Download Initiation, elongation, and termination strategies in polyketide and

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Initiation, elongation, and termination strategies in polyketide
and polypeptide antibiotic biosynthesis
Thomas A Keating and Christopher T Walsh*
Progress in sequence analysis of biosynthetic gene clusters
encoding polyketides and nonribosomal peptides and in the
reconstitution of in vitro activities continues to reveal new
insights into the growth of these natural products’ acyl chains,
which have been revealed as a series of elongating, covalent,
acyl enzyme intermediates on their multimodular scaffolds.
Studies that focus on the three stages of natural product
biosynthesis – initiation, elongation, and termination – have
yielded crucial information on monomer substrate specificity,
domain and module portability, and product release
mechanisms, all of which are important not only for an
understanding of this exquisite enzymatic machinery, but also
for the rational construction of new, functional synthetases and
synthases that are a goal of combinatorial biosynthesis.
Addresses
Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115,
USA
*e-mail: [email protected]
Current Opinion in Chemical Biology 1999, 3:598–606
1367-5931/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
A
adenylation
ACP
acyl carrier protein
ArCP
aryl carrier protein
AT
acyl transferase
C
condensation
Cy
cyclization
DHB-Ser N-(2,3-dihydroxybenzoate)-L-serine
E
epimerization
KR
ketoreductase
KS
ketosynthase
NRPS
nonribosomal peptide synthetase
PCP
peptidyl carrier protein
PKS
polyketide synthase
P-pant
phosphopantetheine
PPTase
phosphopantetheinyl transferase
TE
thioesterase
xCP
unspecified carrier protein
Introduction
The polyketide synthases (PKSs) [1,2] and the nonribosomal peptide synthetases (NRPSs) [3,4] are responsible for
the biosynthesis of some of the most important therapeutic natural products in use today, including vancomycin,
erythromycin, penicillin, and bacitracin, among many others. The type I PKSs and the NRPSs are large,
multifunctional proteins or protein complexes that employ
a common biosynthetic strategy to yield their products.
(The type II PKSs, not discussed here, differ in that each
catalytic site is located on a separate protein.) Both the
synthases and the synthetases can be visualized as ‘assembly line’ enzymes: the monomers that make up the final
product are selected from the cellular pool, are covalently
tethered to specific locations on the enzyme, and are then
condensed and modified in a linear, stepwise fashion. The
completed chain, having incorporated the last monomer
tethered near the carboxyl terminus of the enzyme, is then
released, by hydrolysis or cyclization, according to the specific product. The primary distinction between the PKS
and NRPS systems lies in their choice of monomers: the
PKSs select activated fatty acids (acyl-Coenzyme A
thioesters), while the NRPSs employ ATP to activate
amino acids as AMP–esters in situ.
In keeping with the assembly line analogy for PKS and
NRPS systems, the synthases and synthetases are made up
of ‘modules’, which comprise the minimum functions of
monomer attachment, monomer loading, and coupling. It
is the iterative cycle of module function, then, that constitutes the synthase/synthetase activity. Each module can be
further devolved into individual ‘domains’, each of which
has a single function, alluded to above (carrier, loading, or
condensation). Domain and module organization is illustrated schematically in Figure 1.
The organization of functional domains into modules and
of modules into synthases/synthetases serves as a template
for monomer loading and condensation [5]. The amino
acid sequence of functionally similar domains and modules
are homologous, and thus, familiarity with the synthetase
building blocks of modules and domains permits the prediction of the ‘assembly line’ organization from the
structure of a known natural product and vice versa [6]. It is
also this modular organization that has inspired efforts to
attempt rational construction of unnatural synthases that
biosynthesize novel compounds [7]. The organization of
the PKS and NRPS systems dictates our discussion below,
which is divided into the three functional stages of synthase/synthetase activity: initiation (Figure 1), elongation
(Figure 2), and termination (Figure 3).
Before beginning, we issue a brief comment about the posttranslational modification, or ‘priming’, of the PKS and
NRPS enzymes. Every carrier protein domain (ArCP, PCP,
ACP: aryl, peptidyl and acyl carrier proteins, respectively)
must be converted from an inactive apo form to an active
holo form by covalent attachment of a Coenzyme-A-derived
phosphopantetheine (P-pant) group to a specific serine
sidechain found in every type of carrier protein (xCP) [8].
This modification, which is required only once (holo
enzymes are competent for multiple turnovers of natural
product synthesis), is catalyzed by a member of the phosphopantetheinyl transferase (PPTase) enzyme family [9].
The PPTases, of which there are more than 20 (as identified
by homology searches), can be divided into two subtypes:
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Figure 1
Core NRPS domains
(a)
Core PKS domains
PCP
C
A
HO
HO
HO
HO
ACP
PCP
Domain
KS
ACP
AT
Domain
Module
Module
Coenzyme A
Coenzyme A
PPTase
PPTase
3', 5'-ADP
3', 5'-ADP
(b)
O
O
HO
H
N
SH
O
H
N
O
O
O P O–
HO
O
PCP
C
A
H
N
SH
H
N
O
O
O
O P O–
HO
H
N
SH
O
H
N
O
O
O P O–
O
O
PCP
ACP
HO
H
N
SH
H
N
O
O P O–
O
KS
AT
ACP
Current Opinion in Chemical Biology
Priming. The core set of elongation domains is shown. Each core can
complete one iteration of chain extension when the appropriate
monomers are present. (a) Apo proteins. Each PCP and ACP has a
conserved serine residue (represented by the hydroxyl group, OH) that
serves as the location of a phosphopantetheine post-translational
modification. Apo proteins are unable to participate in chain elongation.
(b) Holo proteins. The apo proteins are post-translationally modified
with a phosphopantetheine prosthetic arm (derived from Coenzyme A)
that is attached by a phosphopantetheinyl transferase (PPTase). The
holo proteins are ‘primed’ and thus competent for chain elongation
after monomers have been covalently attached to a
phosphopantetheine tether through a thioester bond (not shown).
those that modify the ACPs of fatty acid synthases and
PKSs, and those that modify the PCPs of NRPSs. P-pant
attachment supplies the 20 Å long flexible ‘arm’ that terminates in a nucleophilic thiol (Figure 1), which serves as the
point of monomer attachment by thioester linkage. In this
review, we will elaborate on the three stages of polyketide
and polypeptide biosynthesis, which are reflected in both
the structure and the function of the synthases and synthetases. We shall begin, as the enzymes do, with a loading
unit that selects and covalently loads the first monomer (initiation), proceed through a variable number of modules that
add subsequent monomers to the growing natural product
(elongation), and end with the release of the mature product
by a thioesterase (termination).
specifically activates free L-phenylalanine as an AMP-ester
at the adenylation (A) domain, covalently attaches the
amino acid to the thiol group of the P-pant prosthetic arm
located on the PCP domain, and finally epimerizes the substrate to produce the D-phenylalanine thioester, an action
catalyzed by the epimerization (E) domain. The PCPloaded D-phenylalanine is then prepared to serve as donor
for the next module in the assembly line.
Initiation: activation and/or loading of starter
acyl groups
Natural product biosynthesis by type I PKSs and by NRPSs
proceeds in a linear, stepwise fashion that begins with a
loading unit that resides at the amino terminus of a synthetase. As shown in Figure 2, the specific domains differ in
NRPS and PKS systems. GrsA, the first protein of the
gramicidin S synthetase complex, is an archetypal NRPS
starting unit [10]. Also referred to as ‘PheATE’ (phenylalanine adenylation-thioesterification-epimerization), GrsA
One might expect a 1:1 stoichiometry between amino acid
loading and ATP consumption, but a study of this issue has
revealed more complex behavior [11]. Analysis of the penicillin precursor α-aminoadipoyl-cysteine-valine (ACV)
synthetase uncovered excess energy consumption (ATPase
activity) under suboptimal amino acid substrate concentrations or elevated thiol concentrations. The results could
point to several sources of nonproductive ATP use: slow
(3 min-1) hydrolysis of P-pant thioester intermediates
(requiring reloading), thiolysis of the same, and/or postulated conformational changes in the absence of one or more
substrates that could increase hydrolysis rates.
A variant of the NRPS loading architecture can be seen in
the starter unit of yersiniabactin synthetase (Figure 2b).
Here, the adenylation domain is a separate protein (YbtE)
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Mechanisms
Figure 2
(a) NRPS starter unit
(b) Aryl-N-capped NRPS starter unit
SH
A
PCP
(c) PKS starter unit
SH
SH
E
GrsA
ArCP
A
YbtE
(gramicidin S)
HMWP2
propionyl-CoA
ATP
-Phe
L
DEBS1
(6-deoxyerythronolide B)
(yersiniabactin)
ATP
ACP
AT
salicylic ac id
ADP
ADP
coenzyme A
OH O
O
O
S
S
S
NH2
A
PCP
GrsA
E
A
YbtE
ArCP
HMWP2
AT
ACP
DEBS1
Current Opinion in Chemical Biology
Initiation. Holo starter units of NRPS, PKS, and aryl-N-capped NRPS
systems are shown. (a) The first protein in gramicidin S biosynthesis,
GrsA, consists of an L-phenylalanine-specific adenylation domain, a
PCP, and an E domain that converts the L-phenylalanine thioester to
D-phenylalanine in preparation for the next module. (b) A variant of the
NRPS starter unit, yersiniabactin synthetase has a separate protein,
YbtE, which is a salicylate adenylating enzyme, to load the aminoterminal ArCP of HMWP2. (c) Loading module of DEBS1, the first
protein in 6-deoxyerythronolide B biosynthesis, employs a propionylCoA specific acyltransferase domain to load the adjoining ACP.
from the amino-terminal ArCP that begins the starter unit
HMWP2 and, thus, this unit operates in trans. This architecture is common among a group of virulence-conferring
siderophore synthetases, including enterobactin
(Escherichia coli) [12••], yersiniabactin (Yersinia pestis) [13],
mycobactin (Mycobacterium tuberculosis) [14], actinomycin
(an antineoplastic agent from Streptomyces sp.) [15] and
pyochelin (Pseudomonas aeruginosa) [16]. This aryl ‘N-capping’ strategy uses a monofunctional initial substrate
(adenylated salicylate) to cap the peptide chain. Control of
initiation of chain growth is an unresolved issue; simply,
why does the synthetase not begin initiation somewhere in
the middle of the assembly line? It has been proposed
[17••] that the condensation domains responsible for subsequent chain elongation may have active sites into which
the P-pant-attached substrates bind, the implication being
that only a properly constituted donor chain can serve as a
substrate for condensation at each point in the assembly
line, thus enforcing a linear, stepwise synthesis.
acyl transferase (AT) domain selects propionyl-CoA and
transfers the already-activated propionyl group to the
P-pant of the adjacent ACP. This ACP will donate the propionyl group to the next linear module.
The PKS system shown in Figure 2c, involving the starter
unit of the DEBS1 module of 6-deoxyerythronolide B synthase [2], consists of the first two domains of the larger
DEBS1 protein, unlike the separate proteins of GrsA and
GrsB (the first two in gramicidin S synthetase), but follows
logic similar to that of the NRPS system (Figure 2a). The
Elongation: condensation, translocation, and
chain modification
In both NRPS and PKS systems, the loading domains A
and AT serve as the ‘gatekeepers’, activating and/or loading the required substrates with high specificity. (For
evidence that this specificity is not absolute, however, and
may vary even within a synthase, see [18].) A systematic
study of 160 A domains, using the crystal structure of the
GrsA A domain [19] as a guide, has revealed variable
regions in the NRPSs that correlate with amino acid substrate specificity; these predictions have been borne out by
mutagenesis data [20]. Likewise, a study of the AT
domains of DEBS and RAPS (rapamycin synthase) has
shown that specificity in PKS can be altered by AT domain
substitution, and that the substrate specificity is conferred
by a short segment of the AT domain [21].
The core domains and iterative chemistry for chain elongation are depicted in Figure 3. As shown, the minimum
set of domains for an elongation cycle consists of a module,
plus an upstream carrier domain that supplies the donor
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Figure 3
(a) Core NRPS domains
(b) Core PKS domains
O
R
O
S
ACP
S
AT
ACP
O
O
R 1HN
KS
O
–O
HS
H 2N
S
R2
S
R3
PCP
C
A
PCP
ACP
R2
SH
O
PCP
C
A
R
S
S
KS
ACP
AT
O
H
N
R 1HN
O
O
SH
S
R3
PCP
O
SH
ACP
R
HS
KS
AT
O
S
ACP
Current Opinion in Chemical Biology
Elongation. (a) In NRPS, elongation is mediated through an as-yetunknown mechanism by C domains. There is no evidence for or
against a covalent intermediate to the C domain. (b) In PKS, the KS
domain accepts the growing chain from the upstream ACP (on the left)
onto a conserved cysteine residue (represented by its thiol group, SH).
The KS also decarboxylates the downstream (on the right) ACPattached malonyl (or methylmalonyl) thioester, creating a carbanion
(middle stage, shown as the group with the minus sign) that results in
chain translocation and extension.
chain. Key unsolved issues in elongation involve the
mechanism and selectivity of the central condensation (C,
in NRPS) and ketosynthase (KS, in PKS) domains, as well
as the structural requirements for getting the acyl chains
on two distinct, upstream and downstream PCP or ACP
domains to converge on one C or KS active site.
product chain to the downstream ACP, which in turn
becomes the upstream donor for the next module.
The catalytic strategy of the C domains has not yet been
revealed (Figure 3a): unlike the situation with the KS
domain (see below), there is no evidence for or against a
covalent acyl–C domain intermediate, although there is a
strongly conserved double histidine (HH) motif present
that has been proven essential by mutagenesis [22•]. When
a mutation in this motif was introduced, the condensation
reaction was abolished [22•].
The KS catalytic strategy is stepwise (Figure 3b): first the
upstream acyl group is moved into the KS active site via
nucleophilic attack by a conserved KS cysteine, thus preserving the thermodynamic activation of the thioester. The
KS domain then decarboxylates the downstream
(methyl)malonyl-S-ACP, generating a carbanion (the enolate nucleophile) that elongates and translocates the
An unresolved issue is the source of substrate selectivity,
which could be mediated either by protein–protein interactions (i.e. ACP–KS–ACP recognition in cis or in trans) or
by acyl chain recognition by the KS (i.e. a specificity function in addition to that exercised by the AT domains). The
analogous NRPS selectivity system also remains opaque,
although a recent study [17••] bypassed the A domain
gatekeeper by loading synthetic aminoacyl CoAs via a
PPTase onto the apo-PCP domains of GrsA and the first,
proline-specific condensation-adenylation-thioesterification module (ProCAT) of TycB of tyrocidine synthetase to
demonstrate some latitude for substrates at the upstream
donor, with much less at the downstream acceptor.
Likewise, module substitution in the DEBS PKS system
has uncovered the presence of ‘linkers’, short peptide
stretches that connect modules and that can permit the
rational construction of novel synthases that assemble
novel products [23••]. Gokale et al. [23••] conclude that
there is considerable tolerance for incoming, donor substrates, and suggest that ‘rewiring’ synthases by swapping
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Mechanisms
entire modules may prove more fruitful in combinatorial
approaches than domain swapping.
If an assembly line model is correct, monomers can be
reloaded and a second chain initiated once the first has
moved downstream from the loading unit. In the steady
state, multiple intermediates, one docked at each carrier
protein, could be present on a single synthase or synthetase. Evidence that this is indeed the case arises from
mutations of the final rifF gene in the rifamycin synthase:
covalent intermediates accumulate, as the synthase cannot
release the mature product [24•]. Hydrolysis and analysis
revealed that all intermediate stages are present, including
some cyclized forms, indicating that chain modification
occurs concurrently with chain elongation.
An infrequently appearing variant of the C domain in
NRPS is the cyclization (Cy) domain. Using a downstream
cysteine or serine acceptor, the Cy domain combines the
condensation function of the C domain with additional
heterocyclization and dehydration functions (Figure 4). Cy
domains were first noted in the bacitracin synthetase [25]
and were directly validated in the 170 kDa yersiniabactin
HMWP2 fragment shown schematically in Figure 4 [26•].
The signature HHxxxDGxS motif (where x is any amino
acid, and residues are represented by single-letter code for
amino acids) of the C domain is modified to DxxxxDxxS
in the Cy domain, the aspartate residues (D) of which have
also been shown to be critical (C Walsh, unpublished
data). Other systems with Cy domains include mycobactin
[14] and pyochelin [16], whose two Cy domains yield a
4,2 thiazoline–thiazolidine molecule reminiscent of the
DNA-intercalating bithiazole of the antitumor agent
bleomycin [27].
Chain modifications, catalyzed by additional domains
inserted in the basic module, can occur during elongation
cycles. Classic examples of insertion in PKS, recognizable
from products of fatty acid synthesis, include insertion of
ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER) domains to accomplish the net four-electron
reduction of a β-ketoacyl intermediate to the saturated
methylene. Inactivation or absence of one or more of the
KR, DH, or ER domains preserves the product of a partial
reduction (β-keto, β-hydroxyl, or α,β-unsaturated) in the
polyketide. Domain substitution and/or site-directed
mutations in specific elongation cycles are often accepted
by the PKS machinery to produce ‘unnatural’ natural products [23••]. Recent work with the DEBS system has led to
a 100-variant library for the erythromycin aglycone [28••].
Inspection of the amino acid sequence of the mixed
NRPS/PKS yersiniabactin synthetase revealed two
methyltransferase-encoding domains in the irp1 gene that
are in the expected positions in the protein product for
C,C-dimethylation of a malonyl moiety followed by
Figure 4
(a)
SH
A
ArCP
Heterocyclization variant produced by
alternate initiation and elongation steps for
aryl-N-capped NRPS in the yersiniabactin
synthetase system (starter unit HMWP2
residues 1–1491). The xCP domains are
shown already phosphopantetheinylated.
(a) The salicylate-adenylating enzyme YbtE
loads the amino-terminal ArCP, while the
internal L-cysteine-specific A domain loads the
PCP. (b) Through an unknown mechanism,
the Cy domain then catalyzes condensation
and (c) the cyclodehydration to the thiazoline
before (d) the product (2-hydroxyphenylthiazoline) is released into solution by
nucleophilic water or L-cysteine.
SH
Cy
PCP
A
HMWP2 1-1491
YbtE
ATP
Salicylic acid
L -cysteine
ADP
O
OH O
H 2N
S
S
HS
A
ArCP
Cy
PCP
A
(b)
H
N
SH
O
S
OH O HS
A
ArCP
Cy
A
PCP
(c)
(d)
O
N
SH
OH
S
S
OH
N
COR
S
A
ArCP
Cy
A
PCP
R = L-cysteine, –OH
Current Opinion in Chemical Biology
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Polyketide and polypeptide antibiotic biosynthesis Keating and Walsh
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Figure 5
(a)
(b)
SH
COOH
H 2N
O
N
H
OH OH O
OH OH O
O
H
N
S
S
HO
HO
O
PCP
DEBS3
AcvA
OH OH O
SH
COOH
O
H 2N
PCP
OH OH O
O
H
N
N
H
O
O
O
E
O
OH
SH
O
H
N
N
H
H 2N
O
O
OH
OH
O
O
OH
α-Amino-adipoyl-cysteine-valine
(c)
TE
ACP
TE
H2O
COOH
TE
ACP
TE
E
6-Deoxyerythronolide B
OH
OH
H
N
O
OH
O
OH
H
N
S
HO
NH2
O
PCP
TE
HO
O
O
S
O
NH2
O
OH
OH
N
H
TE
PCP
EntF
OH
OH
OH
OH
OH
O
HN
OH
HO
OH
H
N
O
O
O
NH2
PCP
O
O
N
H
OH
O
SH
OH
HN
O
O
O
O
S
OH
H
N
O
O
O
OH
O
O
TE
PCP
O
N
H
OH
OH
TE
OH
OH
H
N
O
O
OH
O
HO
H
N
O
O
O
O
HN
HO
O
O
Enterobactin
HO
Current Opinion in Chemical Biology
Termination and integrated thioesterase (TE) domain function. TEs
possess a conserved serine residue (represented by its hydroxyl
group) that acts as a nucleophile to accept transfer of the mature
natural product from the last carrier protein domain. (a) AcvA TE has
esterase and hydrolase functions. The β-lactam precursor
α-aminoadipoyl–cysteine–valine is released via water hydrolysis. Other
TEs of this type are used to release vancomycin and yersiniabactin.
(b) DEBS3 TE has both esterase and cyclase functions, which act to
release 6-deoxyerythronolide B. DEBS3 represents a class of TEs that
release the final product via cyclization (others include FK506 and
bacitracin). (c) Assembly of enterobactin by the TE of EntF. The
function of the TE domain not only encompasses TE and cyclase
duties, but also acts as a carrier domain to allow the linear trimer of
N-2,3-dihydroxybenzoylserine to accumulate before macrolactonization
and enterobactin release.
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α-methylation of the cysteine that forms the final thiazoline in yersiniabactin [13]. C-methylation most likely
occurs via similar domains in products such as bleomycin
and epothilone. Identification of such domains will enable
development of strategies to test their portability in combinatorial biosynthesis studies.
Termination: specialized domains catalyze
release of mature NRPS and PKS products
Once the iterative elongation process has moved the product chain to the final carrier domain at the terminus of the
last module, a specialized thioesterase (TE) domain, located after this carrier domain, catalyzes product release [29].
The TEs, which bear homology to the TEs of fatty acid
synthases, accomplish this via internal product transfer
from the xCP P-pant to a conserved serine in the TE by
nucleophilic hydroxyl attack (Figure 5). This transfer converts the acyl-S-xCP thioester to an acyl-O-TE oxoester in
preparation for release.
Two general outcomes are known for chain release from
the TE domains: intermolecular hydrolysis releases the
free acid (e.g. vancomycin synthetase, and the ACV synthetase shown in Figure 5a), while intramolecular capture
by a nucleophile leads to macrolactonization with concomitant release (e.g. erythromycin, FK506, rapamycin
and DEBS3 in Figure 5b). With an amine nucleophile in
the chain, a macrolactam (e.g. bacitracin and cyclosporin)
can result. For these intramolecular cyclizations to occur,
the TE domain must exercise kinetic control over competing water molecules while adopting the proper
conformation to correctly position the nucleophile.
Some TE specificity questions have been addressed in the
DEBS PKS system [30,31] and the surfactin NRPS system
[32] by transporting the TE to upstream sites, where it has
been active for lactonizing (DEBS) or hydrolyzing (surfactin) a shorter, incomplete chain [33]. When the DEBS
TE was employed as an independent domain and assayed
for its activity in trans, however, only hydrolase activity was
observed, suggesting that some unknown domain interaction is critical for lactonization. In addition to the
carboxy-terminal TE domains found in NRPSs and PKSs,
many systems, including surfactin [29], tylosin [34],
lichenysin [35], and yersiniabactin [13], contain an additional TE as a separate protein. In some cases [29,34,36]
but not others [32], inactivating mutations in this external
TE reduce in vivo nonribosomal protein or polyketide production by >90%. Butler et al. [34] suggested that the
external TEs may perform a housekeeping function by
hydrolyzing mis-acylated monomers or acyl chains stalled
on carrier protein domains. Xue et al. [36] observed that
deletion
of
the
external
TE
from
the
methymycin/pikromycin PKS influences the ratio of 12membered to 14-membered macrolide products.
An additional role for the TE in EntF, a component of enterobactin synthetase [12••], has recently been uncovered
[37•]. Enterobactin is a cyclotrimer of N-(2,3-dihydroxybenzoate)-L-serine (DHB-Ser). As shown in Figure 5c, the TE
domain serves as an additional carrier domain for the units
of DHB-Ser condensed by the upstream domains of EntF.
After three units of DHB-Ser have reached the TE, macrolactonization occurs to release enterobactin. Data obtained
through mutagenesis of TE active site residues and mass
spectrometric characterization of a DHB-Ser dimer attached
to the TE argue strongly for these dual roles.
Three variants of the covalent disconnection for release of
full-length chains that do not involve a TE domain have
recently been described. Firstly, D-lysergic acid peptides
in ergot peptide synthetases are assembled as D-lysergylAla-Phe-Pro-S-enzyme tripeptides and, there being no TE
domain, are released by diketopiperazine formation arising
from attack of the phenylalanine amide nitrogen on the
P-pant thioester [38]. Secondly, in rifamycin biosynthesis,
a separate amide synthase (RifF) is employed to catalyze
intramolecular lactam formation and chain release, as there
is no carboxy-terminal TE domain [24•]. This strategy is
also present in the structurally similar naphthomycin PKS
[39]. Thirdly, two known systems, the yeast Lys2
α-aminoadipate reductase and the saframycin synthetase,
terminate in an NAD(P)H reductase domain instead of a
TE. Lys2 reduces a P-pant-tethered α-aminoadipoyl
thioester with hydride to the easily hydrolyzed thiohemiaminal [40]; identical logic is postulated [40] for reductive
release of the saframycin precursor Ala–Gly–Tyr–Tyr
tetrapeptide that then cyclizes to a hemiaminal found in
the natural product [41].
Finally, we call attention to a novel architecture unearthed
in the syringomycin synthetase [42•]. Before the terminal
TE on the eight module SyrE protein, a C–PCP two
domain unit is inserted after the last module, with no in cis
means of aminoacylation of this PCP. Biochemical evidence points to in trans loading of this last PCP by an A
domain on SyrB, a separate protein positioned upstream of
SyrE. Syringomycin synthetase thus constitutes a counterexample to the colinearity of the domain organization
with the final product.
Acknowledgements
Work in this laboratory on nonribosomal peptide synthetases has been
supported by grants from the National Institutes of Health. TAK is a Fellow
of the Cancer Research Fund of the Damon Runyon–Walter Winchell
Foundation, DRG-1483.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Katz L: Manipulation of modular polyketide synthases. Chem Rev
1997, 97:2557-2575.
2.
Khosla C: Harnessing the biosynthetic potential of modular
polyketide synthases. Chem Rev 1997, 97:2577-2590.
3.
Marahiel MA, Stachelhaus T, Mootz HD: Modular peptide
synthetases involved in nonribosomal peptide synthesis.
Chem Rev 1997, 97:2651-2673.
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Polyketide and polypeptide antibiotic biosynthesis Keating and Walsh
4.
von Döhren H, Keller U, Vater J, Zocher R: Multifunctional peptide
synthetases. Chem Rev 1997, 97:2675-2705.
5.
Stein T, Vater J, Kruft V, Otto A, Wittmann-Liebold B, Franke P,
Panico M, McDowell R, Morris HR: The multiple carrier model of
nonribosomal peptide biosynthesis at modular multienzymatic
templates. J Biol Chem 1996, 271:15428-15435.
6.
Konz D, Marahiel MA: How do peptide synthetases generate
structural diversity? Chem Biol 1999, 6:R39-R48.
7.
Cane DE, Walsh CT, Khosla C: Harnessing the biosynthetic code:
combinations, permutations, and mutations. Science 1998,
282:63-68.
8.
Walsh CT, Gehring AM, Weinreb PH, Quadri LEN, Flugel RS: Posttranslational modification of polyketide and nonribosomal peptide
synthases. Curr Opin Chem Biol 1997, 1:309-315.
9.
Lambalot RH, Gehring AM, Flugel RS, Zuber P, LaCelle M, Marahiel MA,
Reid R, Khosla C, Walsh CT: A new enzyme superfamily — the
phosphopantetheinyl transferases. Chem Biol 1996, 3:923-936.
10. Krätzschmar J, Krause M, Marahiel MA: Gramicidin S biosynthesis
operon containing the structural genes grsA and grsB has an
open reading frame encoding a protein homologous to fatty acid
thioesterases. J Bacteriol 1989, 171:5422-5429.
11. Kallow W, von Döhren H, Kleinkauf H: Penicillin biosynthesis:
βenergy requirement for tripeptide precursor formation by ∆-(L-β
aminoadipyl)-L-cysteinyl-D-valine synthetase from Acremonium
chrysogenum. Biochemistry 1998, 37:5947-5952.
12. Gehring AM, Mori I, Walsh CT: Reconstitution and characterization
•• of the Escherichia coli enterobactin synthetase from EntB, EntE,
and EntF. Biochemistry 1998, 37:2648-2659.
This paper describes the in vitro reconstitution of the biosynthesis of the
siderophore enterobactin, a cyclic trilactone of N-(2,3-dihydroxybenzoyl)-Lserine. The necessary components were holo-EntB (isochorismate
lyase–ArCP), EntE (dihydroxybenzoate-AMP ligase), and holo-EntF (C-APCP-TE synthetase). The holo enzymes were produced by the covalent
attachment of a P-pant group catalyzed by EntD, which is a phosphopantetheinyl transferase. Mutation of the active site serine in the TE of EntF abolished enterobactin synthetase activity.
13. Gehring AM, DeMoll E, Fetherston JD, Mori I, Mayhew GF,
Blattner FR, Walsh CT, Perry RD: Iron acquisition in plague:
modular logic in enzymatic biogenesis of yersiniabactin by
Yersinia pestis. Chem Biol 1998, 5:573-586.
14. Quadri LEN, Sello J, Keating TA, Weinreb PH, Walsh CT:
Identification of a Mycobacterium tuberculosis gene cluster
encoding the biosynthetic enzymes for assembly of the virulenceconferring siderophore mycobactin. Chem Biol 1998, 5:631-645.
15. Pfennig F, Schauwecker F, Keller U: Molecular characterization of
the genes of actinomycin synthetase I and of a 4-methyl-3hydroxyanthranilic acid carrier protein involved in the assembly of
the acylpeptide chain of actinomycin in Streptomyces. J Biol Chem
1999, 274:12508-12516.
16. Reimmann C, Serino L, Beyeler M, Haas D: Dihydroaeruginoic acid
synthetase and pyochelin synthetase, products of the pchEF
genes, are induced by extracellular pyochelin in Pseudomonas
aeruginosa. Microbiology 1998, 144:3135-3148.
17.
••
Belshaw PJ, Walsh CT, Stachelhaus T: Aminoacyl-CoAs as probes
of condensation domain selectivity in nonribosomal peptide
synthesis. Science 1999, 284:486-489.
This paper describes a method for bypassing the amino acid substrate
specificity of the adenylation domains in NRPSs. After chemically synthesizing aminoacylated-Coenzyme A analogs, a PPTase can be used to directly
modify an apo-xCP domain with the P-pant tether carrying the desired
monomer. These monomers can then test the specificity of the condensation
domain toward the upstream and downstream substrates. The authors found
much greater selectivity for the the downstream monomer than for the
upstream one.
18. Hunziker D, Yu T-W, Hutchinson CR, Floss HG, Khosla C: Primer unit
specificity in rifamycin biosynthesis principally resides in the later
stages of the biosynthetic pathway. J Am Chem Soc 1998,
120:1092-1093.
19. Conti E, Stachelhaus T, Marahiel MA, Brick P: Structural basis for
the activation of phenylalanine in the non-ribosomal biosynthesis
of gramicidin S. EMBO J 1997, 16:4174-4183.
20. Stachelhaus T, Mootz HD, Marahiel MA: The specificity-conferring
code of adenylation domains in nonribosomal peptide
synthetases. Chem Biol 1999, 6:493-505.
605
21. Lau J, Fu H, Cane DE, Khosla C: Dissecting the role of
acyltransferase domains of modular polyketide synthases in the
choice and stereochemical fate of extender units. Biochemistry
1999, 38:1643-1651.
22. Stachelhaus T, Mootz HD, Bergendahl V, Marahiel MA: Peptide bond
•
formation in nonribosomal peptide biosynthesis. J Biol Chem
1998, 273:22773-22781.
This paper describes an assay for condensation domain function based on
dipeptide formation and release by two modules: GrsA (PheATE) from gramicidin S synthetase, and truncated TycB (ProCAT) from tyrocidin synthetase.
A functional condensation domain catalyzes amide bond formation, with
noncatalytic release of a diketopiperazine molecule providing an assayable
product. A mutation in the proposed active site motif of the condensation
domain abolished the condensation reaction.
23. Gokhale RS, Tsuji SY, Cane DE, Khosla C: Dissecting and exploiting
•• intermodular communication in polyketide synthases. Science
1999, 284:482-485.
The authors demonstrate that the individual modules of the DEBS PKS are
tolerant toward the identity of the upstream acyl chain in each condensation
cycle. They also describe linkers, short sequences that lie within and
between the individual polypeptides, that are critical for retaining function
when modules are swapped out of their original position. Proper engineering of linkers allows transfer of intermediates between unnaturally linked
modules, and thus the catalytic production of ‘unnatural natural products’.
24. Yu T-w, Shen Y, Doi-Katayama Y, Tang L, Park C, Moore BS,
•
Hutchinson CR, Floss HG: Direct evidence that the rifamycin
polyketide synthase assembles polyketide chains processively.
Proc Natl Acad Sci USA 1999, 96:9051-9056.
The antibiotic rifamycin is an undecaketide assembled by the rifA–rifE
genes, and cyclized and released by the separate amide synthase encoded
by rifF. Inactivation of this thioesterase abolishes rifamycin production, and
instead leads to the accumulation of covalently attached linear polyketides at
each carrier protein waystation, thus indicating that rifamycin synthase can
simultaneously process multiple acyl chains.
25. Konz D, Klens A, Schörgendorfer K, Marahiel MA: The bacitracin
biosynthesis operon of Bacillus licheniformis ATCC 10716:
molecular characterization of three multi-modular peptide
synthetases. Chem Biol 1997, 4:927-937.
26. Gehring AM, Mori I, Perry RD, Walsh CT: The nonribosomal peptide
•
synthetase HMWP2 forms a thiazoline ring during biogenesis of
yersiniabactin, an iron-chelating virulence factor of Yersinia pestis.
Biochemistry 1998, 37:11637-11650.
This paper describes the first biochemical characterization of a cyclization
domain, the first of three in yersiniabactin synthetase. The amino-terminal
two-thirds of the synthetase HMWP2 is able to catalyze bond formation
between salicylate and cysteine, and then to cyclize and dehydrate this intermediate to a hydroxyphenyl-thiazoline-carboxylate. This product is released
from the enzyme fragment by hydrolysis or by attack of cysteine.
27.
Shen B, Du L, Sanchez C, Chen M, Edwards DJ: Bleomycin
biosynthesis in Streptomyces verticillus ATCC15003: a model of
hybrid peptide and polyketide biosynthesis. Bioorg Chem 1999,
27:155-171.
28. McDaniel R, Thamchaipenet A, Gustafsson C, Fu H, Betlach M,
•• Ashley G: Multiple genetic modifications of the erythromycin
polyketide synthase to produce a library of novel ‘unnatural’
natural products. Proc Natl Acad Sci USA 1999, 96:1846-1851.
Beginning with the DEBS PKS system, the authors have described manipulation of several elements that enable combinatorial biosynthesis of the
6-deoxyerythronolide B (6-dEB) core structure – AT substitution (affecting
monomer identity), KR deletion and addition (affecting the oxidation state of
a carbon center), and KR stereochemical alteration (affecting sterochemistry
of a carbon center). By making single, double, and triple substitutions in the
PKS, over 100 novel 6-dEB analogs have been produced, although the titers
of the new products were uneven. The authors emphasize the inherent ‘plasticity’ of the PKS, as demonstrated by their successful modifications of each
of the six modules of DEBS.
29. Schneider A, Marahiel MA: Genetic evidence for a role of
thioesterase domains, integrated in or associated with peptide
synthetases, in non-ribosomal peptide biosynthesis in Bacillus
subtilis. Arch Microbiol 1998, 169:404-410.
30. Kao CM, Luo G, Katz L, Cane DE, Khosla C: Engineered
biosynthesis of structurally diverse tetraketides by a trimodular
polyketide synthase. J Am Chem Soc 1996, 118:9184-9185.
31. Kao CM, Luo G, Katz L, Cane DE, Khosla C: Manipulation of
macrolide ring size by directed mutagenesis of a modular
polyketide synthase. J Am Chem Soc 1995, 117:9105-9106.
32. de Ferra F, Rodriguez F, Tortora O, Tosi C, Grandi G: Engineering of
peptide synthetases. Key role of the thioesterase-like domain for
efficient production of recombinant peptides. J Biol Chem 1997,
272:25304-25309.
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33. Cortés J, Wiesmann KEH, Roberts GA, Brown MJB, Staunton J,
Leadley PF: Repositioning of a domain in a modular polyketide
synthase to promote specific chain cleavage. Science 1995,
268:1487-1489.
34. Butler AR, Bate N, Cundliffe E: Impact of thioesterase activity on
tylosin biosynthesis in Streptomyces fradiae. Chem Biol 1999,
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35. Konz D, Doekel S, Marahiel MA: Molecular and biochemical
characterization of the protein template controlling biosynthesis
of the lipopeptide lichenysin. J Bacteriol 1999, 181:133-140.
36. Xue Y, Zhao L, Liu H-w, Sherman DH: A gene cluster for macrolide
antibiotic biosynthesis in Streptomyces venezuelae: architecture
of metabolic diversity. Proc Natl Acad Sci USA 1998,
95:12111-12116.
37.
•
Shaw-Reid CA, Kelleher NL, Losey HC, Gehring AM, Berg C,
Walsh CT: Assembly line enzymology by multimodular
nonribosomal peptide synthetases: the thioesterase domain of
E. coli EntF catalyzes both elongation and cyclolactonization.
Chem Biol 1999, 6:385-400.
Through thioesterase mutagenesis and mass spectrometry of trapped intermediates, this paper confirms a dual role for the TE domain of enterobactin
synthetase: it not only cyclolactonizes the trimer of N-(2,3-dihydroxybenzoate)-L-serine to yield enterobactin as the final step, but it also serves as the
last waystation for elongation. In this way, enterobactin synthetase displays
a novel architecture; for an NRPS constructing a product composed of six
monomers, five modules corresponding to each condensation event would
be expected. However, enterobactin synthetase has only one module, which
requires the TE to accept three units of N-(2,3-dibydroxybenzoate)-L-serine
before cyclolactonization and release.
38. Walzel B, Riederer B, Keller U: Mechanism of alkaloid cyclopeptide
synthesis in the ergot fungus Claviceps purpurea. Chem Biol
1997, 4:223-230.
39. Chen S, von Bamberg D, Hale V, Breuer M, Hardt B, Müller R, Floss HG,
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and naphthomycin. Identification and analysis of two separate
biosynthetic gene clusters in Streptomyces collinus Tü 1892. Eur J
Biochem 1999, 261:98-107.
40. Ehmann DE, Gehring AM, Walsh CT: Lysine biosynthesis in
Saccharomyces cerevisiae: mechanism of α-aminoadipate
reductase (Lys2) involves posttranslational
phosphopantetheinylation by Lys5. Biochemistry 1999,
38:6171-6177.
41. Pospiech A, Bietenhader J, Schupp T: Two multifunctional peptide
synthetases and an O-methyltransferase are involved in the
biosynthesis of the DNA-binding antibiotic and antitumour agent
saframycin Mx1 from Myxococcus xanthus. Microbiology 1996,
142:741-746.
42. Guenzi E, Galli G, Grgurina I, Gross DC, Grandi G: Characterization
•
of the syringomycin synthetase gene cluster. J Biol Chem 1998,
273:32857-32863.
The authors describe the synthetase of syringomycin, a lipodepsipeptide
with phytotoxic properties. Syringomycin synthetase is unique in its architecture in that the ‘colinearity rule’ describing the assembly line process is
not followed. There are nine monomers incorporated into syringomycin; the
adenylation domain for threonine, the last amino acid in the chain, is not
located in the last module upstream of the TE domain, where it would be
expected, but rather on a separate protein, SyrB, located upstream of the
main SyrE polypeptide. This implies that, at least, a specific in trans threonylAMP transfer must occur from the SyrB protein to the last PCP in SyrE.