Supplementary knowledge about E domains is limited to timing of condensation and epimerization [20,21] and to the ability of the TycB3-E domain, originally embedded in a Phe-activating e
Trang 1Utility of epimerization domains for the redesign of
nonribosomal peptide synthetases
Daniel B Stein, Uwe Linne and Mohamed A Marahiel
Fachbereich Chemie ⁄ Biochemie, Philipps-Universita¨t Marburg, Germany
d-Configured amino acids are an important
particular-ity occurring in various natural bioactive peptide
com-pounds These are, for example, peptides secreted by
certain animals such as amphibians and spiders [1] or
the antimicrobial lantibiotics which are synthesized
ribosomally in bacteria [2] Another group of
numer-ous pharmacologically interesting peptides containing
d-amino acids is produced in bacteria and fungi by a class of huge multienzymes, the modular nonribosomal peptide synthetases (NRPSs) [3,4]
Generally, NRPS assembly lines are constructed by
a certain sequence of domains with specific catalytic functions Essential core domains are the adenylation (A) domain for the selective acivation of amino acids
Keywords
epimerization; multienzyme; nonribosomal
peptide synthetase; protein engineering
Correspondence
M A Marahiel, Fachbereich
Chemie ⁄ Biochemie, Philipps-Universita¨t
Marburg, Hans-Meerwein-Straße,
35032 Marburg, Germany
Fax: +49 6421 2822191
Tel: +49 6421 2825722
E-mail: marahiel@chemie.uni-marburg.de
(Received 23 May 2005, revised 11 July
2005, accepted 18 July 2005)
doi:10.1111/j.1742-4658.2005.04871.x
Many pharmacologically important agents are assembled on multimodular nonribosomal peptide synthetases (NRPSs) whose modules comprise a set
of core domains with all essential catalytic functions necessary for the incorporation and modification of one building block Very often, d-amino acids are found in such products which, with few exceptions, are generated
by the action of NRPS integrated epimerization (E) domains that alter the stereochemistry of the corresponding peptidyl carrier protein (PCP) bound
l-intermediate In this study we present a quantitative investigation of substrate specificity of four different E domains (two ‘peptidyl-’ and two
‘aminoacyl-’E domains) derived from different NRPSs towards PCP bound peptides The respective PCP-E bidomain apo-proteins (TycB3-, FenD2-, TycA- and GrsA-PCP-E) were primed with various peptidyl-CoA precur-sors by utilizing the promiscuous phosphopantetheinyl transferase Sfp PCP bound peptidyl-S-Ppant epimerization products were chemically cleaved and analyzed for their l⁄ d-ratios by LCMS We were able to show that all four E domains tolerate a broad variety of peptidyl-S-Ppant-sub-strates as evaluated by kobs values and final l⁄ d-product equilibria deter-mined for each reaction The two C-terminal amino acids of the substrate seem to be recognized by ‘peptidyl-’E domains Interestingly, the ‘amino-acyl-’E domains GrsA- and TycA-E were also able to convert the elongated intermediates All four E domains accepted an N-methylated precursor as well and epimerized this substrate with high efficiency Finally, we could demonstrate that the condensation (C) domain of TycB1 is also able to process peptidyl substrates transferred by TycA In conclusion, these find-ings are of great impact on future engineering attempts
Abbreviations
A, adenylation domain; aminoacyl- or peptidyl-S-Ppant, aminoacylated thioester form of cofactor Ppant bound to the strictly conserved serine residue of PCPs; C, condensation domain; DKP, diketopiperazine; E, epimerization domain; ICR, ion cyclotron resonance; NRPS,
nonribosomal peptide synthetase; PCP, peptidyl carrier protein (also refered to as T); PCP C , PCP normally localized in front of a C domain; PCPE, PCP naturally connected to an E domain; Ppant, 4¢-phosphopantetheine; SIM, single ion mode; T, thiolation domain – also refered to
as PCP but used for protein descriptions (‘one letter–one domain’ nomenclature of NRPSs); TE, thioesterase domain; TFA, trifluoroacetic acid.
Trang 2as aminoacyl-O-AMP under ATP consumption [5],
and the following peptidyl carrier protein [PCP, often
also denoted as thiolation (T) domain] [6,7]
Post-translational introduction of an 4¢-phosphopantetheine
(Ppant) cofactor forms the active holo-PCP which
accepts the adenylated amino acid to yield an
amino-acyl-S-Ppant-PCP [8,9] Other core domains are the
condensation (C) domain carrying out the peptide
bond formation between two PCP bound S-Ppant
intermediates [10], and a C-terminally located
termin-ation domain for product release which is in most
systems a thioesterase (TE) domain [11]
Besides other optional modifications, the
incorpor-ation of d-amino acids into the product is a special
fea-ture appearing in nonribosomally synthesized peptides
For NRPS d-configured amino acids can be provided
by external racemases, then selectively be recognized,
and activated by a specific A domain Nevertheless,
more commonly epimerization (E) domains [12,13]
integrated in a module catalyze the conversion of a
PCP bound, Ppant thioesterified l⁄ d-amino acid (in
ini-tiation modules) or l⁄ d-peptidyl (in elongation
mod-ules) moiety by de- and reprotonating the Ca atom of
the substrate The epimerization reaction is reversible
and finds its end in the adjustment of an equilibrium
between both isomers [14] However, only the
d-ami-noacyl- or peptidyl-S-Ppant is singled out by the
stereo-selective donor site of the downstream C domain for
condensation with the next building block [15,16]
Bac-terial NRPS systems most often consist of several
dis-tributed enzymes that, with only few exceptions, carry
E domains at their C-terminal end Such E domains
were shown to be involved in the specific and ordered
recognition of synthetases within NRPS assembly lines
[17], a finding that makes them interesting candidates
for the engineering of new hybrid enzymes Short
com-munication-mediating (COM) domains as part of E
domains were recently identified to be responsible for
this selective interaction [18] Although the function of
E domains was studied extensively by a mutational
approach, only a little insight was afforded into the
exact mechanistic process of the catalysis [14] Other
studies addressed the portability of E domains by
con-structing fusion proteins of the type A⁄ PCP-E The
main limitation of this immense time consuming genetic
approach is the low throughput Though, it led to the
information that the E domain originating from the
Phe-activating intiation module TycA is also able to
convert Trp, Ile and Val with slightly decreased
effi-ciency [13] Aminoacyl-pantetheine derivatives were
reported to be accepted as soluble substrates by the E
domain of GrsA-ATE (PheATE) [19] In doing so, very
high Kmvalues were determined indicating that it is not
possible to gain information about the native substrate tolerance of E domains this way Supplementary knowledge about E domains is limited to timing of condensation and epimerization [20,21] and to the ability of the TycB3-E domain, originally embedded in
a Phe-activating elongation module of the tyrocidine synthetase, to epimerize the aminoacyl-Phe-S-Ppant substrate instead of the cognate enzyme bound tetra-peptide with lower efficiency [20] On the other hand, nothing is known about the ability of an aminoacyl-S-Ppant epimerizing E domain to convert peptidyl-S-Ppant substrates This could be of special interest for the engineering of NRPSs by module extension As
in all previous approaches only the amino acid being epimerized was varied and mainly aminoacyl sub-strates were investigated, it remains unclear if there is
a strong specialization of E domains for recognizing distinct sites of their cognate substrate and therefore if one can strictly distinguish between ‘aminoacyl-’ and
‘peptidyl-’E domains
In this study, we accomplished an investigation of the substrate specificity of E domains by using various peptidyl-CoA precursors The peptidyl-S-Pant moiety
of these CoAs was transferred onto four different PCP-E bidomain constructs (TycB3-, FenD2-, TycA-and GrsA-PCP-E) (known to be the smallest working system for E domains [14]) under exploitation of the promiscuity shown by the 4¢-Ppant transferase Sfp (Fig 1) Subsequently, velocity of the catalyzed epi-merization and the final l to d equilibrium were ana-lyzed by chemical cleavage of the reaction products from the enzyme after quenching the reaction By this chemoenzymatic approach, which forms a new mini-mal system for the investigation of native E domain specificity, we could reveal a broad substrate tolerance
of the C-terminal E domains used here Most interest-ingly, we observed the ability of E domains, naturally located in initiation modules, to epimerize peptidyl-S-Ppant substrates Likewise, we detected acceptance and elongation of peptidyl substrates, first loaded onto TycA-PCP-E and allowed to epimerize, by the TycB1
-C domain which naturally follows an initiation module and there exclusively connects two aminoacyl residues
Results
Generation and purification of the recombinant enzymes
A set of four recombinant PCP-E bidomain proteins derived from different NRPS systems was constructed (Fig 2) The enzymes chosen for this study harbour E domains which are all located at the C termini of the
Trang 3NRPSs Two constructs thereof [TycA-PCP-E
(65.6 kDa) and GrsA-PCP-E (67.6 kDa)] contain E
domains from initiation modules (both activating and
incorporating Phe) which only epimerize
aminoacyl-S-Ppant substrates in their natural context The two
other constructs [TycB3-PCP-E (63.9 kDa) and FenD2
-PCP-E (66.8 kDa)] in contrast contain E domains
from elongation modules (TycB3is also activating and
incorporating Phe, FenD2 is activating and
incorpor-ating Thr) both naturally epimerizing
tetrapeptidyl-S-Ppant substrates The individual enzymes were
successfully expressed as C-terminal His6-tagged
fusions in the heterologous host Escherichia coli M15⁄ pREP4 and could be purified to homogeneity by single-step Ni2+ affinity chromatography as confirmed
by SDS⁄ PAGE (data not shown)
Use of synthetic peptidyl-CoAs with Sfp and PCP-E bidomain constructs for assaying epimerization activity
The first aim of this study was to develop an assay for the investigation of E domain substrate specificity with synthetic CoA precursors and HPLC based analysis of
Fig 1 Schematic representation of the assay system for the investigation of E domain substrate specificity The PCP dom-ain of the PCP-E bidomdom-ain protein is primed with the peptidyl-Ppant moiety of the CoA substrate under catalytic action of Sfp This initiates the catalytic conversion by the
E domain leading to the formation of an equilibrium between the two enzyme bound
L ⁄ D -peptidyl-S-Ppant intermediates (the Ppant moiety is illustrated by the wavy line).
Fig 2 Schematic representation of the biosynthetic operons for (1) tyrocidine from B brevis ATCC 8185 (2) gramicidin from B brevis ATCC
9999 and (3) fengycin from B subtilis F29-3 The gene fragments cloned for construction of the recombinant PCP-E-bidomain proteins used
in this study are presented in consideration of their relative location underneath each system.
Trang 4reaction products The preparation and use of
pep-tidyl- instead of aminoacyl-CoAs was preferred because
E domains had not been investigated with elongated
S-Ppant-substrates before In addition, proceeding this
way should enable one to identify amino acid residues
of the substrate, other than the one actually
epimer-ized, which might reveal an influence on the
epimeriza-tion reacepimeriza-tion
Initial experiments showed that the peptidyl-S-Ppant
moiety of synthetically prepared peptidyl-CoAs (for all
precursors synthesized in this study see Fig 3) can be
loaded onto the recombinant PCP-E constructs
util-izing 15 lm of the 4¢-Ppant transferase Sfp for a fast
modification (as described in Experimental
Proce-dures) The reaction was quenched at defined time points after the addition of Sfp by adding 10% (v⁄ v) tricholoroacetic acid (TCA) to the samples This led to precipitation of the proteins including enzyme bound peptidyl-S-Ppant intermediates After separation of excess substrate by washing, reaction products were set free under alkaline conditions and could be applied to HPLC-MS First measurements revealed that the load-ing reaction was almost completed after 15 s (earliest point of time assessed) as estimated by comparing the intensity of MS signals belonging to peptides regained from samples which were taken at different time points When Sfp was omitted in the reaction mixture,
no detectable amount of the corresponding peptide was regained by the sample work up (data not shown) Both diastereomers resulting from the epimerization reactions were separated by reversed phase HPLC (Fig 4) The relative values of l-⁄ d-isomers were cal-culated easily by determining the area of the ion extracted MS signals Therefore, the use of an internal standard was not necessary for quantification Pro-ceeding like this, it was possible to resolve formation
of converted peptidyl substrates in dependence of time (Fig 5) (the margin of error, as estimated by up to four independent determinations of each time curve, was approximately ± 5%) Apparent kobsvalues could
be calculated from these data in analogy to the radio-TLC assays earlier reported [13,14] The decisive advantage of this chemoenzymatic approach is the bypassing of natural substrate activation and conden-sation which allows loading of any peptidyl precursor With these fundamental notices it was possible to establish this new minimal system for investigation of
E domain specificity
Assuming that ‘peptidyl-’E domains mainly recog-nize the C-terminal amino acid residue of their pep-tidyl-S-Ppant substrate (the directly thioester bound and actually epimerized moiety), in a set of peptidyl-CoAs ) besides the cognate precursor for TycB3
-PCP-E, fPFF-CoA (1) [d-Phe-l-Pro-l-Phe-l-Phe-S-Ppant;
we will use the shorter one letter abbreviation for amino acids (capital letter for l-amino acids, lower-case letter for d-amino acids) throughout this text], and the corresponding shortened one, FF-S-Ppant (2)) this decisive site (R2) was varied while keeping the N-terminal Phe (R1) constant To test if the final equilibrium position reached in the reaction with FF-CoA (2, conversion from l to d) is the same when converting from d to l, Ff-CoA (3) was designed Fur-ther synthesized CoAs include variations of aromatic [FY-CoA, F(p-fluoro) F-CoA, FW-CoA (4–6)], alipha-tic [FL-CoA (7)], neutral hydrophilic [FN-CoA, FS-CoA, FT-CoA (8–10)], acidic [FD-CoA (11)], and
Fig 3 Presentation of all peptidyl-CoAs synthesized in this study.
Compound 1 is the mimic of the natural substrate for TycB3-E and
2 the corresponding shortened dipeptidyl-CoA In precursor 3, the
C-terminal Phe (R 2 ) is D -configured While R 1 was kept constant
(Phe) in compounds 4–13, R2was varied In contrast, CoA 14
con-tains a constant Phe for R2and a Ser for R1 Compound 15–17
were designed for investigation of E domain tolerance towards
N-methylated substrates.
Trang 5basic [FH-CoA (12), FK-CoA (13)] amino acid
resi-dues for R2 Then, for evaluating the influence of the
N-terminal substrate part in SF-CoA (14) the
C-ter-minal Phe (R2) was kept constant while residue R1was
changed to Ser
The peptidyl-CoAs synthesized in this study (Fig 3) were basically designed to be suitable for investigating the TycB3-E domain specificity (construct TycB3 -PCP-E) In its natural environment within the tyrocidine biosynthetic template this E domain is responsible for epimerizing the tetrapeptidyl substrate fPFF-S-Ppant For keeping synthesis efforts as simple as possible we wanted to restrict ourselves to the utilization of dipept-idyl-CoA precursors Consequently, we first tested if the cognate fPS-Ppant and the shortened one FF-S-Ppant are converted with comparable efficiencies from l to d The corresponding CoA substrates 1 and
2 were used to load fPFF-S-Ppant and FF-S-Ppant onto TycB3-PCP-E with the help of Sfp, after which the epimerization reaction was followed up It could
be observed that both velocity of the l to d conversion and the portion of d-isomer regained from the enzyme after equilibration are comparable (data below and Table 1) This indicated that at least the two N-ter-minal amino acid residues of the original tetrapeptidyl substrate are not significantly involved in substrate recognition by the TycB3-E domain, allowing us to pursue our work with dipeptidyl-CoAs as model precursors
The E domain of FenD2 is originally embedded in a Thr activating module and thus should represent an epimerase with an opponent substrate specificity in comparison with TycB3-E [expected to be Phe (R2) optimized] FenD2-E in its natural environment within the fengycin synthetic template [22] (Fig 2) converts the tetrapeptidyl substrate EoYT-S-Ppant (l-Glu-d-Orn-l-Tyr-l-Thr-S-Ppant) Thus, the shortened dipep-tidyl-mimic (two C-terminal residues) of the natural substrate for FenD2-E is YT-S-Ppant Because Tyr and Phe are analogues belonging to the group of aro-matic amino acids, the synthesized dipeptidyl-CoAs
Fig 4 HPLC-MS analysis of dipeptides regained from the PCP-E-enzyme exemplified by the reaction of TycB3-PCP-E with Sfp and FF-CoA (1) After priming of the protein with the precursor utilizing Sfp the reaction was quenched in time dependent manner by the addition of 10% TCA Reaction products were released from the enzyme by thioester cleavage with 0.1 M KOH and applied to HPLC (A) Chromato-grams of the extracted [M + H] + mass signal, which is shown in (B), of separated L -Phe- L -Phe and L -Phe- D -Phe The traces show analyses of hydrolysed FF-CoA and samples taken at time points listed in the figure.
Fig 5 2D-Plot for illustration of epimerization activity exemplified
by the reaction of TycB 3 -PCP-E with different substrates After
loading of the corresponding peptidyl-S-Ppant moiety of the
CoA-precursors (shown in the legend underneath the plot) by the help
of Sfp, reactions were quenched at different points of time with
10% TCA Products were regained by alkaline cleavage of the
thio-ester and analysed by HPLC The amount of each isomer was
quantified and the formation of the D (R2)-peptides (in percentage)
presented in dependence of time.
Trang 6with N-terminal Phe should also be suitable for
inves-tigating this system
Nothing was known so far about the ability of E
domains deriving from initiation modules to convert
S-Ppants In general, loading the
peptidyl-S-Ppant moiety of the synthesized CoAs onto PCP-E
constructs is an elegant method of determining
toler-ance of the TycA- and GrsA-E domains (cognate
sub-strate Phe-S-Ppant) for elongated subsub-strates
In addition, the use of CoA precursors enabled us to
investigate the tolerance of E domains for
N-methyla-ted (alkylaN-methyla-ted) peptidyl substrates So far, this had
been tested with fusion proteins derived from the
actinomycin biosynthetic system [23] which did not lead
to the detection of converted product In this study we
applied the tripeptidyl precursor FFMeF-CoA (17)
because utilizing the dipeptidyl substrates FP-CoA (15)
and FMeF-CoA (16) resulted in exceedingly fast
forma-tion of the corresponding DKPs (diketopiperazines) for
noted reasons [24] as soon as loaded onto the protein
with Sfp or when hydrolysed in control reactions
Epimerization activity of TycB3- and FenD2-PCP-E
The cognate substrate of E domains localized in
elon-gation modules (‘peptidyl-’E domains) is a
nonribo-somally assembled peptidyl-S-Ppant intermediate At this, catalytic conversion from l to d only concerns the C-terminal amino acid residue of the enzyme bound substrate It had already been shown that the TycB3-E domain (probed with TycB3-ATE) converts
an l-Phe-S-Ppant substrate with reduced efficiency resulting in about 40% d-Phe [20] The epimerization velocity of TycB3-E with its cognate substrate (fPFF-S-Ppant) had not been published so far but the final
l⁄ d ratio of products was reported to be around 1 : 1 [16] We tested the epimerization activity of both the TycB3- and FenD2-E domain with various peptidyl-CoA precursors to acquire a conception of ‘peptidyl-’E domain substrate tolerance and to investigate the influ-ence of additional N-terminal amino acids on substrate recognition The results are summarized in Table 1 First investigations with the TycB3-PCP-E bidomain (as described above) revealed that the cognate tetra-peptidyl-S-Ppant substrate and its shortened dipeptidyl mimic are epimerized equally fast (both 1.9 min)1) and final d-portions of the regained peptides were also found to be in corresponding ranges (fPFf, 56%; Ff, 63%) With the same velocity a comparable equilib-rium (61% regained Ff) is reached when TycB3-E converts substrate Ff-S-Ppant (loading of 3) from d
to l By utilization of precursors FY-CoA (4) and
Table 1 Results of epimerization activity assay nd, not determined.
Precursor a
Bidomain system
kobs (min)1)
D ⁄ ( D + L ) (%) b
kobs (min)1)
D ⁄ ( D + L ) (%)
kobs (min)1)
D ⁄ ( D + L ) (%)
kobs (min)1)
D ⁄ ( D + L ) (%)
a Shown in Fig 4 b Measured after 15 min incubation c +, epimerization activity could be detected but not be quantified due to analytical reasons d As a consequence from the basic sample work up, the products detected here were not the linear peptides FK and Fk but the cyclic peptides resulting after nucleophilic attack of the Lys-e-amino group to the carbonyl group of the peptide-S-Ppant thioester Yet this has no effect on the observed Ca stereochemistry.
Trang 7F(p-fluoro)F-CoA (5) it was demonstrated that TycB3
-E is also tolerant towards unusual substituted Phe
ana-logues On the other hand, decreased activity
(1.2 min)1) was observed when TycB3-PCP-E was
primed with FW-CoA (6) and Sfp indicating that there
is no general preference for aromatic amino acid
resi-dues at the C-terminal position (R2) of the substrate
Instead, the sterical pretension of Trp might be
respon-sible for slowing down the conversion to the final
equi-librium (59% Fw) Likewise, other variations for R2
affected the epimerization velocity [1.2–1.5 min)1 when
using precursors FL-CoA (7), FN-CoA (8), and
FS-CoA (9)] but not the equilibrium position ( 60%
d-isomer in all cases) Similar results obtained by using
SF-CoA (14) permit speculations that the second
amino acid residue of the peptidyl-S-Pant substrate
(R1) is also involved in the recognition by this E
domain to some extent Peptides regained from the
enzyme after assaying the epimerization activity with
the S-Ppant precursors FD-CoA (11) and FH-CoA
(12) could only be analyzed qualitatively by the
des-cribed HPLC-MS methods It was possible to detect
converted products but poor separation and low signal
quality did not allow for a quantification of the results
The strongest decrease of efficiency was observed with
FK-S-Ppant (loading of 13, 0.8 min)1) even having an
effect on the portion of d-isomer regained from the
enzyme after equilibration (only 30% Fk) This clearly
indicates that a charged residue for R2 somehow
blocks up substrate recognition or correct binding in
the active site of TycB3-E and substrate tolerance is
limited
Because FenD2-E derived from a Thr activating
module, it was expected to have a preference for
substrates with hydrophilic C-terminal amino acid
residues Nevertheless, the catalytic conversion of
fPFF-S-Ppant, after loading of substrate 1 onto
FenD2-PCP-E, was carried out with an efficiency
(1.8 min)1, 56% fPFf) comparable to that of TycB3-E
(see above) Surprisingly, epimerization activity tested
with the precursors containing C-terminal variations of
aromatic amino acids (2–5) was only slightly decreased
[1.5–1.8 min)1, about 60% regained d(R2)-peptides] in
comparison to TycB3-E However, when FW-CoA (6)
was loaded onto FenD2-PCP-E (Trp for R2) the
effi-ciency of catalytic conversion leading to the final
equi-librium (55% Fw) again was lowered to 0.8 min)1 [see
assay with TycB3-PCP-E (1.2 min)1)] Unexpectedly,
FenD2-E showed no preference towards substrates
with C-terminal variations of hydrophilic and aliphatic
amino acids [use of FN-CoA (8) and FL-CoA (7),
1.2–1.3 min)1, 55% regained d-form], only FS-S-Ppant
[FS-CoA (9)] was converted more effectively
(1.8 min)1, 61% Fs) when compared to TycB3-E Nev-ertheless, FenD2-E showed even less efficient epimeri-zation (1.3 min)1) with the mimic of its cognate substrate FT-S-Ppant [FT-CoA (10)] resulting in only 41% Ft Apparently, substrate tolerance of ‘peptidyl-’E domains does not totally conform to the specificity of the module from which the E domain derives Activity after loading of FD-CoA (11) and FH-CoA (12) could
be observed but not be quantified (see above) As seen with TycB3-PCP-E, catalysis of epimerization after loading FK-CoA (13) onto FenD2-PCP-E was strongly impaired (0.9 min)1, 26% Fk) Also, a much slower reaction was observed when using SF-CoA (14) (0.9 min)1) resulting in 55% Sf-S-Ppant Here, catalysis seems to be affected due to the fact that both amino acid residues of the substrate are not cognate, which underlines the presumption that the two C-terminal amino acids are involved in recognition by ‘peptidyl-’E domains
Epimerization activity of TycA- and GrsA-PCP-E The cognate substrate of E domains deriving from ini-tiation modules is a PCP bound aminoacyl-S-Ppant precursor It has been reported that TycA-E and especially GrsA-E, investigated with rapid quench methods, are very efficient enzymes converting l-Phe-S-Ppant enzyme exceedingly fast to a final 2 : 1 (d-⁄ l-S-Ppant) equilibrium [13,14,19] Still, up to now
it has not been reported if the function of these ‘ami-noacyl-’E domains is decided by their position within the NRPS system or if their substrate specificity really differs from ‘peptidyl-’E domains In this study, we addressed this important question by loading peptidyl-S-Pant moieties of CoAs onto TycA- and
GrsA-PCP-E and were able to show epimerization activity of
‘aminoacyl-’E domains with elongated enzyme bound S-Ppant substrates This can be of great impact for future engineering approaches The results are summarized in Table 1
In general, TycA- and GrsA-E behave very simi-larly Both were able to convert FF-S-Ppant [loading
of FF-CoA (2)] almost as efficiently as TycB3-E (TycA-E, 1.8 min)1; GrsA-E, 1.7 min)1) equilibrating
to a slightly reduced amount of 55% Ff-S-Ppant when compared to the d-⁄ l-ratio with their cognate substrate Phe (see above) Surprisingly, a shifted equilibrium position was reached when the ‘amino-acyl-’E domains epimerize from d to l after loading
of Ff-CoA (3) onto the PCP-E bidomains (TycA, 71% Ff; GrsA, 61% Ff) The catalytic efficiency was further impaired by longer peptidyl substrates as seen when using fPFF-CoA (1) (TycA, 1.0 min)1;
Trang 8GrsA, 1.5 min)1) but this finding did not concern
the final product ratio (about 60% d-form)
Although the tested ‘aminoacyl-’E domains were
act-ive with peptidyl substrates, their tolerance towards
distinct S-Ppant precursors apparently deviates from
that of ‘peptidyl-’E domains Both constructs very
efficiently converted FY-S-Ppant (about 2.0 min)1) to
yield 60% Fy-S-Ppant and accepted
F(p-fluoro)F-S-Ppant (substrate 5) as well The use of FW-CoA
(6) not only showed a strong effect on the
epimeri-zation velocity (TycA, 0.8 min)1; GrsA, 1.3 min)1)
but also on produced amount of the corresponding
d-isomer (TycA, 35% Fw; GrsA 46% Fw) The
‘aminoacyl-’E domains seem to be impaired even
more by the bulky C-terminal Trp residue of the
substrate than the tested ‘peptidyl-’E domains When
converting enzyme bound FL-S-Ppant, the same
velocity (1.3–1.4 min)1) and final equilibrium of 60%
Fl-S-Ppant were observed, compared to TycB3- and
FenD2-E In contrast to TycB3-E, TycA- and
GrsA-E are more tolerant towards hydrophilic amino acids
for R2, displayed in strong activity with FN- (both
TycA- and GrsA-E, 1.9 min)1, 68% Fn) and
FS-S-Ppant intermediates (both > 2.0 min)1; TycA, 63%
Fs; GrsA, 68% Fs) Activity was also detected when
FD-CoA (11) and FH-CoA (12) were applied in the
assay, but results again could not be quantified due
to reasons described before As observed after
load-ing FK-CoA (13), the velocity of the catalytic
con-version was decreased (TycA, 1.0 min)1; GrsA,
0.9 min)1) as seen before with the ‘peptidyl-’E
domains Nevertheless, conversion by the ‘aminoacyl-’E
domains resulted in an increased portion of d-isomer
after equilibration (TycA, 52% Fk; GrsA, 54%
Fk) Obviously, TycA- and GrsA-E, in contrast to
TycB3- and FenD2-E, are preferably able to
coordi-nate and epimerize substrates with charged
C-ter-minal amino acid residues The epimerization activity
with SF-S-Ppant-enzyme [loading of SF-CoA (14)]
varied from results with FF-CoA (TycA, > 2.0
min)1; GrsA, 1.7 min)1, about 50% regained Sf) in
approximately the same order of magnitude as when using the precursor FS-CoA (9) This indicates that variations in both parts of the substrate can equally affect the epimerization activity of ‘aminoacyl-’E domains
Epimerization activity with N-methylated peptidyl substrates
Out of previous studies with fusion proteins [23] the question arose if E domains are compatible with pre-ceding N-methylation Theoretically, a methyl (alkyl) group attached to the nitrogen atom of the first pep-tide bond within the substrate should not cause hindrance of Ca-proton abstraction needed for the catalytic conversion Although a prolific interaction
of E domains with M domains on the enzymatic level remains an open question we could show that epime-rization is occurring after loading the Ppant moiety
of precursor FFMeF-CoA (17, i.e l-Phe-l-Phe-N-Me-l-Phe-CoA) onto all PCP-E-bidomain constructs utilized in this study (Table 1 and Fig 6) The N-methylated S-Ppant intermediate was converted very effectively by all enzymes (> 2.0 min)1); only TycA-E showed a slightly lowered activity (1.6 min)1) Additionally, a high portion of d-isomer (FFMef) was regained from the enzymes after adjust-ment of the final equilibrium (about 70% FFMef in all cases) Obviously, E domains are compatible with this class of precursors and furthermore N-methyla-tion of peptidyl-S-Ppant substrates seems even to support the catalytic conversion
Elongation of peptidyl substrates by the C domain of TycB1
Our results showed that E domains deriving from initi-ation modules are able to convert peptidyl-S-Ppant substrates instead of the cognate aminoacyl intermedi-ate To evaluate this finding in the context of biocom-binatorial approaches, an interesting question was if a
B
A
Fig 6 Separation of the N-methylated
trip-eptides FFMeF ⁄ FFMef by chiral HPLC The
peptides were regained from TycB3-PCP-E
after quenching the reaction at the points of
time following each trace Illustrated in
(A) are the corresponding signals for the
mass ([M + H] + ) shown in (B) obtained by
SIM-MS analysis.
Trang 9downstream C domain interacting in the native system
with an ‘aminoacyl-’E domain is able to accept and
elongate these enzyme bound peptidyl precursors The
upstream electrophilic donor site of the TycB1-C
domain showed in previous studies a relaxed specificity
concerning the transferred amino acid substrate, but
d-configuration is measurably preferred [15,25] In this
study, we also could reveal tolerance of the TycB1-C
domain for peptidyl substrates by using the well
estab-lished system TycA⁄ TycB1[10] TycA-PCP-E therefore
was primed with the peptidyl-S-Ppant moiety of
selec-ted CoAs with Sfp and preincubaselec-ted (see Experimental
procedures) for epimerization of the bound
intermedi-ates Separately, the acceptor enzyme TycB1-CAT⁄
TEsrf, chosen because it seemed to be a promising
candidate for fast product release [24], was
preincubat-ed to activate and covalently load l-Pro Product for-mation was initiated by mixing equal volumes of the protein solutions and stopped by enzyme precipitation with methanol Products contained in the supernatant were identified by HPLC-MS analysis
All detected products (as expected after condensa-tion with Pro) are shown in Table 2 As expected, only one product was observed when TycA-PCP-E was loa-ded with either FF- or Ff-CoA (2 and 3) and peptides transferred to TycB1-CAT⁄ TEsrf for elongation Cata-lytic release presumably yielded FfP with a detected mass of 410.3 gÆmol)1([M + H]+) No products were found when ATP was omitted from the assay (Fig 7) Also, formation of only one distinct product was detected when TycB1-CAT⁄ TEsrf processed the pep-tides transferred by TycA-PCP-E after loading of FL-and fPFF-CoA (7 FL-and 1) Thus, TycB1-C is able to accept and elongate all of these peptidyl-S-Ppants up
to a length of four amino acids (substrate 1 was the longest used in our study) Although we have not com-pared the formed tripeptides and pentapeptides to chemical standard compounds, the detection of only one product indicates that the stereo-selectivity of the donor site is retained when TycB1-C accepts these peptidyl substrates Yet, the C-terminal part of the peptidyl-S-Ppant substrate accepted by TycB1-C seems
to be involved in recognition, as seen before with the tested E domains After using FT-CoA (10) and FK-CoA (13) in the elongation assay, no detectable products with the expected mass (FtP, [M + H]+¼
364 gÆmol)1; FkP, [M + H]+¼ 391 gÆmol)1) were released by TycB1-CAT⁄ Tesrf, clearly indicating that
Table 2 Identification of elongated peptidyl products investigated
in the system TycA ⁄ TycB1 nd, no product detected.
Precursor for
TycA-PCPE a
Expected
product
Ionization method Species
Observed mass (calculated mass) [g mol)1] FF-CoA (2) FfP ESI [M + H] + 410.3 (410.2)
Ff-CoA (3) FfP ESI [M + H] + 410.3 (410.2)
FL-CoA (7) FlP ESI [M + H]+ 376.3 (376.2)
FS-CoA (9) FsP ESI [M + H] + 350.1 b (350.2)
FT-CoA (10) FtP ESI [M + H] + nd (364.2)
FK-CoA (13) FkP ESI [M + H]+ nd (391.2)
SF-CoA (14) SfP ESI [M + H] + 350.1 d (350.2)
fPFF-CoA (1) fPFfP ESI [M + H] + 654.3 (654.3)
a Shown in Fig 4 b A product mixture of 1 : 1 (presumably
FsP ⁄ FSP) was detected c A product mixture of 5.4 : 1 (presumably
SfP ⁄ SFP) was detected.
Fig 7 HPLC analysis of peptidyl substrates elongated by the C domain of TycB1 As an example, the formation of the tripeptide FfP is illus-trated in (A) TycA-PCP-E was primed with FF-S-Ppant by help of Sfp and FF-CoA (2), allowed to equilibrate, and incubated with TycB1-CAT ⁄
TE srf [24], which had been preincubated to load Pro FF was presumably produced by in trans action of the thioesterase of TycB 1 -CAT ⁄ TE srf
with FF-CoA Nevertheless, TycB1-C accepts and elongates Ff to form FfP (black trace) that was released by the acceptor enzyme and could
be identified by detection of the corresponding [M + H] + mass signal shown in (B) No product was detected when ATP was omitted in the assay (grey trace).
Trang 10the TycB1-C donor site discriminates noncognate
C-terminal amino acid residues of the S-Ppant
sub-strate The assay with FS-CoA (9) resulted in a 1 : 1
mixture of products with the corresponding mass
(m⁄ z) of 350 ([M + H]+) as expected for FsP⁄ FSP
Likewise, a 5.4 : 1 mixture of products was obtained
after transfer of SF [loading of SF-CoA (14) onto
TycA-PCP-E] to TycB1-CAT⁄ TEsrf Although, due to
missing chemical standards, the actual stereochemistry
of these products remains obscure, considering the
results of the epimerization assay with TycA-PCP-E,
we expect the portion of 5.4 (84%) to be SfP This
indicates that the second amino acid residue (R1) of
the peptidyl-S-Ppant substrate coming from
TycA-PCP-E influences stereo-selection by the TycB1-C
domain donor site only slightly
Discussion
In this study, we gained new information on substrate
recognition and specificity of E domains focusing onto
aspects of potential decisive sites (amino acid residues)
of a peptidyl-substrate recognized by an E domain with
the aim to utilize E-domains for the redesign of
non-ribosomal peptides The main issue to be addressed was
if the substrate specificity of E domains is concordant
with the specificity of the module (activated amino acid)
it originates from Besides this, an interesting question
was if E domains of initiation modules are generally
able to epimerize peptidyl- instead of their cognate
aminoacyl-substrates (‘aminoacyl-’ vs ‘peptidyl-’E
domains) For this purpose, the use of CoA precursors
and different apo-PCP-E bidomain constructs turned
out to be very useful for following up epimerization
reactions and evaluating the activities of E domains
The approach had already been used in similar manner
for the characterization of C [15,16] and TE domains
[26] We accomplished covalent loading of the
peptidyl-S-Ppant moiety of the peptidyl-CoAs onto the invariant
Ser of each PCP domain by exploiting the promiscuous
Ppant transferase Sfp [8,9] The advantage of this new
minimal system for the investigation of E domain
spe-cificity is not only the circumvention of natural
sub-strate activation but also the HPLC based analysis of
reaction products in contrast to the previously used
radio-TLC assay [13,14,20] Also, the E domains stay in
native connection to their respective PCPE, which was
shown to be essential for E domain activity [13], and
consequently should behave as if they are in their native
enzymatic environment
We investigated substrate recognition and tolerance
of two ‘peptidyl-’E domains (proteins TycB3- and
FenD2-PCP-E) and two ‘aminoacyl-’E domains
(proteins TycA- and GrsA-PCP-E), respectively As expected, TycB3-E shows a preference for peptidyl-S-Ppant substrates with C-terminal Phe and Phe ana-logues Nevertheless, the use of precursor 6 with a Trp
in position R2revealed a discrimination of this aroma-tic residue This finding was surprising because the module TycB3 was reported to be activating Trp as well with great efficiency [27] Therefore, one would have concluded that TycB3-E should be similarly able
to epimerize a peptidyl-S-Ppant substrate with a C-ter-minal Trp However, TycB3-E is tolerant towards a broad variety of peptidyl-S-Ppant substrates with altered C-terminal amino acid residues
Assuming that the specificity of an E domain (recog-nition of the C-terminal site of the substrate) is identi-cal with the specificity of the module it originates from, we decided to investigate FenD2-E as first exam-ple of an E domain contained in a module which is not naturally Phe but Thr specific As described earlier, the synthetic CoA precursors were also suitable for this system Interestingly, FenD2-E showed no general enhanced activity with substrates containing C-ter-minal hydrophilic amino acid residues Instead, even a slightly reduced rate (1.3 min)1) was detected when we used the mimic-precursor of the cognate substrate (FT-CoA, 10) Moreover, this reaction resulted in only 41% Ft Seemingly, there is no direct correlation between the substrate specificity of E domains and the specificity of the module they originate from In sum-mary, the substrate tolerances of the two different
‘peptidyl-’E domains investigated in this study conform more with each other and the substrate specificities are less evolved than expected In general, both E domains tolerate a broad variety of altered substrates
Within the scope of the investigations reported here,
we also discovered acitvity of the two ‘aminoacyl-’E domains (TycA- and GrsA-E) with peptidyl-S-Ppant substrates In contrast, applying another strategy with fusion proteins and natural substrate activation as reported before for the investigation of tolerance towards altered aminoacyl substrates [13] would require the construction of large and complex protein systems This is again emphasizing the convenience of our minimized assay approach utilizing PCP-E bi-domains with synthetic peptidyl-CoAs and Sfp Both PheATE initiation modules, TycA- and especially GrsA-ATE [19] have been very well characterized in the past, so far most often investigated in the so called DKP assay [10], and known to resemble each other in many characteristics Thus, we expected and observed great resemblance between their two ‘aminoacyl-’E domains Two striking differences were found in con-trast to the tested ‘peptidyl-’E domains First, when