On the substrate specificity of dehydration by lacticin 481 synthetase

Article abstract of DOI:10.1021/ja067672v

Dehydroamino acids are valuable building blocks that are a challenge to incorporate synthetically into unprotected peptides. Lantibiotic synthetases possess dehydration activity that converts Ser and Thr residues in their peptide substrates into dehydroal

Full text of DOI:10.1021/ja067672v

Published on Web 02/01/2007
On the Substrate Specificity of Dehydration by Lacticin 481 Synthetase
Xingang Zhang and Wilfred A. van der Donk*
Roger Adams Laboratory, Department of Chemistry, UniVersity of Illinois at UrbanasChampaign,
600 South Mathews AVenue, Urbana, Illinois 61801
Received November 2, 2006; E-mail: vddonk@uiuc.edu
Dehydroamino acids are found in many cyclic peptide natural
products,1 and they enhance proteolytic stability and biological
activity of linear peptides.2 Furthermore, dehydroamino acids can
be used as precursors to peptide conjugates3 and unnatural amino
acids.4-6 Incorporation of dehydroamino acids into unprotected
peptides using synthetic methods is currently challenging.7 One
promising route to these structures is through the biosynthetic
machinery for lantibiotics. These compounds are ribosomally
synthesized and post-translationally modified antimicrobial pep-
tides.5 The first step in the modification process involves the
dehydration of Ser and Thr residues to dehydroalanines and
Z-dehydrobutyrines, respectively. This reaction was recently re-
constituted in vitro for lacticin 481 synthetase LctM (Figure 1) and
the haloduracin synthetases HalM.8,9 In this study, we report on
the substrate selectivity of the enzymatic dehydration reaction by
LctM using various Thr analogues, showing that several dehy-
droamino acids are readily accessible using this approach.
Several studies have recently shown that the lantibiotic dehy-
dratases can process non-lantibiotic substrates as long as they are
attached at the C-terminus of a leader peptide (Figure 1).10-12 During
lantibiotic biosynthesis, this leader peptide is removed by a protease
after completion of the post-translational modifications. The
specificity of the dehydratases with respect to the structure of the
hydroxyl-bearing amino acid has not been investigated to date. To
evaluate this question, we prepared a series of potential substrate
analogues (Scheme 1). These structures were all prepared suitably
protected for Fmoc-based solid-phase peptide synthesis (SPPS).13
The key step in the synthesis of the Thr analogues 1-8 involved
the addition of organozinc or organocopper reagents to D-Garner
aldehyde,14 which took place with excellent diastereoselectivities
and without affecting the existing stereocenter as shown by chiral
SFC analysis.15
Using SPPS, the protected amino acids were then incorporated
into the synthetic heptapeptides CysAsnMetAsnXxxTrpAla corre-
sponding to residues 38-44 of the LctA substrate for LctM. The
amino acids 1-9 replaced a Ser in these peptides that is usually
present at position 42 of LctA (Figure 1). The synthetic peptides
were then ligated to a truncated LctA peptide corresponding to
residues 1-37 containing a thioester at its C-terminus. This peptide
was obtained by expression in Escherichia coli fused at the
C-terminus to an intein-chitin binding domain, subsequent puri-
fication using affinity chromatography, and elution with the sodium
salt of mercaptoethanesulfonic acid (MES).16-18 The resulting MES
thioester was then ligated with the synthetic heptapeptides using
native chemical ligation.19,20
Figure 1. Dehydration of LctA catalyzed by lacticin 481 synthetase (LctM).
The truncated substrate used in this work is boxed.
Scheme 1
was first incorporated at position 42 of the truncated LctA peptide
(LctA1-43-S42T). Clean conversion to a product with a mass
decreased by 54 Da (3 H2O) with respect to the substrate was
observed, resulting from dehydration of Thr33, Ser35, and Thr42
(see Figure S1 in the Supporting Information). Substitution of Ser42
with (R)-3-ethylserine also led to clean 3-fold dehydration (Figure
2A), demonstrating that LctM can tolerate an ethyl group and
thereby install dehydronorvalines into peptides. However, both
propyl and isopropyl groups proved too large for the enzyme as
the LctA analogues with (R)-3-propylserine (2) and (R)-3-isopro-
pylserine (3) at position 42 resulted in just two dehydrations after
incubation with LctM (Figures S2 and S3). Treatment of the 2-fold
dehydrated product with cyanogen bromide, which cleaves the
peptide after Met1 and Met40, and subsequent analysis by MALDI-
MS conclusively showed that analogues 2 and 3 at position 42 were
not dehydrated (Figures S6 and S7). On the other hand, a three-
carbon substituent that is less sterically demanding, such as the
propynyl group in 6, was accepted by the enzyme (Figure 2B) as
were the vinyl and ethynyl analogues 4 and 5 (e.g., Figure 2C for
ethynyl). Upon dehydration, these substrates result in interesting
R,â-γ,δ-unsaturated amino acids with extended conjugation.
After HPLC purification, the LctA analogues that correspond to
LctA1-43 were incubated with LctM in the presence of ATP and
Mg2+. Previous studies have shown that the enzyme uses ATP for
phosphorylation of the hydroxyl groups of Ser and Thr that undergo
elimination.18 The assays with the LctA analogues were then
analyzed by MALDI-TOF mass spectrometry. As a control, Thr
9
2212
J. AM. CHEM. SOC. 2007, 129, 2212-2213
10.1021/ja067672v CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Acknowledgment. This work was supported by the National
Institutes of Health (GM58822). We thank S.E. Denmark for use
of the SFC equipment.
Supporting Information Available: Synthetic routes and experi-
mental procedures for all transformations that produced previously
unknown compounds as well as their full spectral characterization. This
material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 2. MALDI-MS spectra of assays in which analogues of a truncated
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In addition to evaluating the tolerance to variation of the methyl
group of Thr, the importance of its stereochemistry was investigated.
Dehydration of Thr results in Z-dehydrobutyrine in all lantibiotics
investigated to date, indicating an anti elimination mechanism.5
Substitution of Ser42 with allo-Thr, if tolerated by the enzyme,
would result in formation of an E-dehydrobutyrine. In the event, a
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