Mutational analysis of the C-domain in nonribosomal peptide
synthesis
Veit Bergendahl*, Uwe Linne and Mohamed A. Marahiel
Biochemie/Fachbereich Chemie, Philipps-Universita
Èt Marburg, Germany
The initial condensation event in the nonribosomal biosyn-
thesis of the peptide antibiotics gramicidin S and tyrocidine A
takes place between a phenylalanine activating racemase
GrsA/TycA and the ®rst proline-activating module of GrsB/
TycB. Recently we established a minimal in vitro model
system for NRPS with recombinant His
6
-tagged GrsA
(GrsA
Phe
-ATE; 127 kDa) and TycB1 (TycB1
Pro
-CAT;
120 kDa) and demonstrated the catalytic function of the
C-domaininTycB1
Pro
-CAT to form a peptide bond
between phenylalanine and proline during diketopiperazine
formation (DKP). In this work we took advantage of this
system to identify catalytically important residues in the
C-domain of TycB1
Pro
-CAT using site-directed mutagenesis
and peptide mapping. Mutations in TycB1
Pro
-CAT of 10
strictly conserved residues among 80 other C-domains with
potential catalytic function, revealed that only R62A,
H147R and D151N are impaired in peptide-bond
formation. All other mutations led to either unaected
(Q19A, C154A/S, Y166F/W and R284A) or insoluble
proteins (H146A, R67A and W202L). Although 100 n
M
of
theserineproteaseinhibitorsN-a-tosyl-
L
-phenylalanylchlo-
romethane or phenylmethanesulfonyl ¯uoride completely
abolished DKP synthesis, no covalently bound inhibitor
derivatives in the C-domain could be identi®ed by peptide
mapping using HPLC-MS. Though the results do not reveal
a particular mechanism for the C-domain, they exhibit a
possible way of catalysis analogous to the functionally
related enzymes chloramphenicol acetyltransferase and
dihydrolipoyl transacetylase. Based on this, we propose a
mechanism in which one catalytic residue (H147) and two
other structural residues (R62 and D151) are involved in
amino-acid condensation.
Keywords: nonribosomal peptide synthesis; nonribosomal
peptide synthetases; peptide synthetases; condensation
domain; chloramphenicolacetyltransferase.
A broad range of organisms utilize nonribosomal peptide
synthesis to produce an immense spectrum of bioactive
peptides (antibiotics, siderophores, biosurfactants and
immunosuppressants, as well as antitumor and antiviral
agents). For that purpose, they avail themselves a large
number of amino and carboxy acids as substrates. The
biosynthesis of these pharmacological signi®cant agents is
performed by nonribosomal peptide synthetases (NRPS),
which in their modular organization are related to poly-
ketide synthases (PKS) [1,2]. These large multifunctional
enzymes are arranged in assembly lines with specialized
units completely equipped for the correct activation and
incorporation of a single substrate. Such catalytic units,
referred to as modules, are composed of functionally speci®c
and independent domains, each of them responsible for
catalyzing one single reaction. Remarkably, the order of the
modules (with a repetitious assembly of domains) is
predominantly colinear to the ®nal product [3,4].
The C-domain, a 450-amino-acid expanding region at the
N-terminus of each elongating module, was attributed after
extensive sequence analysis with the condensation activity.
It was previously con®rmed to be responsible for the
catalysis of peptide bond formation by the development of a
minimized system representative for this family of enzymes
[5]. Furthermore recent ®ndings indicate that the C-domains
are bearing signi®cant substrate selectivity for the nucleo-
philic acceptor amino acid and an enatioselectivity for the
electrophilic donor substrate [6]. The inherent selectivity at
the acceptor site has been shown to prevent internal
misinitiation of the biosynthetic process and to control the
timing of substrate epimerization [7]. Recognition and
activation of the substrate amino acid are facilitated by the
A-domain [8] through carboxy adenylation of the substrate.
Hence, the substrate selectivity of the A-domains [9]
simultaneously determines the primary sequence of the
product. Subsequently the activated amino acid is tethered
to the terminal thiol moiety of a 4¢-phosphopantetheinyl
(Ppant) group [10,11]. This Ppant-cofactor itself is post-
translationally transferred to a conserved serine residue of
the T-domain also designated as PCP (peptidyl carrier
protein) by a special class of CoASH binding 4¢-Ppant-
transferases [12±14]. Besides these three domains (C-A-T),
which are essential for a functional elongating module, there
are some optional domains for further modi®cation of the
Correspondence to M. A. Marahiel, Biochemie/Fachbereich Chemie,
Philipps-Universita
Èt Marburg, Hans-Meerwein-Straûe, 35032 Mar-
burg, Germany. Fax: + 49 6421 2822191, Tel.: + 49 6421 2825722,
E-mail: marahiel@chemie.uni-marburg.de
Abbreviations: A-domain, adenylation domain; C-domain, condensa-
tion domain, DKP,
D
-Phe-
L
-Pro-diketopiperazine; E-domain, epi-
merization domain; IPTG, isopropyl thio-b-
D
-galactoside; LSC, liquid
scintillation counting; NRPS, nonribosomal peptide synthetases;
PKS, polyketide synthases; Ppant, 4¢-phosphopantetheine; PP
i
,inor-
ganic pyrophosphate; T-domain, thiolation domain (also described as
PCP, peptidyl-carrier-protein).
*Present address: McArdle Laboratory for Cancer Research, Univer-
sity of Wisconsin, Medical School, 1400 University Avenue, Madison,
WI 53706, USA.
(Received 16 August 2001, revised 15 November 2001, accepted 20
November 2001)
Eur. J. Biochem. 269, 620±629 (2002) ÓFEBS 2002
substrate amino acids. Those domains are the epimerization
domain (E-domain) for C
a
-epimerization [15], the methyl-
transferase domain (M-domain) for N-methylation [16] and
the cyclization domain (Cy-domain) [17]. These latter
domains are related to the C-domains, as they catalyze the
simultaneous condensation and heterocyclization of two
aminoacyl or peptidyl substrates. Release of the ®nal
product is catalyzed by a thioesterase (Te)-like domain
found at the C-terminal terminating module of NRPSs
templates [18,19].
The enormous size and complexity of most peptide
synthetases (up to 1.6 MDa [20]) have signi®cantly
restrained a more detailed study of the C-domain function
in the past. Therefore, we previously established a mini-
mized NRPS in vitro system [5], which comprises of the
initiation module GrsA
Phe
-ATE (phenylalanine-activating
module; A-, T- and E-domain) from the gramicidin S
system and the ®rst module in the second peptide synthetase
of the tyrocidine A system TycB1
Pro
-CAT (proline-activat-
ing module; C-, A-, and T-domain; see Fig. 1). Both
proteins can be obtained in active form as recombinant
His
6
-tag fusions by overexpression in E. coli [5]. By applying
previously described in vitro assays, it is now possible to
monitor the condensation of their cognate substrates
L
-Phe
and
L
-Pro, and the presumably uncatalyzed intramolecular
cyclization that ends up in the release of the cyclic dipeptide
D
-Phe-
L
-Pro-diketopiperazine (DKP; see Fig. 2). The same
system was also utilized to demonstrate that C-domains
possess an intrinsic editing function for the incoming
aminoacyl moiety [6]. By using aminoacyl-S-CoA as probes
it was shown that the ®rst C-domain of the tyrocidine
synthetase complex possesses an enantioselectivity at the
electrophilic donor site (
D
-Phe) and a substrate speci®city at
the nucleophilic acceptor site (
L
-Pro) in the formation of the
chain-initiating
D
-Phe-
L
-Pro dipeptidyl intermediate. The
knowledge about the architecture that creates this selectivity
and the residues, which are involved in catalysis of peptide-
bond formation remained largely unclear.
Sequence analysis revealed a highly conserved motif
HHxxxDGx(S/C), commonly called ÔHis-motifÕ,thatwas
suspected to participate in the catalysis. This hypothesis was
supported by the ®nding that a single mutation of the
Fig. 1. Genes and domain organizations of
gramicidin S and tyrocidine A synthetases.
These genes lead to the minimal system com-
prising the NRPSs GrsA
Phe
-ATE (from grsA)
and TycB1
Pro
-CAT (from tycA). Domains are
depicted to illustrate the module structure. The
amino-acid selectivity of each A-domain is
indicated using the three-letter code. TycA and
GrsA are highly similar to each other and can
be used interchangeably, just as GrsB1 and
TycB1.
Fig. 2. Currently accepted model of the con-
densation of
L
-Phe and
L
-Pro as catalyzed by
the peptide synthetases GrsA
Phe
-ATE and
TycB1
Pro
-CAT. The crucial known steps of
peptide bond formation in NRPSs are illus-
trated for the current system using the texture
code of Fig. 1. First the substrate amino acids
are activated under ATP hydrolysis as
an adenylate and then enzyme bound on the
PpantmoietyofeachT-domainasa
thioester.
L
-Phe-S-Ppant is then epimerized by
the E-domain of GrsA
Phe
-ATE, but
only
D
-Phe-S-Ppant undergoes condensation
with
L
-Pro-S-Ppant presented at the C-do-
main of TycB1
Pro
-CAT. Free GrsA
Phe
-ATE
can now be reloaded whereas
D
-Phe-
L
-Pro-
DKP can be released by intramolecular cycli-
zation on TycB1
Pro
-CAT.
ÓFEBS 2002 Catalysis of nonribosomal peptide-bond formation (Eur. J. Biochem. 269) 621
second histidine residue (italic) in TycB1
Pro
-CAT (H147V)
was suf®cient to abolish peptide bond and DKP formation
with GrsA
Phe
-ATE [5]. Furthermore, a chromosomal point
mutation in srfA-B, changing a D to A in the His-motif
(italic) of the corresponding Asp domain of the surfactin
synthetase was found to abolish surfactin production in
the B. subtilis producer strain [21]. Accordingly, the pres-
ence of a catalytic diad or triad [22] was discussed on the
basis of sequence analysis and in a proposed analogy to the
family of dihydrolipoyl transacetylases or chloramphenicol
acetyltransferases [23,24]. Among the questions about
C-domain function are (a) what (additional) residues are
important for catalysis of peptide-bond formation, (b) how
are these residues arranged in the catalytic center, and (c)
whether the growing aminoacyl (or peptidyl) donor is
getting (at least at same point) covalently tethered to the
C-domain? To address these questions, mutants in residues
conserved across 80 NRPS C-domains were constructed in
the C-domain of TycB1
Pro
-CAT and assayed for their
ability to catalyze peptide bond formation between
D
-Phe-
S-Ppant-GrsA
Phe
-ATE and
L
-Pro-S-Ppant-TycB1
Pro
-
CAT. Furthermore, the minimal system GrsA
Phe
-ATE/
TycB1
Pro
-CAT was compromised with two inhibitors
(phenylmethanesulfonyl ¯uoride and N-a-tosyl-
L
-phenyl-
alanylchloromethane), and analyzed for the appearance
of possible covalent C-domain/inhibitor complexes in order
to ®nd a catalytic triade similar to the one in serine
proteases.
EXPERIMENTAL PROCEDURES
Sequence alignments for the identi®cation of highly
conserved residues
The sequences of more than 80 C-domains were retrieved
from publicly accessable databases (NCBI, SwissProt, etc.).
Sequences used were derived from biosynthesis systems of
gramicidin S (Grs), tyrocidin A (Tyc), surfactin (Srf),
lichenysin (Lic), bacitracin (Bac), fengycin (Fen), entero-
bactin (Ent), chloroeremomycin (Cep), pristinamycin (Snb),
enniatin (Esyn), HC-toxin (Hts1), penicillin (Acv) and
cyclosporin (SimA). After outlining the 450-amino-acid
stretches, the sequences were aligned using the program
MEGALIGN
from the DNA Star package, applying the Jotun
Hein algorithm with default parameters.
Site-directed mutagenesis and cloning
All TycB1
Pro
-CAT mutants were constructed by site-
directed mutagenesis of pProCAT [5] using either the so-
called ÔMegaprimer-PCRÕmethod [25] with the Expand
long-range PCR system
TM
(Bo
Èhringer Mannheim, Germa-
ny) or the QuickChange
TM
Site-Directed Mutagenesis Kit
(Stratagene). Restriction sites for subsequent cloning and
screening were introduced with PCR oligonucleotides
(MWG-Biotech, Germany) summarized in Table 1. Quick-
Change
TM
mutagenesis was carried out in accordance with
the manufacturer's protocol. For the ÔMegaprimer-PCRÕ,
PCR products were puri®ed with the QIAquick-spin PCR
puri®cation kit (Qiagen, Germany), digested with NcoIand
ligated. Standard procedures were applied for all DNA
manipulations [26] and the Escherichia coli strain XL1-Blue
[27] was used for cloning. The mutant TycB1
Pro
-
CAT(H147V) was obtained by site-directed mutagenesis
as described previously [5]. Together with the desired point
mutations, additional silent mutations were introduced in
order to generate a new restriction site, which allowed a
simple detection of all mutated plasmids. The fusion sites
between vector and insert, as well as the mutation-site of the
plasmids pProCAT were con®rmed by DNA sequencing
using an ABI-Prism 310 Genetic Analyzer with standard
protocols described by ABI (Applied Biosystems,
Germany).
Expression and puri®cation of functional holo-peptide
synthetase fragments
To achieve in vivo production of functional holo-peptide
synthetase modules, the Ppant-transferase gene gsp was
coexpressed with the pQE60 plasmids carrying the DNA
fragments for the TycB1
Pro
-CAT mutants. Expression and
puri®cation using single-step Ni
2+
-af®nity chromatography
was performed according to previously published proce-
dures [5]. Purity of the proteins was judged by SDS/PAGE
(data not shown). Fractions containing the recombinant
proteins were pooled and dialyzed against assay buffer
(50 m
M
Hepes, pH 8.0, 100 m
M
sodium chloride, 10 m
M
magnesium chloride, 2 m
M
dithioerythritol and 1 m
M
EDTA). After addition of 10% glycerol (v/v), the proteins
could be stored at )80 °C. Protein concentrations were
determined using the calculated extinction coef®cients for
the A
280
of the proteins: 138 690
M
)1
ácm
)1
for GrsA
Phe
-
ATE and 92 230
M
)1
ácm
)1
for TycB1
Pro
-CAT and its
mutants.
ATP-pyrophosphate exchange assay
The ATP-pyrophosphate exchange reaction was carried out
to examine the adenylation activity of all recombinant
peptide synthetase fragments puri®ed. Reaction mixtures
contained (®nal volume: 100 lL): 50 m
M
Hepes, pH 8.0,
100 m
M
sodium chloride, 10 m
M
magnesium chloride,
1m
M
EDTA, 1 m
M
amino acid and 300 n
M
enzyme. The
reaction was initiated by the addition of 2 m
M
ATP, 0.2 m
M
tetrasodium pyrophosphate and 0.15 lCi of tetrasodium
[
32
P]pyrophosphate (NEN/DuPont) and incubated at 37 °C
for 10 min. Reactions were quenched by adding 0.5 mL of a
stop mix containing 1.2% (w/v) activated charcoal, 100 m
M
tetrasodium pyrophosphate and 350 m
M
perchloric acid.
Subsequently, the charcoal was pelleted by centrifugation,
washed once with 1 mL water and resuspended in 0.5 mL
water. After addition of 3.5 mL of liquid scintillation ¯uid
(Rotiscint Eco Plus; Roth, Germany), the charcoal-bound
radioactivity was determined by liquid scintillation counting
(LSC) with a 1900CA Tri-Carb liquid scintilliaton analyzer
(Packard).
Thioester formation: radioassay for the detection
of covalent amino-acid incorporation
Reaction mixtures in assay buffer (50 m
M
Hepes, pH 8.0,
100 m
M
sodium chloride, 10 m
M
magnesium chloride,
1m
M
EDTA) contained 500 n
M
enzyme, 2 m
M
ATP and
2l
M
[
14
C]-amino acid (Hartmann, Germany). The thio-
esteri®cation was initiated upon the addition of the radio-
labeled amino acid and the reaction was quenched after
622 V. Bergendahl et al. (Eur. J. Biochem. 269)ÓFEBS 2002
10 min by the addition of 1 mL chilled 10% (w/v)
trichloroacetic acid and incubated on ice for 15 min. The
precipitates were pelleted by centrifugation (4 °C, 16 000 g)
and washed two times with 1 mL 10% trichloroacetic acid
(w/v). The pellet containing the acid-stable label was then
dissolved in 150 lL formic acid and quanti®ed by LSC as
described above.
Method of normalization for values in condensation
assays
A common margin of error is made when determining a
protein concentration by the calculated extinction coef®-
cient. According to our experience, the current set of
proteins has shown that thioesteri®cation activities may
vary signi®cantly (up to threefold) depending on the batch
of protein utilized in the assays. In the present work, we tried
to take this behaviour into consideration by normalizing the
values with the thiolation activity as an internal standard for
amount of active protein. We normalized the values
obtained in all the assays for dipeptide formation and for
DKP production by multiplying the counts in the radioac-
tive assays and the area in the HPLC-assays by the ratio of
counts in the thiolation assay of mutant over wild-type.
Values in the elongation assay were expressed as relative
values to the value at t0 which was set as 100%. DKP
amounts were also expressed as relative values (percent of
wild-type value).
Radio assay for the detection of elongation
500 n
M
of holo-enzyme (GrsA
Phe
-ATE and TycB1
Pro
-
CAT) were preincubated seperately in assay buffer with
their substrate amino acids [2 l
M
[
14
C]
L
-Phe (450 mCiám-
mol
)1
), 100 l
ML
-Pro] and ATP (2 m
M
). After 3 min,
product formation was initiated by mixing equal volumes
of reaction mixtures. At various time-points, 200 lL
aliquots were taken and immediately quenched by addition
of 1 mL ice-cold trichloroacetic acid (10%). After 15 min
on ice, samples were centrifuged (4 °C, 16 000 g)for
20 min, washed two times with 1 mL ice-cold trichloro-
acetic acid, redissolved in 150 lL formic acid and quan-
ti®ed by LSC.
DKP formation: indirect assay for
D
-Phe-
L
-Pro dipeptide
formation
The formation of the dipeptide was analyzed using GrsA
Phe
-
ATE and the different C-domain mutants of TycB1
Pro
-
CAT. To ensure a complete acylation of the peptide
synthetase fragments with their cognate amino acids, a
preincubation was carried out for 3 min at 37 °C; 1 l
M
GrsA
Phe
-ATE was incubated in assay buffer containing
2m
M
ATP and 0.5
M
phenylalanine (mixture A), and
TycB1
Pro
-CAT mutants were incubated in assay buffer
containing 1 l
M
enzyme, 2 m
M
ATP and 0.5 m
M
proline
(mixture B). For reactions with N-a-tosyl-
L
-phenylalanyl-
chloromethane and phenylmethanesulfonyl ¯uoride the
inhibitor was suspended in ethanol and added to mixture B
without exceeding 1% ethanol content in the reaction
mixture. The condensation reaction was initiated by the
addition of 1 vol. of mixture B to 1 vol. of mixture A and
incubated 45 min at 37 °C. In order to analyze the nature of
the product(s), the reaction mixture (1 mL) was diluted by
adding 4 mL water and immediately extracted with buta-
nol/chloroform [4 : 1; (v/v)]. The organic phases were
Table 1. Primers used for mutagenesis destinguished by the two dierent PCR techniques applied. The restriction sites indicated were introduced to
ease the screening for mutated plasmids after cloning. OP refers to outer primer and MP to megaprimer. Also the desired mutation is indicated in
the name of each primer. (lower case: modi®ed sequences; bold: restriction sites).
Name of primer Sequence
Restriction
enzyme
Megaprimer PCR OP1 5¢-cat gCC ATG GGT GTA TTT AGC-3¢NcoI
OP2 5¢-cat gCC ATG GTT AAT TTC TCC TCT TTA ATG-3¢NcoI
MP(D151N) 5¢-GAA GCA CCA GCC GTt CAT GAG GAT GTG-3¢BspHI
MP(C154S) 5¢-AAT GCT GAA GgA CCA GCC GTC CAT GAG-3¢AvaII
MP(C154A) 5¢-AAT GCT GAA tgc CCA GCC GTC CAT GAG-3¢BsmI
MP(H146A) 5¢-CCA TGA GGA TGT Gcg cAA AGC TCC A-3¢FspI
MP(H147R) 5¢-CCA TGA GGA Tcc GAT GAA AGC TCC A-3¢BamHI
MP(Y166W) 5¢-GCA AGG ACA Acc AtA TGG CAA GCA AGT C-3¢NdeI
MP(Y166F) 5¢-GCA AGG ACA Aga AgA TGG CAA GCA AGT C-3¢
QuickChange
TM
3¢Q19A 5¢-GCG TTG ACC CCG ATG gcc GAG GGG ATG CTG TTT CAC-3¢CfrI
5¢Q19A 5¢-GTG AAA CAG CAT CCC CTC ggc CAT CGG GGT CAA CGC-3¢CfrI
5¢R62A 5¢-CTG CAT GTG CTG GTA GAG gcc TAC GAT GTA TTC CGC ACG-3¢AatI
3¢R62A 5¢-CGT GCG GAA TAC ATC GTA ggc CTC TAC CAG CAC ATG CAG-3¢AatI
5¢R67A 5¢-GGT AGA GAG ATA CGA TGT ATT Cgc gAC GTT GTT TAT CTA TGA
AAA GC-3¢
NruI
3¢R67A 5¢-GCT TTT CAT AGA GAA ACA ACG Tcg cGA ATA CAT CGT ATC TCT
CTA CC-3¢
NruI
5¢W202L 5¢-CAG GCC GCT CTC AAC TAc Tta AGC GAC TAT CTG GAA GCC-3¢A¯II
3¢W202L 5¢-GGC TTC CAG ATA GTC GCT taA gTA GTT GAG AGC GGC CTG-3¢A¯II
5¢R284A 5¢-GGC TCT GTT GTA TCC GGA gct CCT ACA GAC ATC GTC GG-3¢SacI
3¢R284A 5¢-CCG ACG ATG TCT GTA GGa gcT CCG GAT ACA ACA GAG CC-3¢SacI
ÓFEBS 2002 Catalysis of nonribosomal peptide-bond formation (Eur. J. Biochem. 269) 623
transferred to fresh tubes and washed once with 5 mL of
0.1
M
sodium chloride. After removal of the solvent under
vacuum, the reminders of each reaction were further
investigated by HPLC or HPLC-MS. The samples prepared
were resolved in 200 lL 10% of buffer B and the products
separated by using a C18 reversed-phase column (Nucleosil
3mm´250 mm, pore-size 120 A
Ê, particle-size 3 lm;
Macherey & Nagel) on a HP1100 HPLC-MS system
(Agilent Technologies) with simultaneous monitoring at
detector wavelengths of 214 and 256 nm. The following
gradient pro®le was used at a ¯ow-rate of 0.35 mLámin
)1
:
loading (10% buffer B), linear gradient to 30% buffer B in
1 min, followed by a linear gradient to 100% buffer B in
20 min, and then holding 100% buffer B for 10 min
(buffer A, 0.05% formic acid in H
2
O; buffer B, 0.04%
formic acid in methanol).
Peptide mapping of trypsin-digested TcyB1
Pro
-CAT
Peptide mapping of TycB1
Pro
-CAT was performed accord-
ing to the manufacturer's protocol using the Sequencing
Grade Modi®ed Trypsin Kit (Promega). Treatment of
protein with N-a-tosyl-
L
-phenylalanylchloromethane or
phenylmethanesulfonyl ¯uoride was performed before the
digest by incubating a 10-fold excess of inhibitor with
TycB1
Pro
-CAT (1 l
M
)for10minat25°C. N-a-Tosyl-
L
-phenylalanylchloromethane and phenylmethanesulfonyl
¯uoride treated protein (1 mg) was precipitated and washed
once with acetone to eliminate residual inhibitor that might
interfere with the digest [28]. HPLC conditions were as
described by Promega except for using a HP1100 HPLC-MS
system and a C18 reversed-phase column (Nucleosil
3´250 mm, pore size 120 A
Ê,particlesize3lm; Macherey
& Nagel).
RESULTS
Homology searches to select targets for the site-directed
mutagenesis of the C-domain in TycB1
Pro
-CAT
For a fairly long time, virtually no biochemical data were
available on C domains, and consequently, this 450-
amino-acid stretch was considered to be only a spacer
between consecutive, amino-acid-activating A-T bi-domains
[29]. Recently, however, it could be demonstrated that this
region is actually responsible for catalysis of peptide bond-
formation [5]. Furthermore, it was noted that C-domains
share the signature sequence motif HHxxxDGxSW (the
so-called ÔHis motifÕ) with a superfamily of acyl transferases,
and that the second His and the Asp residues are
indispensable for C-domain activity [5,21]. To identify
additional catalytic key residues, we started our study with
alignments of the primary sequences of NRPSs C-domains.
A scan of 80 C-domains revealed an overall similarity
ranking from 60% (between TycB1 and GrsB1) to < 20%
(between TycB1 and Hts1), with an average percentage of
similarity of 35% (data not shown). Among all C-domains
investigated, we found eight absolutely invariant residues, of
which all have functionalized side chains (carboxyl, amino,
amine, guanidino, sulfhydryl or hydroxyl groups). These
latter residues (namely Q19, R62, R67, H146, H147, D151,
W202 and R284 within TycB1
Pro
-CAT) and additionally
C154 and Y166 (> 95% conserved and discussed as
potentially involved in catalysis) were selected as targets
for the subsequent mutational analysis.
Generation and puri®cation of the recombinant
enzymes
In this study, we constructed a set of 12 TycB1
Pro
-CAT [5]
single mutants (Q19A, R62A, R67A, H146A, H147R,
D151N, C154S, C154A, Y166W, Y151F, W202L and
R284A). Mutations other than to alanine were intentionally
designed to show residual activity for similar functional
groups (H/R, D/N, C/S and Y/W) as opposed to residues
with no functionalized group (H/V, C/A and Y/F). In case
of a catalytic triade Asp-His-Ser or Asp-His-Tyr similar
functionalized groups were expected to exhibit residual
activity, whereas unfunctionalized groups would have none.
All mutants were individually expressed as C-terminal His6-
tag fusions in the heterologous host E. coli and puri®ed by
Ni
2+
-af®nity chromatography. As judged by SDS, all
proteins could be puri®ed to homogeneity (data not shown),
although in general, the solubility of the recombinant
proteins appeared not to be very high (as estimated on
< 30% by comparison after SDS/PAGE of pellet and
supernatant after cell lysis). Highest amounts of soluble
protein comparable to wild-type level were obtained for
mutants Q19A, R62A, H147V, H147R, C154S and
C154A. Preparation of mutants D151N, Y166W, Y151F,
R284A revealed slightly lower solubilities. Mutants R67A,
H146A and W202L gave less than 0.5 mg soluble protein
per L culture indicating a misfolding induced by the
mutation. In order to test for correct folding, all mutants
were subjected to ATP±PP
i
exchange assay, which assesses
the activity and selectivity of the A domain embedded
within the C- and T-domains of all tridomain TycB1
Pro
-
CAT derivatives. Although A domain activity indicates a
correct folding of the entire TycB1
Pro
-CAT enzymes, it
cannot be excluded that the connected C- and T-domains
domains are somehow impaired in folding. The assay
revealed that within an error-margin of < 5%, the same
wild-type amino-acid dependent activity in the ATP±PP
i
-
exchange assay could be obtained for most mutants,
indicating that their structure cannot have changed much
if at all. Three TycB1
Pro
-CAT mutants, R67A, H146A and
W202l sustained a drop in adenylation activity to less than
5% wild-type activity and therefore are likely to be affected
in folding (see below).
Dipeptidyl-
S
-Ppant- and DKP-formation
Prior studies revealed that C-domains are the peptide bond-
forming catalysts, which raises the question of how
ef®ciently the C-domain mutants of TycB1
Pro
-CAT transfer
the
D
-Phe moiety from GrsA
Phe
-ATE. To assay for the
formation of the dipeptidyl-S-Ppant nascent product,
L
-Pro
was allowed to load onto holo-TycB1
Pro
-CAT mutants in
the presence of ATP. Subsequently, the
L
-Pro-S-Ppant-
enzymes were mixed, respectively, with holo-GrsA
Phe
-ATE,
which had been loaded in a preincubation with ATP and
radiolabeled
L
-[
14
C]Phe. Once translocation of
D
-[
14
C]Phe
from GrsA
Phe
-ATE to TycB1
Pro
-CAT occurs by C-domain
catalysis, the vacant holo-GrsA
Phe
-ATE is rapidly reloaded
with surplus
L
-[
14
C]Phe. Thus, if samples are taken at
de®ned time points and immediately quenched by the
624 V. Bergendahl et al. (Eur. J. Biochem. 269)ÓFEBS 2002