Nine enzymes are required for assembly of the pacidamycin group of peptidyl nucleoside antibiotics

Article abstract of DOI:10.1021/ja2011109

Pacidamycins are a family of uridyl peptide antibiotics that inhibit the translocase MraY, an essential enzyme in bacterial cell wall biosynthesis that to date has not been clinically targeted. The pacidamycin structural skeleton contains a doubly inverted peptidyl chain with a β-peptide and a ureido linkage as well as a 3′-deoxyuridine nucleoside attached to DABA ₃ of the peptidyl chain via an enamide linkage. Although the biosynthetic gene cluster for pacidamycins was identified recently, the assembly line of this group of peptidyl nucleoside antibiotics remained poorly understood because of the highly dissociated nature of the encoded nonribosomal peptide synthetase (NRPS) domains and modules. This work has identified a minimum set of enzymes needed for generation of the pacidamycin scaffold from amino acid and nucleoside monomers, highlighting a freestanding thiolation (T) domain (PacH) as a key carrier component in the peptidyl chain assembly as well as a freestanding condensation (C) domain (PacI) catalyzing the release of the assembled peptide by a nucleoside moiety. On the basis of the substrate promiscuity of this enzymatic assembly line, several pacidamycin analogues were produced using in vitro total biosynthesis.

Full text of DOI:10.1021/ja2011109

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Nine Enzymes Are Required for Assembly of the Pacidamycin Group of
Peptidyl Nucleoside Antibiotics
Wenjun Zhang,†,‡ Ioanna Ntai,§ Megan L. Bolla,|| Steven J. Malcolmson,† Daniel Kahne,|| Neil L. Kelleher,§
and Christopher T. Walsh*,†
†Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,
United States
‡Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720, United States
§Departments of Chemistry and Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
S Supporting Information
b
(Ala/m-Tyr2), a bicyclic heterocycle, or a dipeptide (Ala/Gly1-m-
ABSTRACT: Pacidamycins are a family of uridyl peptide
antibiotics that inhibit the translocase MraY, an essential
enzyme in bacterial cell wall biosynthesis that to date has
not been clinically targeted. The pacidamycin structural
skeleton contains a doubly inverted peptidyl chain with a β-
peptide and a ureido linkage as well as a 30-deoxyuridine
nucleoside attached to DABA3 of the peptidyl chain via an
enamide linkage. Although the biosynthetic gene cluster for
pacidamycins was identified recently, the assembly line of
this group of peptidyl nucleoside antibiotics remained poorly
understood because of the highly dissociated nature of the
encoded nonribosomal peptide synthetase (NRPS) do-
mains and modules. This work has identified a minimum
set of enzymes needed for generation of the pacidamycin
scaffold from amino acid and nucleoside monomers, high-
lighting a freestanding thiolation (T) domain (PacH) as a
key carrier component in the peptidyl chain assembly as well
as a freestanding condensation (C) domain (PacI) catalyz-
ing the release of the assembled peptide by a nucleoside
moiety. On the basis of the substrate promiscuity of this
enzymatic assembly line, several pacidamycin analogues
were produced using in vitro total biosynthesis.
Tyr2) is linked to the β-amino group of DABA at the N-terminus.
Thus, in the pacidamycin framework, the tetra/pentapeptidyl
chain reverses direction twice, at Ala/m-Tyr2-DABA3 and at
Ala4-Phe/Trp/m-Tyr5 via a β-peptide and a ureido linkage,
respectively (Figure 1). All of these nonstandard connectivities
suggest novel chemical logic and enzymatic machinery for
assembly of the pacidamycin scaffold.
The biosynthetic gene cluster for pacidamycins was reported
recently,4,5 allowing for the heterologous production of pacida-
mycin D/S, the uridyl tetrapeptides containing a single N-term-
inal Ala tethered to the β-amino group of DABA (Figure 1).
Highly dissociated nonribosomal peptide synthetase (NRPS)
modules were encoded, presenting a challenge in dissecting the
peptidyl core assembly (Figure S1 in the Supporting Information).
We characterized the substrate specificities of several encoded
adenylation (A) domains (PacLOP) and reconstituted the for-
mation of the C-terminal ureido dipeptide (Ala4-Phe/Trp/m-
Tyr5) using PacJLNO.5 In this work, the functions of six addi-
tional proteins (PacDHIUVW) have been defined by in vitro
characterization, and a picture of the complete assembly line for
generation of the unusual uridyl tetrapeptide scaffold has been
provided.
PacP has previously demonstrated a strong preference for the
activation of L-2,3-diaminopropionate (DAP), indicating that it
would be the DABA activation enzyme.5 Therefore, (2S,3S)- and
(2R,3R)-DABA were chemically synthesized using the published
methods6 (Figure S2) and tested in the ATPꢀ[32P]PPi exchange
assay. As expected, PacP exhibited a preference for reversible
formation of (2S,3S)-DABA-AMP; no substantial activation of
(2R,3R)-DABA was detected, validating the stereoselectivity of
PacP (Figure 2a). PacV, a standalone methyltransferase encoded
by the gene cluster, was predicted to catalyze the N-methylation
of DABA. The purified PacV indeed transferred the methyl group
from S-adenosylmethionine (SAM) to holo-PacP (∼90 kD), as
demonstrated by SDS-PAGE autoradiography, only after DABA
had been activated by the A domain of PacP and presumably
loaded onto the in cis thiolation (T) domain (Figure 2b). No
methylation of free DABA/DAP catalyzed by PacV could be
acidamycins are members of a large class of uridyl peptide
P
antibiotics that also includes mureidomycins, napsamycins,
and sansanmycins.1,2 They are sufficient structural mimics of the
UDP-N-acetylmuramoyl pentapeptide intermediate in the bac-
terial cell wall assembly to inhibit the translocase MraY, a
clinically unexploited target in the development of new anti-
bacterial drugs.3 Elucidation of the enzymatic mechanism for the
pacidamycin scaffold assembly will therefore facilitate the gen-
eration of new MraY-targeted peptidyl nucleoside antibiotics
through combinatorial biosynthesis.
More than 10 related pacidamycin family compounds have
been isolated from Streptomyces coeruleorubidus,1 all of which
share a common structural skeleton having a (2S,3S)-diamino-
butyric acid (DABA) residue serving as a connection point for
the 30-deoxyuridine moiety via a 40,50-enamide linkage. A ureido
dipeptide (Ala4-Phe/Trp/m-Tyr5) is attached to the R-amino
group of DABA at the C-terminus, and a single amino acid
Received: February 4, 2011
Published: March 18, 2011
r
2011 American Chemical Society
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Figure 2. Characterization of NRPSs. (a) A-domain activities of PacP,
PacU, and PacW. Abbreviations: NE, no enzyme; DABA, diaminobu-
tyric acid; DAP, diaminopropionate; m-Tyr, m-Tyrosine (DL). A relative
activity of 100% for L-2,3-DAP-, L-Ala-, and m-Tyr-dependent exchange
corresponds to 282k, 70k, and 141k cpm, respectively. (b) Autoradio-
graph of one SDS-PAGE gel illustrating the covalent loading of 14C-
labeled substrate (highlighted in red). Enzymes and substrates used in
each lane are indicated in the table (highlighted in gray).
Figure 1. Structures of selected pacidamycins and proposed biosyn-
thetic scheme for pacidamycin S. Domain notation: T, thiolation; A,
adenylation; C, condensation; TE, thioesterase.
detected by liquid chromatographyꢀmass spectrometry (LCꢀMS),
and the preincubation of DABA/DAP with PacP significantly
increased the initial rate of methylation (Figure S3), indicating
that PacP-tethered DABA could be the natural substrate for the
SAM-dependent methyltransferase PacV to install a methyl
moiety on the β-amino group of DABA.
well with the in vivo PacU disruption result. L-[14C]Ala was
further used in the loading assays to trace the covalent aminoacyl-
S-thiolation intermediate. Radioactivity migrated with PacH but
not PacP and was completely dependent on the presence of PacU
and DABA (Figure 2b). These results suggested that Ala was
condensed to DABA-S-PacH but not DABA-S-PacP after activa-
tion as Ala-AMP by PacU. The radioactivity signal remained the
same in the absence of methyltransferase PacV and SAM, indicat-
ing that N-methylation of DABA is not a prerequisite for the
aminoacylation of DABA-S-PacH by Ala. Indeed, the formation of
Ala2-DABA3-S-PacH was confirmed by FTMS (Figure 3b). PacV
and SAM were then excluded in the following assays to simplify the
in vitro reconstitution system. It is notable that we also identified a
digene cassette (designated pacWX) on the genome of S. coeruleor-
ubidus(GenBank accession no. HQ874646) encoding a standalone
A-domain protein and a phenylalanine hydroxylase. PacW shows
high sequence similarity to PacU (87% similarity) and upon
purification demonstrated a strong preference for the activation
of m-Tyr over other aromatic amino acids (Figure 2a). PacW is
therefore hypothesized to be involved in the activation of N-term-
inal m-Tyr found in pacidamycin 4/5/5T (Figure 1).
Upon the addition of a freestanding holo-T protein (PacH,
∼10 kD), the radioactive [14C]CH3 derived from SAM was
transferred from PacP to PacH (Figure 2b). The PacH homo-
logue from Streptomyces roseosporus (88% similarity), which was
also purified in holo form, exhibited higher loading efficiency and
was used throughout subsequent studies. Transfer of aminoacyl
groups between T domains to facilitate subsequent tailoring has
been observed previously, promoted by an acyltransferase of the
R,β-hydrolase family, such as CmaE in coronamic acid biosyn-
thesis.7 However, the thioesterase (TE) domain of PacP lacks the
typical catalytic triad of this family, and no obvious in trans
acyltransferase candidate could be found encoded in the gene
cluster; the aminoacyl moiety may be shuttled between the HS-
pantetheinyl prosthetic groups of PacP and PacH by sponta-
neous transthiolation. In addition, transfer of the DABA moiety
was not dependent on the N-methylation, as DABA-S-PacH
could be formed and detected by Fourier transform MS (FTMS)
(Figure 3a).
We then probed the mechanism for attachment of the N-term-
inal Ala to the β-amino group of thioester-tethered DABA in
pacidamycin D/S biosynthesis. PacU, a standalone A-domain
protein, was indicated to be specifically related to the activation
of N-terminal Ala by targeted gene disruption, although it failed
to activate L-Ala in vitro in our initial trials.5 PacU was later
repurified from Escherichia coli and exhibited a strong preference
toward reversible formation of L-Ala-AMP (Figure 2a), correlating
The enzymatic mechanism and timing of the incorporation of
the ureido dipeptide onto the R-amino group of DABA was also
investigated using SDS-PAGE autoradiography and FTMS. Our
prior work showed that the ureido dipeptide (Ala4-Phe/Trp/m-
Tyr5) could be formed and tethered on the pantetheinyl arm of
PacN through the activity of PacJLNO.5 When PacN-S-Ala4-
CO-[14C]Phe5 and DABA-S-PacH were combined, 14C-labeled
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Figure 4. Extracted ion chromatograms showing production of paci-
damycin analogues using 50-aminouridine as an offloading substrate.
The calculated mass with 10 ppm mass error tolerance was used. The
assay components are shown in Table S2; the high-resolution MS (HR-
MS) and HR-MS/MS analyses of each compound are shown in Figures
S6ꢀS8. The MS/MS fragments confirmed the attachment of the uridine
moiety to DABA3 carbonyl through an amide linkage as well as the
regiospecific modifications of the DABA3 amino group (NR vs Nβ).
Figure 3. Detection of PacH-bound biosynthetic intermediates by
FTMS after trypsin digestion. ATP, L-Phe, L-Ala, DABA, and PacPH
were present in all assays. PacU was present in assays (b) and (d), while
PacDJLNO were present in assays (c) and (d). Theoretical mass
calculations are shown in Figure S4.
L-Phe was transferred from PacN (∼63 kD) to PacH only in the
presence of a standalone condensation (C)-domain protein
PacD (Figure 2b). The PacH-S-DABA3-Ala4-CO-Phe5 species
was further detected by FTMS (Figure 3c), confirming that PacD
catalyzes the nucleophilic attack of the R-amino group of DABA
on the PacN-tethered thioester of the C-terminal ureido dipep-
tide. No radioactive L-Phe was transferred to DABA-S-PacP,
suggesting that PacH, not PacP, was one of the cognate loading
domains for PacD recognition. It could also be deduced that the
aminoacylation of the R- and β-amino groups of DABA could
take place independently.
The tetrapeptidyl thioester of PacH (Figure 3d) was stable
under incubation conditions, presumably awaiting offloading to
the 50-amino group of a deoxyuridine species to generate pacid-
amycins. PacI, the remaining standalone C-domain protein (∼47
kD), was anticipated to release the tetrapeptidyl chain from the
thioester linkage on PacH by a uridine derivative. Because 30-
deoxy-40,50-enaminouridine was not available, 50-aminouridine
was chemically synthesized8 and tested as a surrogate substrate in
the in vitro assays to probe the function of PacI. In fact, PacI
catalyzed the offloading of the tetrapeptidyl chain from PacH by
50-aminouridine via an amide linkage (Figure 4 and Figures
S6ꢀS8 and S19). PacI shows low sequence similarity to any
other known proteins and is the first identified C-domain protein
shown to condense peptide and nucleoside substrates. In addi-
tion, PacI could also utilize uridine and 30-deoxyuridine as alter-
native substrates, forming an oxoester rather than the natural
amide linkage (Figure 5 and Figures S9ꢀS18). The relaxed
substrate specificities of PacI together with other catalytic
domains in the assembly line could facilitate the enzymatic
synthesis of pacidamycin analogues. For example, nine analo-
gues (1ꢀ2, 5ꢀ8, and 10ꢀ12) varied at the C-terminal aro-
matic amino acid, central diamino acid, and uridine moieties
were generated in vitro using the nine proteins PacDHIJLNO-
PU (Figures 4 and 5). In addition, when PacU was replaced by
PacW, only products with N-terminal m-Tyr (3ꢀ4, 9) were
Figure 5. Extracted ion chromatograms showing the production of
pacidamycin ester analogues. The calculated mass with a 10 ppm mass
error tolerance was used. The assay components are shown in Table S2;
the HR-MS and HR-MS/MS analyses of each compound are shown in
Figures S9ꢀS18.
formed (Figure 4 and 5), confirming the orthogonal functions
of PacU and PacW in the activation of N-terminal Ala and m-
Tyr, respectively.
In summary, we have dissected the distributed 10-protein
assembly line that builds the scaffold of tetrapeptidyl nucleoside
pacidamycin antibiotics. The trifunctional (2S,3S)-DABA is the
central building block, and it is activated by and loaded onto
PacP, methylated by PacV, and transferred to PacH. The tetra-
peptide framework is then assembled on PacH, perhaps reflect-
ing the participation of that 10 kD T-domain protein scaffold in
recognition by the downstream enzymes. The assembly includes
attachment of the N-terminal Ala (activated by PacU) or m-Tyr
(activated by PacW) to the β-amino group of DABA and attach-
ment of the C-terminal ureido dipeptide (formed by PacJLNO)
to the R-amino group of DABA promoted by PacD. Finally, PacI
catalyzes the release of the tetrapeptidyl intermediate from PacH
by uridines (Figure 1 and Figure S20). Our work provides the
basis for rational reprogramming of various uridyl peptide anti-
biotic assembly lines, including pacidamycins, mureidomycins,
and napsamycins, as the recently identified biosynthetic gene
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cluster for napsamycins encodes the highly homologous discrete
NRPSs.9 We will next scale up the in vitro total biosynthesis of
various pacidamycin analogues to obtain material to test for
antibiotic activities and evaluate structureꢀactivity relationships.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures, SDS-
b
PAGE analysis of purified proteins, synthesis of DABA and amino-
uridine, methylation time course, MS calculation of PacH-bound
biosynthetic intermediates, compound characterizations, and bio-
synthetic pathway schemes. This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
Christopher_walsh@hms.harvard.edu
’ ACKNOWLEDGMENT
This work was supported by NIH Grants GM49338 (C.T.W.),
GM067725-08 (N.L.K.) and GM066174 (D.K.).
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