Transcript Barbara Soldo

EXPLORING THE ROLE OF A CONSERVED MOTIF IN THE
ADENYLATION DOMAIN OF A NON-RIBOSOMAL PEPTIDE
SYNTHETASE FROM BACILLUS BREVIS
V. Bučević-Popović, M. Šprung, B. Soldo, S. Orhanović, M. Pavela-Vrančić
Faculty of Science, Department of Chemistry, Teslina 12, Split, CROATIA
Introduction
module 1
Nonribosomal peptide synthetases (NRPS) are highly sophisticated natural nanomachines that catalyze the
synthesis of small peptides with antibiotic, immunosuppressant, and anticancer activities. NRPS usually contain
one module for each amino acid incorporated into the final peptide product. Each module consists of several
catalytic domains that catalyze the activation of specific amino acids (adenylation (A) domain), covalent thioester
binding (peptidyl-carrier-protein (PCP) domain), formation of peptide bond (condensation (C) domain), and
optionally, various substrate modifications (Fig. 1) (1).
E
A
PCP
Phe
TycA
module 2
module 4
module 3
C
A
PCP C
Pro
A
PCP C
Phe
A
PCP
Phe
module 6
module 5
E
C
PCP
Te
A9
Figure 3. A9 core motif. Sequence alignment of representative Adomains showing the region surrounding A9 core motif. The sequences
correspond to the following proteins: P09095, tyrocidine synthetase 1
from Bacillus brevis; Q01757, ACV synthetase from Streptomyces
clavuligerus; P19828, AngR protein from Vibrio anguillarum; P11454,
enterobactin synthetase component F from Escherichia coli; P0C062,
gramicidin S synthetase 1 from Bacillus brevis; Q08787, surfactin
synthetase C from Bacillus subtilis.
adenylate-forming
conformation
C-terminal
subdomain
rotation
140
Activity (%)
120
100
80
60
40
20
0
A
WT
L484A
P485A
P485G
Y487A
M488A
P490A
P490G
S491R
A9
160
L-Phe
140
Activity (%)
120
D-Phe
100
80
60
40
20
0
B
WT
L484A
P485A
P485G
Y487A
M488A
P490A
P490G
S491R
thioester-forming
conformation
A
PCP C
Tyr
A
PCP C
Val
A
PCP C
Orn
A
PCP Te
Leu
-adenylation domain
C
- condensation domain
-peptidiyl carrier protein
E
- epimerisation domain
-thioesterase domain
antibiotic tyrocidine biosynthetic system from bacterium Bacillus brevis.
Full assembly consists of three NRPS, each composed of one (TycA),
three (TycB), and six (TycC) modules.
Results and Discussion
Phe-AMP
PPi
A
PCP C
Gln
module 9
Figure 1. Tyrocidine synthetase 1 (TycA) is the first component of the
A9
ATP
A
PCP C
Asn
module 10
TycC
Adenylation (A) domain of NRPS catalyzes the two-step reaction of ATP-driven activation of amino acid and
its transfer to the PCP domain. Together with the with acyl- and aryl-CoA synthetases and firefly luciferase, Adomains constitute the superfamily of adenylate-forming enzymes. It has recently been proposed that
adenylate-forming enzymes use a 140° domain rotation to present opposing faces of the C-terminal subdomain
to the active site for the different partial reactions (2) (Fig. 2). Sequence alignment of A-domains allowed
identification of 10 ‘core motifs’ (A1-A10), most of which were assigned particular functions in substrate binding
and/or catalysis, based on structural and mutagenesis studies. This study was aimed at elucidating the role of
A9 core motif (Fig. 3), whose function has not been established so far, using tyrocidine synthetase 1 (TycA)
from B. brevis as a model.
Phe
module 7
TycB
A
A
module 8
Figure 2.
Two-step reaction catalyzed
by TycA adenylation domain.
In the first half-reaction, L-Phe
is converted into enzymebound L-Phe-adenylate (L-PheAMP). In the second step, the
activated L-Phe is transferred
to the thiol group of PCP-linked
4’-phosphopantethein cofactor.
Homology models for TycA Adomain were derived by
MODELLER using A-domain
from gramicidin S synthetase 1
(PDB ID: 1amuA) for adenylateand and D-alanyl carrier protein
ligase for thioester-forming
conformation (PDB ID: 3e7w)
as templates.
N-terminal subdomain is shown
in yellow, C-terminal
subdomain in blue. Several
highly conserved motifs that
form the active site are shown
in red, while the motif
comprising conserved Asp that
serves as a hinge for domain
rotation is shown in green. Due
to subdomain movement, A9
motif (shown in dotted van der
Waals surface) occupies
different positions relative to the
active site.
Generation and Purification of A9 Mutants of TycA. To examine the role of
A9 conserved motif in A-domain of TycA, a set of eight mutant enzymes
carrying single amino acid substitutions was created. Five residues in A9
motif (Fig. 3), L484, P485, Y487, M488 and P490, were mutated to Ala or
Ala and Gly, while S491 following immediately after A9 region was mutated
to Arg. The wild type and mutant enzymes were expressed in E. coli and
purified to homogeneity according to Pfeifer et al. (3). Two mutants, P490A
and P490G revealed a significantly decreased solubility, indicating that
P490 residue might play a structural role.
Limited Proteolysis of TycA. To evaluate the effect of A9 mutation on protein
conformation, we employed partial proteolytic digestion with trypsin (Fig.
4.). In agreement with previous work (4), tryptic cleavage of wild type TycA,
produces four main fragments resulting from cleavage in linker regions
between domains or subdomains. However, tryptic digest of P490 mutants,
lack the largest fragment containing C-terminal subdomain. Fast proteolytic
degradation indicates a less compact fold of C-terminal subdomain as a
result of mutation.
wt TycA
t (min)
0
5
10
15
30
P490A TycA
60
90
t (min)
A
AC
PCP
E
PCP
E
0
5
10
15
30
60
90
PCP
E
E
E
AN
AN
PCP
A
P490ATycA were performed in 50 mM, pH 7.5, at protein:protease ratio 50:1 (w:w).
in the absence of substrates. In the presence of substrates (L-Phe and ATP), the
rate of proteolysis was decreased but the proteolytic pattern remained the same.
activity. Adenylation activity was measured by ATP-[32P]PPi
exchange assay (A) using L-Phe as a substrate and continuous
spectrophotometric pyrophosphate assay (B) with either L-Phe or
D-Phe. Reaction mixtures contained saturating substrate
concentration (1 mM). Activities are expressed relative to the
value measured with the wild type TycA.
Adenylation Activity of A9 Core Motif Mutants. The aminoacyladenylate formation was first assayed by the amino-acid
dependent ATP-[32P]PPi exchange assay in the presence of
natural substrate L-Phe (Fig. 5A). The rates of the exchange
indicate that, with the exception of P490 mutants, single amino
acid substitutions in A9 region have no significant influence on
TycA adenylation activity. The effect of P490 mutation is
especially evident in P490G enzyme, in which substitution of
rigid proline with flexible glycine results in drastic reduction of
enzymatic acitivtiy. Essentially the same results were obtained
with complementary continuous spectophotometric
pyrophosphate assay (Fig. 5B). In the absence of an activated
amino acid acceptor (e. g. a holo-PCP domain), PPi release rate
is limited by the slow leakage of amino-acyl adenylate from the
active site (5). Although faster PPi release was measured in
presence of non-cognate D-Phe (approximately fivefold), relative
activities of A9 mutants compared to the wild type were
comparable irrespective of the stereoisomer used.
Figure 4. Patrial tryptic digestion of TycA. Proteolytic digestions of wild type and
PPi Release Activity in the Presence of Amino-acyl Acceptor.
In order to monitor the complete A-domain catalyzed reaction,
we attempted to measure acceleration of PPi release as a result
of interdomain transfer of aminoacyl to TycA holo-PCP domain
added in trans. TycA PCP domain was coexpressed together
with Srf protein, catalyzing its in vivo posttranslational priming
with 4’-phosphopantetein cofactor. Although, HPLC analysis (not
shown) indicates that the majority of PCP protein is in its holo
form, the acceleration of PPi release upon addition of holo-PCP
was not observed.
PPi release was also monitored in the presence of CoA, that
may serve as a mimic of a 4’-phophopanthetein cofactor bound
to PCP (6). CoA stimulates PPi release activity of TycA in
presence of D-Phe (Fig. 6), while it has no effect when assayed
in presence of TycA natural substrate, L-Phe. In the presence of
natural substrate, enzyme probably adopts the conformation
that is selectively suited for transfer of amino acid to holo-PCP.
Whether slightly reduced ability to transfer activated amino acid
to CoA in presence of non-cognate amino acid reflects the
influence of mutations in A9 motif on the second half-reaction of
A-domain catalytic cycle, remains to be further examined.
Acknowledgements
This work is supported by the grant from the Croatian Ministry
of Science, Education and Sports 177-0000000-2962
Figure 6. PPi release rate in the
presence of CoA. The most prominent
increase was observed for S491R enzyme,
designed to resemble post-A9 region of 4chlorobenzoate:CoA ligase, which uses
CoA as an acceptor cosubstrate in the
second half-reaction (7). The increase of
PPi release rate by CoA was smaller than
in wild type enzyme for all of the other
mutant proteins. Due to the low
adenylation activity, P490G mutant was not
assayed with CoA. The PPi release rate
was assayed with D-Phe as amino acid
substrate.
● WT
■ L484A
○ P485A
● P485G
□ Y487A
● M488A
■ P490A
■ S491R
Initial velocity (min-1)
Figure 5. Effect of mutations in A9 motif on TycA adenylation
SH
Phe-AMP
[CoA] (mM)
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