꢀtRNAIle reaction, with both being significantly slower than the reaction
containing LeuꢀtRNALeu (Figure 2). Thus, at minimum the identity of the amino acid is important
for product formation by DhpHꢀC.
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We investigated whether DhpHꢀC identifies recognition elements on its tRNALeu
substrates using a different strategy. For this series of experiments, we kept Leu as the amino
acid and determined whether DhpHꢀC has a preference for select isoacceptors of E. coli
tRNALeu. Isoacceptor tRNAs were obtained by in vitro transcription and will be named herein
based on their anticodon sequence. Previous studies on the CDPS AlbC showed that the enzyme
prefers tRNA substrates with a G1ꢀC72 base pair match in the acceptor stem.11 To investigate
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whether DhpHꢀC shares this feature, the tRNALeu (UAG) and tRNALeu (GAG) sequences were
used as substrates, since their acceptor stems contain a GꢀC match and a GꢀU mismatch,
respectively (Figure 3a). We observed that DhpHꢀC does display a clear preference, since the
reaction was complete with nearly 100% product formation in about 1 h with the tRNALeu (UAG)
isoacceptor, but less than 20% product was formed in 6 h with the tRNALeu (GAG) isoacceptor
(Figure 3b). Importantly, generation of the tRNALeu (GAG) U72C isoacceptor mutant to
introduce the G1ꢀC72 base pair match resulted in the rescue of activity back to levels
commensurate with the tRNALeu (UAG) substrate (Figure 3b). Such a drastic change in product
formation levels with the mutation of just one nucleotide corroborates the hypothesis that the
acceptor stem is important for recognition of the tRNA substrate,11, 12, 24 and that the antiꢀcodon
is not likely to be involved in substrate recognition. Interestingly, DhpHꢀC and AlbC are not
related by structure or sequence, yet they have converged towards similar modes of tRNA
substrate recognition.
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Finally, to further probe the G1ꢀC72 recognition element of DhpHꢀC, we compared a
series of variant tRNALeu (GAG) substrates: U72C (GC pair), G1A (AU pair), and G1AꢀU72C
(AC pair). FemX has been shown to require C72 for substantial product formation, with a G1ꢀ
C72 match being most effective.24, 25 For DhpHꢀC, none of the additional variants showed
activity equal to the GAG (GC) substrate (Figure 3b). Thus, the presence of the G1ꢀC72 base
pair match on the acceptor stem is important for DhpHꢀC activity. It is possible that the enzyme
makes specific contacts with G1 and C72, or that the strong base pair helps position the acceptor
stem for enzyme interaction with the amino acid.
In conclusion, we have investigated the molecular basis of DhpHꢀC interaction with its
aaꢀtRNA substrate by determining key residues important for enzyme activity and essential
nucleotides of the tRNA acceptor stem that aid in enzyme recognition. Our substrate scope
analysis with hydrophobic amino acids revealed that DhpHꢀC can produce multiple dipeptide
phosphonates. This tolerance could potentially be leveraged to generate new phosphonate
analogs chemoenzymatically. The strong preference displayed for Leu may explain why thus far
no congeners of dehydrophos have been observed to be produced by S. luridus. Our results also
highlight the similarities among aaꢀtRNAꢀdependent enzymes in natural product biosynthesis,
despite the three distinct structural folds. Like the CDPS AlbC and dehydratases involved in
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modifying ribosomal peptide natural products,11, 12 DhpHꢀC recognizes nucleotides in the tRNA
acceptor stem.
Methods
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Activity Assays by 31P NMR Spectroscopy
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NMR experiments were performed on an Agilent 600 MHz spectrometer equipped with an
OneNMR probe. 31P spectra were recorded in 20% D2O at room temperature. A typical reaction
contained 50 mM HEPES pH 7.5, 150 mM KCl, 15 mM MgCl2, 6 mM ATP, 6 mM LꢀLeu, 5
mM LꢀAlaP, 8 µM LeuRS, 10 µM DhpHꢀC WT and variants, 0.01 U/µL TIPP, and 3 mg/mL
total E. coli tRNA (Sigma Aldrich/Roche) or 16 µM in vitro transcribed tRNALeu [8 µM for
GAG (AU)] in 500 µL. Based on aminoacylation studies, the amount of total tRNA in each aaRS
reaction was increased as follows: IleRS (1.7x), ValRS (1.3x), PheRS (3.2x), and LeuRSm*
(1.8x). Reactions with various synthetases (8 µM) were incubated at room temperature with
components needed for aminoacylation before the addition of 35 µM DhpHꢀC WT (10 µM for
the LeuRS sample) and 5 mM LꢀAlaP was added to initiate the amideꢀforming reaction after 15
min (1 h for LeuRS*m). Product formation was followed by collecting spectra every 4 min for 6
h. Spectra were collected for 2 h for the in vitro transcribed tRNALeu (UAG) since the reaction
had gone to completion within that amount of time. Spectra were analyzed using MestReNova
version 8.0.0ꢀ10524 software. Percent product formation was calculated by integration of peak
area. Stages of the reactions in which less than ~10% of the product was formed or substrate
remained were difficult to analyze because of the lowꢀsignalꢀtoꢀnoise. As a result, data for
experiments that ran to completion were not plotted after ~85ꢀ90% product formation. Replicate
endpoint assays were quenched by treatment with Chelex® (Sigma Aldrich, 20 min of shaking at
room temperature) and filtration through a 10 kDa molecular weight cut off Amicon filter (EMD
Millipore). D2O (100 µL) was added before analysis. Results were plotted using Igor Pro version
6.32A.
Supporting Information
The supporting information is available free of charge via the internet at http://pubs.acs.org.
Included in the supporting information are materials used in this study, additional experimental
procedures, and supporting figures.
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Acknowledgements
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Funding was provided by National Institutes of Health Grant P01 GM077596 (to W.A.v.d.D.).
NMR spectra were collected on a 600 MHz NMR spectrometer that was funded by NIH grant
number S10ꢀRR028833 located in the Carl R. Woese Institute for Genomic Biology Core
Facility. We thank Z. Li of the Metabolomics Center, Roy J. Carver Biotechnology Center at the
University of Illinois at UrbanaꢀChampaign for performing the LCMS analysis and K. A. Wang
for preliminary LCMS testing. We thank S. Martinis (UIUC) for providing the plasmid for
LeuRS and LeuRS Y330A/D342A/D345A, Z. Huang for preparation of pETꢀ15b valRS, and M.
Ortega for preparation of T7 RNA polymerase. In addition, we thank M. Saks and A. Banerjee
for helpful discussions and I. Bothwell and K. A. Wang for critically reviewing the manuscript.
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Figure 1. a) The DhpHꢀC reaction and the final structure of dehydrophos. b) Crystal structure of
FemX in complex with the CCA portion of the tRNA acceptor stem and a triazole ring to mimic
the placement of aaꢀtRNA (PDB 4II9).8 Highlighted residues are predicted to be important for
interaction with the tRNA acceptor stem and the amino acid. c) Overlay of FemX (tan) with an Iꢀ
TASSER model of DhpHꢀC (cyan).8, 22 Highlighted residues of DhpHꢀC were targeted for siteꢀ
directed mutagenesis. d-e) Percent product formed plotted against time of the DhpHꢀC reaction
as measured by 31P NMR spectroscopy.
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Figure 2. Percent product formation as measured by 31P NMR spectroscopy plotted against
reaction time for the DhpHꢀC coupled enzyme reaction with various amino acid/aaRS pairs. All
reactions were conducted with total tRNA from E. coli.
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Figure 3. a) Sequences of E. coli tRNALeu acceptor stems named by the sequence of their
anticodon. Mutations are shown in red. b) Percent product formation over time of DhpHꢀC WT
with the various in vitro transcribed tRNALeu variants.
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TOC Graphical Abstract
72x40mm (300 x 300 DPI)
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