Recognition of hybrid PCP/ACP acyl carrier proteins in nonribosomal peptide synthetase modules ...

40 Pages·2002·3.44 MB·English

JBC Papers in Press. Published on February 26, 2002 as Manuscript M200120200
Recognition of hybrid PCP/ACP acyl carrier proteins in nonribosomal peptide
synthetase modules by the 4'-phophopantetheinyl transferases AcpS and Sfp

Mohammad Reza Mofid ¶, Robert Finking ¶, and Mohamed A. Marahiel*

¶ These authors contributed equally to this work.

running title: hybrid PCP/ACP NRPS

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*corresponding author: d
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Prof. Mohamed A. Marahiel http
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Philipps-Universität Marburg w
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Fachbereich Chemie/Biochemie rg
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Hans-Meerwein-Str. ue
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D-35032 Marburg A
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Germany , 2
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[phone]
[phone] (fax)
[email protected]

keywords: Acyl-carrier protein, polyketide synthase, nonribosomal peptide synthetase,
coenzyme A, 4’-phosphopantetheine transferase

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
SUMMARY
The acyl carrier proteins (ACPs) of fatty acid (FAS) and polylketide synthases (PKS)
as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases (NRPSs) are
modified by 4´-phosphopantetheinyl transferases (PPTases) from inactive apo-enzymes to
their active holo-forms by transferring the 4´-phosphopantetheinyl moiety of coenzyme A to a
conserved serine residue of the carrier protein. PPTases have been classified into two types:
the AcpS-type accepts ACPs of FAS and some ACPs of type II PKS as substrates while the
Sfp-type exhibits an extraordinarily broad substrate specificity.
Based on the previously published co-crystal structure of Bacillus subtilis AcpS and
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ACP which provided detailed information about the interacting residues of the two proteins, w
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we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the d
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corresponding helix of B. subtilis ACP that contains the interacting residues. This was http
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performed for the PCP domain as a single protein as well as for the TycA-PCP domain within w
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the NRPS module TycA from B. brevis. rg
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Both resulting proteins, designated hybrid-PCP (hPCP) and hybrid-TycA (hTycA) ue
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were modified in vivo during heterologous expression in Escherichia coli (hPCP: 51%, A
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hTycA: 75%) and in vitro with AcpS as well as Sfp to 100%. , 2
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The designated hTycA module contains two other domains, an adenylation (A)
domain (activating phenylalanine to Phe-AMP and afterwards transferring the Phe to the PCP
domain) and an epimerization (E) domain (converting the PCP-bound L-Phe to D-Phe). We
show here that the modified PCP domain of hTycA communicates with the A domain and that
the cofactor of holo-hPCP is loaded with Phe. However, communication between the hybrid
PCP and the E domain seems to be disabled. Nevertheless, hTycA is recognized by the next
proline activating elongation module TycB1 in vitro and the dipeptide is formed and released
as diketopiperazine.

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ABBREVIATIONS
ACP Acyl-carrier protein
AcpS PPTase involved in FAS, Acyl-carrier protein synthase
AU absorption units
CD Circular dichroism
CoA Coenzyme A
DPM decays per minute
FAS Fatty acid synthase
IPTG Isopropyl-(cid:1)-D-thiogalactopyranoside
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NRPS Nonribosomal peptide synthetase w
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NTA Nitrilotriacetic acid d
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PCP Peptidyl-carrier protein http
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PCR Polymerase chain reaction w
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PKS Polyketide synthase rg
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Ppant 4’-phosphopantetheine ue
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PPTase 4’-phosphopantetheine transferase A
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Sfp PPTase involved in surfactin production , 2
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TCA Trichloracetic acid
TFA Trifluoroacetic acid

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INTRODUCTION
A large number of natural bioactive peptides (e.g. vancomycin, cyclosporine,
tyrocidine and surfactin) are produced by bacteria and fungi via a template-directed, nucleic-
acid-independent mechanism carried out by multienzyme complexes called nonribosomal
peptide synthetases (NRPSs) (1-3). The biosynthetic mechanism of NRPS resembles that of
fatty acid synthases (FASs) and polyketide synthases (PKSs) which catalyse fatty acids
synthesis and production of polyketides like erythromycin and tetracyclin (4,5).
NRPSs have a modular architecture: each module is responsible for the incorporation
of one building block into the nascent peptide chain. A module can be subdivided into
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domains, each representing the single enzymatic activity necessary for synthesis and w
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modification of the peptide chain. The adenylation (A) domain is responsible for substrate d
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recognition and activation as aminoacyladenylate at the expense of ATP (6,7). The http
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downstream PCP domains are the sites of intermediate binding and serve as carrier units (8). w
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The peptide bond formation between covalently bound intermediates on two consecutive rg
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modules is catalyzed by a condensation (C) domain (9,10) (Fig. 1). During this peptide ue
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assembly, optional modifications of the substrates can occur, depending on the presence of A
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modifying domains within the corresponding module. For example, epimerization (E) , 2
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domains catalyze the conversion of incorporated L-amino acids to their D-isomers (2).
NRPSs, FASs and PKSs contain one or more small PCP or ACP domains that are 80-
100 amino acids (aa) in length that carry the building blocks and elongated intermediates to
the different catalytic centers. Depending on the architecture of the multienzyme complex,
these carrier proteins appear as integrated domains or as individual proteins. The peptidyl
carrier protein domains (PCPs) in NRPSs and the acyl carrier proteins (ACPs) in FASs and
PKSs share a conserved sequence motif GX(D,H)S(L,I)(D,K) containing an invariable serine
residue (11). During posttranslational modification the 4´-phosphopantetheine (Ppant)
cofactor is transferred from Coenzyme A and attached to this serine residue converting the
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carrier proteins from the inactive apo to the active holo forms (4) (Fig. 2). Dedicated 4´-
phosphopantetheinyl transferases (PPTases) catalyze the nucleophilic attack of the hydroxyl
sidechain of the conserved carrier protein serine residue onto the 5'-ß-pyrophosphate linkage
of coenzyme A (CoASH) in a magnesium ion dependent reaction (4). During peptide or
polyketide synthesis, the activated substrates and elongation intermediates are covalently
linked as thioesters to the thiol moiety of the 4´-Ppant.
PPTases have been classified into two types based on substrate specificity: the AcpS-
type of primary metabolism and the Sfp-type of secondary metabolism (4,12). PPTases of the
AcpS-type are about 120 aa in length and have a pI value of about 9.6; they are found in
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almost every known microorganism and are responsible for the modification of fatty acid w
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ACPs. Biochemical studies demonstrated that ACPs of type II PKS systems, the D-alanyl d
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carrier protein (DCP) from Lactobacillus casei involved in D-alanylation of lipoteichoic acid http
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(13) and NodF from Rhizobia involved in the acylation of the oligosaccharide-based w
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nodulation factors could be modified by AcpS from E. coli (14), too. rg
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The PPTase Sfp of B. subtilis represents the prototype of the second group of PPTases. ue
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Enzymes of this class are about 240 aa in length with a pI value of about 6 and have mostly A
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been found associated with NRPS gene clusters (15). The well-characterized Sfp exhibits an , 2
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extraordinarily broad substrate specificity and is able to modify all carrier proteins so far
tested including PCPs of NRPSs and ACPs of both FASs and PKSs (16).
The crystal structure of B. subtilis AcpS co-crystallized with CoA and ACP gave
detailed insight into the interactions between this enzyme and its substrates (17). It was
possible to identify those residues of ACP that are responsible for the protein interaction with
AcpS, most of which are provided by helix 3 of the ACP. The solution structure of a
prototype PCP domain showed that PCPs fold into a similar secondary structure as ACPs do
(18).
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In this study, we present the construction of hybrid PCP/ACP carrier proteins through
the replacement of the coding region for helix 2 in the TycC3-PCP with the corresponding
region of ACP from B. subtilis as well as the replacement of this PCP helix in the domain of
the initiation module TycA of the tyrocidine synthetase. Both hybrid proteins were
biochemically characterized in modification assays with the PPTases AcpS and Sfp from B.
subtilis. Using the newly created PCP domain within the NRPS module hTycA, we also show
that hPCP is able to perform the necessary communication with the A and C domains.

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EXPERIMENTAL PROCEDURES w
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General techniques d
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E. coli was grown on LB medium with 10 mM MgCl2. Antibiotics were used at the http
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following concentrations, ampicillin 100 µg/mL, kanamycin 25 µg/mL. For E. coli w
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techniques, such as transformation and plasmid preparation, standard protocols were used rg
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(19). The HerculaseTM Hotstart polymerase (Stratagene, Heidelberg, Germany) was used to ue
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amplify gene fragments for cloning and expression purposes. Oligonucleotides were A
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purchased from MWG Biotech. [3H]-CoA and [14C]-Phe were purchased from Hartmann , 2
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Analytics (Braunschweig, Germany).

Construction of plasmids
Construction of the plasmid pQE70-hPCP: the plasmid pQE70-TycC3-PCP (18)
served as a template in a PCR reaction with oligonucleotides carrying the sequence
information for helix 2 (italic) of ACP from B. subtilis including a SacI restriction site
(underlined) and the sequence from TycC3-PCP (boldface): 5´-
ATAGAGCTCGTTATGGAACTTGAAGACGAGTATCAGGTTGAGCTTCC-3´ and 5´-
ATAGAGCTCAACTACATCTAGGGAATCTCCGCCGATCTGGAAAAAGT-3´ to amplify
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a fragment that contained hPCP, under control of the T5 promotor of pQE70. This fragment
was treated with SacI, the template DNA was digested with DpnI, and the PCR-product was
subsequently re-ligated to pQE70-hPCP (3664 bp) (Fig. 3).
Construction of the plasmid pQE60-hTycA: the plasmid pTycA (20) served as a
template in a PCR reaction with oligonucleotides: 5´-
TTTAACTAGTTATGGAACTTGAAGACTATCAATTGAAGCTAGAGACGAAAGAC-3´
and 5´-ATATACTAGTTCAACTACATCTAGCGAATCTCCGCCGAGCGAG-3´ to amplify a
fragment that contained hTycA. This fragment was treated with SpeI, the template DNA was
digested with DpnI, and the PCR-product was subsequently re-ligated to pQE60-hTycA (6686
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Overproduction and purification of recombinant proteins http
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E. coli M15 which carries the plasmid pREP4 providing the lacI gene for efficient w
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repression of transcription was transformed with the described plasmids. The proteins were rg
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overproduced as previously described (10). Expression was induced with 1 mM IPTG (final ue
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concentration) at an A of 0.6 and the cells were allowed to grow for an additional 2 h A
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before being harvested by centrifugation at 4,500 g at 4° C. The cell pellet was resuspended in , 2
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buffer A (50 mM Hepes, 150 mM NaCl, pH 7.8), and disrupted by three passages through a
cooled French pressure cell. The resulting cell extract was centrifuged at 36,000 g and 4°C for
30 min. Protein purification using Ni2+-NTA-affinity chromatography was carried out as
previously described. Purified protein was dialyzed against the respective assay buffer,
brought to 10 % glycerol (v/v) and stored at –80 °C. TycC3-PCP (hereafter referred to as
PCP), TycA, TycB1, AcpS form B. subtilis and Sfp-His were produced and purified as
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previously described (10). Protein concentrations were determined based on the calculated
extinction coefficient at 280 nm: PCP 9,530 M-1 cm-1, hPCP 9,530 M-1 cm-1 and hTycA-His
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133,580 M-1 cm-1.
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Posttranslational Modification of the Enzymes by Ppant transferase and CoASH in vivo.
Priming of heterologously expressed proteins was achieved by incubation of 1 µM
apo-enzyme with 100 µM CoASH and 25 nM recombinant Bacillus subtilis Ppant transferase
Sfp or AcpS for 30 min at 37 °C (16,21) prior to the addition of any amino acid.

Radioassay for the Detection of Posttranslational Modification of the Enzymes.
AcpS and Sfp activity was assayed by using a radioactive assay method essentially as
described previously (22). This method measures the incorporation of the [3H]-labeled 4’-
phosphopantetheine group from [3H]-CoA into apo-enzymes. Reaction mixtures containing
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75 mM MES/HCl pH 6.0 (in the case of AcpS 50 mM Tris/HCl pH 8.8), 10 mM MgCl , 1 w
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µM of the respective protein, 20 µM CoA, 200 nM [3H]-CoA (specific activity: 40 Ci/mmol, ed
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0.95 mCi/mL), and 2.2 -5.6 nM AcpS of B. subtilis or 25 nM Sfp were incubated at 37 °C for http
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30 min. Reactions were stopped by the addition of 0.8 mL of ice-cold 10 % TCA (w/v) and 15 w
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µL BSA (25 mg/mL). Precipitated protein was collected by centrifugation at 13,000 rpm and rg
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4 °C for 15 min using a microcentrifuge. The pellet was washed twice with 0.8 mL of ice-cold ue
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TCA (w/v) and resuspended in 180 µL formic acid. The resulting suspension was mixed with A
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3.5 mL Rotiszint Eco Plus scintillation fluid (Roth, Karlsruhe, Germany) and counted using a , 2
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1900CA Tri-Carb liquid scintillation analyzer (Packard, Dreieich, Germany).

Kinetic and HPLC analysis of hPCP and PCP.
For kinetic studies, the amount of holo-carrier protein formed was determined by an
HPLC method. 800 µL Reaction mixtures containing, 1-125 µM apo-PCP or 5-135 µM apo-
hPCP, 50 mM Tris/HCl pH 8.8 (75 mM MES/HCl pH 6.0 in the case of Sfp) 12.5 mM
MgCl , 2 mM DTT, 1 mM CoA and 11.2 nM AcpS of B.subtilis or 10 nM Sfp were incubated
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at 37 °C for 10 min. The reaction was stopped and the protein precipitated by the addition of
10 % TCA. Reaction mixtures were centrifuged for 30 min at 13,000 rpm and 4 °C in a
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microcentrifuge. The pellet was subsequently resuspended in 120 µL of 50 mM Tris/HCl pH
8.8. A 100 µL sample of this solution was injected onto a reversed phase HPLC column
(Nucleosil C18, 250 mm, 5 µm, 300 Å, Macherey&Nagel) equilibrated with 60 % solvent A
(H O with 0.1% TFA) in the case of PCP. Apo- and holo-PCP could be separated by applying
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a 15 mL linear gradient 40 to 95% solvent B (acetonitrile with 0.1% TFA) (flowrate 0.5
mL/min and temp. 40 °C). Samples were examined for their A . Under these conditions, the
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holo-carrier protein migrates faster than the apo- form (retention times were 10.67 and 11.95
min respectively). The amount of holo-PCP formed was determined by comparing the peak
area of the holo-PCP formed with those of both apo- and holo-PCP and substracting the
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amount of holo-PCP that was already present after the heterologous expression of the protein w
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in E. coli (see Results). d
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In the case of hPCP, a 1.2 mL linear gradient to 50 % solvent C (methanol with 0.1 % TFA) http
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for 25 min (flowrate 0.5 mL/min and temp. 40 °C) followed by a 7.2 mL linear gradient to w
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90% solvent C at 0.3 mL/min and 40 °C, holo- and apo-hPCP eluted at the respective rg
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retention times 26.1 and 27.7 min. ue
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Covalent Incorporation of [3H]-Phosphopantetheine into TycA and hTycA. , 2
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The incorporation of [3H]-phosphopantetheine was performed as described previously
(22). Reaction mixtures (100 µL) contained 50 mM Tris/HCl pH 8.8 ( in the case of Sfp 75
mM MES/HCl pH 6.0), 10 mM MgCl , 20 µM CoA, 200 nM [3H]-CoA (specific activity: 40
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Ci/mmol, 0.95 mCi/mL), 25 nM ACPS or Sfp and 1 µM apo-TycA or -hTycA. The reactions
were started by the addition of the PPTase and were incubated at 37 °C for 30 min. The
reactions were quenched with 800 µL of 10 % TCA (w/v) and 15 µl of 25 mg/ml BSA. The
mixture was incubated on ice for 15 min. The precipitate was pelleted by centrifugation
(13000 rpm for 30 min in a microcentrifuge), washed twice with 1 mL 10% TCA (w/v) and
subsequently solubilized in 180 µL of concentrated formic acid. The redissolved protein was
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mixed with 3.5 mL Rotiszint Eco Plus scintillation fluid (Roth) and and the amount of
radioactivity incorporated was quantified using a 1900CA Tri-Carb liquid scintillation
(Packard, Dreieich, Germany).

Covalent Incorporation of [14C]-Phenylalanine into TycA and hTycA.
To investigate the interaction between the adenylation domain and the PCP of TycA
and hTycA, both modules were covalently loaded with [14C]-L- or [14C]-D-Phe.
Aminoacylation was performed as described previously (10,23). The synthetases were
aminoacylated in reaction mixtures (100 µL) containing assay buffer (75 mM Tris-HCl, pH
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8.5, 10 mM MgCl , 5 mM ATP), 1 µM holo-TycA or holo-hTycA and 2 µM [14C]-L-Phe w
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(450 mCi/mmol) or 2 µM D-[14C]-Phe (56 mCi/ mmol). The reaction mixtures were incubated ed
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at 37 °C for specific times, samples were taken and quenched with 0.8 mL of ice-cold 10 % http
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TCA (w/v) and 15 µl of a 25 mg/mL BSA. The mixture was incubated on ice for 15 min. The w
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precipitate was pelleted by centrifugation (13000 rpm for 30 min in a microcentrifuge), rg
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washed twice with 1 mL 10 % TCA (w/v) and solubilized in 180 µL of concentrated formic ue
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acid. The redissolved protein was mixed with 3.5 mL liquid scintillation fluid and the amount A
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of incorporated radioactivity was quantified by liquid scintillation analysis (Packard, , 2
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Dreieich, Germany).

Radio-TLC Assay for Detection of Phe-S-Ppant Epimerization.
To investigate the conversion of L-Phe-S-Ppant enzyme to the D-Phe-S-Ppant species
(and vice versa), TycA(PheATE) and hTycA(PheATE) were allowed to activate and
covalently load [14C]-L- or [14C]-D-Phe (24). Reaction mixtures in assay buffer containing 1
µM holo-enzyme as well as 8.9 µM L-[14C]-Phe (450 mCi/mmol) or 71.4 µM D-[14C]-Phe
(56 mCi/ mmol) were incubated for 3 min at 37 °C (final volume 200 µL). At defined time
points samples were taken and quenched with 0.8 mL of ice-cold 10 % TCA (w/v). After 15
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