Báo cáo khoa học: Mutational analysis of the C-domain in nonribosomal peptide synthesis pot

Recently we established a minimal in vitro model system for NRPS with recombinant His¢-tagged GrsA GrsAppe-ATE; 127 kDa and TycBl TycBlp,.-CAT; 120 kDa and demonstrated the catalytic fun

Mutational analysis of the C-domain in nonribosomal peptide

synthesis

Veit Bergendahl*, Uwe Linne and Mohamed A Marahiel

Biochemie/Fachbereich Chemie, Philipps-Universitaét 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 first proline-activating module of GrsB/

TycB Recently we established a minimal in vitro model

system for NRPS with recombinant His¢-tagged GrsA

(GrsAppe-ATE; 127 kDa) and TycBl (TycBlp,.-CAT;

120 kDa) and demonstrated the catalytic function of the

C-domain in TycBlp,.-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 TycB1p,.-CAT using site-directed mutagenesis

and peptide mapping Mutations in TycBlp,.-CAT of 10

strictly conserved residues among 80 other C-domains with

potential catalytic function, revealed that only R62A,

HI47R and DISIN are impaired 1n peptide-bond

formation All other mutations led to either unaffected

(QI9A, CI54A/S, YI66F/W and R284A) or insoluble proteins (H146A, R67A and W202L) Although 100 nm of the serine protease inhibitors N-a-tosyl-L-phenylalanylchlo- romethane or phenylmethanesulfonyl fluoride completely abolished DKP synthesis, no covalently bound inhibitor derivatives in the C-domain could be identified 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 significant 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,

Correspondence to M A Marahiel, Biochemie/Fachbereich Chemie,

Philipps-Universitat Marburg, Hans-Meerwein-StraBe, 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, DK P, p-Phe-L-Pro-diketopiperazine; E-domain, epi-

merization domain; IPTG, isopropyl thio-B-p-galactoside; LSC, liquid

scintillation counting; NRPS, nonribosomal peptide synthetases;

PKS, polyketide synthases; Ppant, 4’-phosphopantetheine; PP;, 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)

referred to as modules, are composed of functionally specific 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 final 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 confirmed 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 findings indicate that the C-domains

are bearing significant 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 modification of the

A-Domain

T-Domain C-Domain E-Domain TE-Domain

(cognate amino acid)

Fig 1 Genes and domain organizations of

gramicidin S and tyrocidine A synthetases

These genes lead to the minimal system com-

prising the NRPSs GrsAp,.-ATE (from grsA)

and TycBlp,.-CAT (from tvcA) 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

TycBl

substrate amino acids Those domains are the epimerization

domain (E-domain) for C,-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 final

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 significantly

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 GrsAp,.-ATE (phenylalanine-activating

module; A-, T- and E-domain) from the gramicidin S$

system and the first module in the second peptide synthetase

of the tyrocidine A system TycB1p,,.-CAT (proline-activat-

ing module; C-, A-, and T-domain; see Fig 1) Both

proteins can be obtained in active form as recombinant

Fig 2 Currently accepted model of the con-

densation of L-Phe and L-Pro as catalyzed by

the peptide synthetases GrsAp,,.-ATE and

TycBlp,,-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

Ppant moiety of each T-domain as a >

thioester L-Phe-S-Ppant is then epimerized by 1

only p-Phe-S-Ppant undergoes condensation dc + ›

with L-Pro-S-Ppant presented at the C-do- om

main of TycBlp,;.-CAT Free GrsApp.-ATE

GrsA/TycA(PheATE)

recycle

—_

GrsB1/TycB1(ProCAT)

His,-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 b-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 first C-domain of the tyrocidine synthetase complex possesses an enantioselectivity at the electrophilic donor site (b-Phe) and a substrate specificity at the nucleophilic acceptor site (L-Pro) in the formation of the chain-initiating p-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 HHAxxxDGx(S/C), commonly called ‘His-motif’, that was suspected to participate in the catalysis This hypothesis was supported by the finding that a single mutation of the

HS

L-Phe L-Pro

+

cyclization

can now be reloaded whereas D-Phe-L-Pro-

DKP can be released by intramolecular cycli-

zation on TycBlp,.-CAT

D-Phe-L-Pro diketopiperazine

second histidine residue (italic) in TycBlp,.-CAT (H147V)

was sufficient to abolish peptide bond and DKP formation

with GrsAp, ATE [5] Furthermore, a chromosomal point

mutation in s7fA-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 TycBlp,.-CAT and assayed for their

ability to catalyze peptide bond formation between p-Phe-

S-Ppant-GrsAp,.-ATE and — L-Pro-S-Ppant-TycBlp,,-

CAT Furthermore, the minimal system GrsAp,.-ATE/

TycBlp,;o>-CAT was compromised with two inhibitors

(phenylmethanesulfonyl fluoride and N-z-tosyl-L-phenyl-

alanylchloromethane), and analyzed for the appearance

of possible covalent C-domain/inhibitor complexes in order

to find a catalytic triade similar to the one in serine

proteases

EXPERIMENTAL PROCEDURES

Sequence alignments for the identification 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 TycBlp,.-CAT mutants were constructed by site-

directed mutagenesis of pProCAT [5] using either the so-

called “Megaprimer-PCR’ method [25] with the Expand

long-range PCR system’ (Bohringer Mannheim, Germa-

ny) or the QuickChange’™ 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™ mutagenesis was carried out in accordance with

the manufacturer’s protocol For the ‘“Megaprimer-PCR’,

PCR products were purified with the QIAquick-spin PCR

purification kit (Qiagen, Germany), digested with NcoI and

ligated Standard procedures were applied for all DNA

manipulations [26] and the Escherichia coli strain XL1-Blue

[27] was used for cloning The mutant TycBlp,o-

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 confirmed by DNA sequencing using an ABI-Prism 310 Genetic Analyzer with standard protocols described by ABI (Applied Biosystems, Germany)

Expression and purification 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 TycBlp,,.-CAT mutants Expression and

purification using single-step Ni? * -affinity 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 mm Hepes, pH 8.0, 100 mm sodium chloride, 10 mm magnesium chloride, 2 mm dithioerythritol and 1 mm EDTA) After addition of 10% glycerol (v/v), the proteins could be stored at —80 °C Protein concentrations were determined using the calculated extinction coefficients for

the A 9 of the proteins: 138 690 M tem"! for GrsAphe- ATE and 92 230m 'cm™! for TycBlp,o-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 purified Reaction mixtures contained (final volume: 100 uwL): 50 mm Hepes, pH 8.0,

100 mm sodium chloride, 10mm magnesium chloride,

1 mm EDTA, | mM amino acid and 300 nm enzyme The reaction was initiated by the addition of 2 mm ATP, 0.2 mm tetrasodium pyrophosphate and 0.15 uCi of tetrasodium [”P]pyrophosphate (NEN/DuPon)) 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 mm tetrasodium pyrophosphate and 350 mm perchloric acid Subsequently, the charcoal was pelleted by centrifugation, washed once with | mL water and resuspended in 0.5 mL water After addition of 3.5 mL of liquid scintillation fluid (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 mm Hepes, pH 8.0,

100 mm sodium chloride, 10 mm magnesium chloride,

1 mm EDTA) contained 500 nm enzyme, 2mm ATP and

2 um ['*C]-amino acid (Hartmann, Germany) The thio- esterification was initiated upon the addition of the radio- labeled amino acid and the reaction was quenched after

Table 1 Primers used for mutagenesis destinguished by the two different 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: modified sequences; bold: restriction sites)

Restriction

MP(Y 166F) 5’-GCA AGG ACA Aga AgA TGG CAA GCA AGT C-3’

AAA GC-3’

CTA CC-3’

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 | mL 10% trichloroacetic acid

(w/v) The pellet containing the acid-stable label was then

dissolved in 150 tL formic acid and quantified 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 coeffi-

cient According to our experience, the current set of

proteins has shown that thioesterification activities may

vary significantly (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 f = 0 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 nm of holo-enzyme (GrsAp,,.-ATE and TycBlp,,-

CAT) were preincubated seperately in assay buffer with

their substrate amino acids [2 um ['*C]L-Phe (450 mCim- mol”), 100 uM r-Pro] and ATP (2 mm) After 3 min, product formation was initiated by mixing equal volumes

of reaction mixtures At various time-points, 200 uL 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 ø) for

20 min, washed two times with 1 mL ice-cold trichloro-

acetic acid, redissolved in 150 uwL formic acid and quan- tified by LSC

DKP formation: indirect assay for p-Phe-.-Pro dipeptide formation

The formation of the dipeptide was analyzed using GrsAppe- ATE and the different C-domain mutants of TycBlp,.-

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 um

GrsAp,. ATE was incubated in assay buffer containing

2mm ATP and 0.5m phenylalanine (mixture A), and

TycBlp,.-CAT mutants were incubated in assay buffer

containing | um enzyme, 2mm ATP and 0.5 mM proline (mixture B) For reactions with N-c-tosyl-L-phenylalanyl- chloromethane and phenylmethanesulfonyl fluoride 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 | 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

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 uL 10% of buffer B and the products

separated by using a C18 reversed-phase column (Nucleosil

3 mm x 250 mm, pore-size 120 A, particle-size 3 um;

Macherey & Nagel) on a HP1100 HPLC-MS system

(Agilent Technologies) with simultaneous monitoring at

detector wavelengths of 214 and 256 nm The following

gradient profile was used at a flow-rate of 0.35 mL-min™:

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,O; buffer B, 0.04%

formic acid in methanol)

Peptide mapping of trypsin-digested TcyB1p,,-CAT

Peptide mapping of TycBlp,.-CAT was performed accord-

ing to the manufacturer’s protocol using the Sequencing

Grade Modified Trypsin Kit (Promega) Treatment of

protein with N-z-tosyl-L-phenylalanylchloromethane or

phenylmethanesulfonyl fluoride was performed before the

digest by incubating a 10-fold excess of inhibitor with

TycBlp,.-CAT (1 um) for 10 min at 25 °C N-o-Tosyl-

L-phenylalanylchloromethane and phenylmethanesulfonyl

fluoride 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 x 250 mm, pore size 120 A, particle size 3 um; Macherey

& Nagel)

RESULTS

Homology searches to select targets for the site-directed

mutagenesis of the C-domain in TycB1p,,.-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 ‘His motif’) 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 TycBlp,.-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 purification of the recombinant enzymes

In this study, we constructed a set of 12 TycBlp,.-CAT [5] single mutants (QI9A, R62A, R67A, H146A, H147R,

DISIN, C1548, CI54A, Y166W, YISIF, 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 purified by Ni- *-affinity chromatography As judged by SDS, all proteins could be purified 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 QI9A, R62A, H147V, H147R, C1548 and

C154A Preparation of mutants DIS5IN, Y166W, YIS5IF,

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; exchange assay, which assesses the activity and selectivity of the A domain embedded

within the C- and T-domains of all tridomain TycB1p,.-

CAT derivatives Although A domain activity indicates a

correct folding of the entire TycBlp,,-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;- exchange assay could be obtained for most mutants, indicating that their structure cannot have changed much

if at all Three TycBlp,,-CAT mutants, R67A, H146A and

W202I 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

efficiently the C-domain mutants of TycBlp,.-CAT transfer the p-Phe moiety from GrsAp,.-ATE To assay for the

formation of the dipeptidyl-S-Ppant nascent product, L-Pro was allowed to load onto holo-TycBlp,,-CAT mutants in the presence of ATP Subsequently, the L-Pro-S-Ppant-

enzymes were mixed, respectively, with holo-GrsAppe-ATE,

which had been loaded in a preincubation with ATP and radiolabeled L-['*C]Phe Once translocation of p-['“C]Phe

from GrsAp,.-ATE to TycBlp,,-CAT occurs by C-domain catalysis, the vacant holo-GrsAp,.-ATE is rapidly reloaded

with surplus 1-['*C]Phe Thus, if samples are taken at defined time points and immediately quenched by the

220 ¬

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addition of 10% (w/v) trichloroacetic acid, the measurable

amount of acid-stable label should increase from one

equivalent to two equivalents, as at that point GrsAp,.-ATE

and TycBlp,,.-CAT are both radiolabeled Simultaneously,

the p-['*C]Phe-L-Pro dipeptide is slowly autoreleased from

TycBlp,.-CAT by cyclization to DKP Consequently, as

soon as all L-['*C]Phe has been consumed, the amount of

acid-stable label will decrease again until all enzyme has

been liberated of radiolabeled substrate

This assay for dipeptide and DKP formation was

performed with wild-type GrsAp,.-ATE and all TycBlp,o-

Fig 3 Time dependence of dipeptide and DKP formation between GrsApne-ATE and C-domain mutants of TycBlp,,-CAT Normalized counts are plotted against the time of sampling The increase in acid- stable label accounts to the translocation of labeled Phe onto TycBlp,.-CAT and rapid reloading of GrsAp,.-ATE Both labeled proteins can be precipitated by 10% trichloroacetic acid solution Mutants Q19A, Y166W and R284A revealed no effect on dipeptide- formation and release of DKP represented by the plot of QI9A The second group (C154A, C1548, Y166F), represented by the plot of Y166F, is only impaired in DKP formation, implied by the slow decresae of the acid stable label The most interesting group, however,

is represented by the plot of R62A It consists of four TycBlp,.-CAT C-domain mutants (R62A, H147R, H147V and DISIN) that are significantly impaired in dipeptide formation, as there is no increase in acid stable label which could indicate a formation of a Phe-Pro dipeptide

CAT C-domain mutants except mutants R67A, H146A and W202L The latter ones were not tested further, as they proved to be insoluble or misfolded As positive and negative controls we performed the assay with wild-type

TycBlp,.-CAT instead of mutants and without TycBlp,.-

CAT [5] On the basis of the results obtained by LSC (summarized in Fig 3), the TycBlp,,-CAT mutants can be categorized in three groups Three mutants (QI9A, Y166W and R284A) revealed no effect on dipeptide-formation and release of DKP (Fig 3A) A second group (C154A, C1545, Y166F) was asymmetrically impaired in DKP release, implying that the release of p-Phe-L-Pro-DKP is somehow influenced by the functional side-chain moieties of C154 and Y166 (Fig 3B) The most interesting group, how-

ever, consists of four TycBlp,.-CAT C-domain mutants

(R62A, HI47R, HI47V and DISIN) that are completely impaired in dipeptide formation (Fig 3C) This indicates that the three residues affected are essential for the

translocation of p-Phe-S-Ppant from GrsAp,,.-ATE to

TycBl p,o-CAT

The product of the condensation assay, DKP, is readily extractable into organic solvent [5] Thus, for a more detailed analysis of the product(s) formed, the assays were performed with nonradiolabeled substrates After 45 min, reactions were extracted with butanol/chloroform and analyzed by HPLC-MS The results of the HPLC-MS analysis, which are summarized in Fig 4, revealed that D-Phe-L-Pro-DKP was synthesized as the only product in the case of the mutants QI9A, C154A/S, Y166W/F and R284A The amount of DKP produced ranged from 30 to

100% of the wild-type TycBlp,.-CAT (normalized for

thiolation activity) Changes in turnover are evident for C154S and R284A, but most significantly for Y166F and Y166W, by a decreased amount of DKP synthesized In contrast, no DKP at all could be detected when using R62A, H147V/R or DISIA

Peptide mapping of “z-tosyl-r-phenylalanylchloro- methane and phenylmethanesulfonyl! fluoride treated TycB1p,,-CAT

Possible catalytic mechanisms for the formation of pep- tide bonds would be for example a catalytic triade as

in serine proteases or catalytic diade as found in

120 4

I

20 +

5s 8 š š È š š 8 & &

ơ 7T T1 6 6 © FS > &

ProCAT mutants

Fig 4 Relative amount of DKP generated with GrsAp,.-ATE and

C-domain mutants of TycBlp,,.-CAT The quantity of DKP synthe-

sized (determined by HPLC-MS) using wild-type TycBlp,.-CAT was

set 100% and errors were calculated from a series of three consecutive

experiments with the same batch of protein

chloramphenicolacetyltransferases [23,24] Most strikingly,

all results obtained so far were in favor of the latter mode of

action, and we also were not able to detect a conserved

serine residue that might act in the catalytic center of

C-domains To further rule out the possibility of a catalytic

triade, we next investigated the inhibition of dipeptide and

DKP formation in the presence of N-a-tosyl-L-phenylalanyl-

chloromethane and phenylmethanesulfonyl fluoride Both

inhibitors form covalent complexes with their target

enzymes via catalytically exposed serine and _ histidine

residues, respectively We found that the minimal system

was susceptible to complete inhibition of DKP synthesis by

100 nm N-a-tosyl-L-phenylalanylchloromethane and phen-

ylmethanesulfonyl fluoride (data not shown) Consequently,

“His"

Q19A R62A R67A H146A H147V/ DI51N C154A/ Y166W/ W202L R284A

H147R C154S Y166F

we next performed a partial digest with trypsin of TycB1p,.- CAT, which had been pretreated with inhibitor Tryptic digests were applied to HPLC and analyzed by a coupled

MS Focusing only on tryptic fragments derived from the C-domain, we were able to separate the resulting peptides by reversed-phase chromatography and to trace out theoretical fragments larger than 500 m/z by MS (calculated by peptide tools, Agilent Technologies; data not shown) However, none of them showed an increase in mass that would occur

by covalent derivatization with N-a-tosyl-L-phenylalanyl- chloromethane or phenylmethanesulfonyl fluoride one would expect in the case of a catalytic triade No further efforts were made to track down the actual target of the inhibitors

DISCUSSION

The purpose of this work was to identify the key residues

in the C-domain and their potential role in the catalytic mechanism For this purpose, sequence alignments of 80 C-domains were made and lead to 10 invariant residues (100% identity except C154 and Y166, who showed 100% similarity by variation with serine in four cases and phenylalanine in one case) These residues were Q19, R62, R67, H146, H147, D151, C154, Y166, W202 and R284

Figure 5 summarizes the results of the site-directed mutagenesis Mutations of residues R67, H146 and W202 seemed to be structurally important as judged by their lack

of solubility Residues Q19, C154, Y166 and R284 were found to be not essential to the condensation activity The condensation reaction requires the action of various domains, so we are dealing with sets of coupled catalyzed and noncatalyzed reactions (adenylation, thiolation, epi- merization and noncatalyzed cyclization) resulting in com- plex kinetic influences Consequently, a more thorough analysis of the mutants does not seem to be possible with the

current methods In contrast, mutation of residues R62,

C5C6 C7

Fig 5 Summary of all observed effects for every TycBlp,,-CAT mutant examined The diagram shows a schematic presentation of the 450-amino-acid C-domain of TycBlp,.-CAT, along with the approximate location of core- motifs Cl—7 [3] Underneath, the effects of Solubility each mutation on solubility and various

activities of each domain are rated by ‘+’ A-Domain (soluble or active) and ~ (insoluble or in-

active) A gray background emphasizes the

particular, as they showed only a loss in con- C-Domain densation activity, while the other domains

appear to be uneffected.

H147 and D151 were found to abolish C-domain activity

completely, while being still active in adenylation and

thiolation activities In previous work, Hisl147 was already

found to be essential for condensation [5] and the

corresponding residue of D151 in the SrfB2,4,.-CAT

surfactin synthetase essential for the synthesis of surfactin

in B subtilis, shown in vivo [21,30] In the present study we

additionally found R62 to be essential for condensation

activity D151 was verified in vitro to be essential in a second

C-domain

Previously, based on sequence alignments, it had been

suggested, that residues C154 or Y166 could also be

involved in the catalytic mechanism and possibly form a

catalytic triad [22] analogous to serine proteases (Asp-His-

Ser) However, the mutation of these two residues led to

enzymes still active in the condensation assay The C154A

mutant showed a DKP-synthesis activity comparable to the

wild-type The C154S mutant showed a reduced amount of

total DKP synthesized when compared to the correspond-

ing alanine mutant In the case of the second potential key

residue, Y166, the two mutants constructed (Y166W and

Y166F) seem to be affected in the DKP-synthesis, but no

significant effect abolishing condensation activity was seen

So we tried to show an inhibitory effect of the typical serine

protease inhibitors N-o-tosyl-L-phenylalanylchloromethane

and phenylmethanesulfonyl fluoride in the condensation

assay However, N-o-tosyl-L-phenylalanylchloromethane

and phenylmethanesulfonyl fluoride abolished product

formation at concentrations of 100 nm, we were not able

to identify their target in the C-domain using peptide

mapping Thus the inhibitory effect may be due to

interference with other reactive residues in NRPS modules

like the thiol group of the 4’-phosphopantethein-moiety or

the aminoacyladenylate of the activated amino acid Fur-

thermore, N-c-tosyl-L-phenylalanylchloromethane could

)

_ 7w

Chloramphenicol acetyltransferase Dihydrolipoyl transacetylase

(Escherichia coli) (Azotobacter vinelandii)

2211 ADERELLLYDMNNIAADAASITILFDELAEL 2241

2214 SOTERVLLIDMHNIISOGASVGVLIEELSKL 2244

also act as a competitive inhibitor by competing with Phe

in binding to the donor site in the C-domain As we could not detect any covalent intermediate of N-œ-tosyl-L-phenyl- alanylchloromethane or phenylmethanesulfonyl fluoride

with residues of the C-domain of TycBlp,o.-CAT, an

analogous mechanism as observed in serine proteases is not supported by our data On the contrary, our data excludes the direct involvement of serine residues in catalysis

of peptide-bond formation as postulated by deCrecy- Lagard et al [22]

Interestingly, in the case of serine proteases, a mutation

in the catalytic triade (Asp-His-Ser — Asp-His-Gly) was recently found by Elliott and coworkers to switch the protease activity into a ligase activity [31] Therefore, a catalytic diad (Asp-His) for condensation domains of NRPSs would be in good agreement with our results and

hence we suggest an alternative mechanism There is a

strong analogy to some acyl transferases, such as the chloramphenicol acetyltransferase or the dihydrolipoyl transacetylase It is the participation of a histidine residue

in almost the same motif [HHxxx(D/N)G] [23,24] These enzymes use Acyl-CoA as a substrate in an acylation reaction and hence share the ability of utilizing a Ppant moiety as an acyl carrier and donor In the crystal structures of both representatives only the second histidine (His195 in chloramphenicol acetyltransferase) was observed to play a central role in catalysis and both show

a very similar architecture (Fig 6) In chloramphenicol acetyltransferase other residues such as Arg18 and Asp199 were found to be significantly distant to the reactive center with the cocrystallized chloramphenicol and therefore were interpreted as less catalytically then structurally important, although an electrostatic interaction to the catalytic center could not have been excluded It is conceivable to find

a similar architecture for the C-domain of peptide

His 195

4¥

Ô Arg 19 N rg sp 199

_

177 DRLLLPLSVQVEHAVCDGEHVARFIMHRIQEL 207

ta

CAT (E.coli)

204 PRLMLPLSLSYDIRVINGAAAARFTERLGOL 243 k2p (Azotobacter Vinelandaa)

135 SQHDYQVIWSFHEILMDOWCPSIIFDDILAI 165

132 DRNKYLLVWSNEHIVMOGWSMGILMERLFQN 162

TycBiprec (Bacillus brevis) LicBivalcC (Bacillus licheniformis) TycB3pheC (Bacillus brevis) SrfA3leuc (Bacillus subtilis)

Fig 6 Structure of chloramphenicol acetyl and dihydrolipoyl transacetylase The horizontal dark gray o helix of the structure includes the His-motif

of which residues His195 and Asp199 are explicitly shown in all pictures A very similar structure can be found in the dihydrolipoyl transacetylase Especially the structure around the catalytic center may be similar to the structure in the C-domain of TycBlp,.-CAT Residues Arg18, His195 and Asp19 are emphasized in the enlarged portion of chloramphenicol acetyltransferase Arg18 (at the light « helix), which forms a salt bridge with Asp199, can be found at a different position in the dihydrolipoyl transacetylase structure and may play a different role there The catalytic centers of chloramphenicol acetyltransferase and dihydrolipoyl transacetylase interact with their substrates bound in the binding pocket of another enzyme molecule within the active homomultimer This could explain the distance in sequence homology with NRPSs C-domains displayed in the alignment of the according regions in chloramphenicol acetyltransferase and dihydrolipoyl transacetylase with the proposed homolog region of four representative C-domains (from tyrocidine, lichenysin and surfactin synthetases), as latter presumably act as in a different structual assambly and on different substrates.

synthetases Accordingly, the residues R62 and D151 in

TycBlp,,.-CAT could form a similar ion pair as found for

R18 and D199 in chloramphenicol acetyltransferase

There, these residues were found to be ‘structurally

important by virtue of being 8 A of the catalytic His’

Following this analogy His147 could act as a general base

It could be similarly stabilized via a tautomeric stabilization

provided by its own carbonyl oxygen from the backbone

rather than a_ side-chain carboxylate group from another

residue [32], such as D151 Accordingly, the effect of

mutations Y166W/F could be interpreted by Y166 being

involved in stabilizing the tetrahedral transition state of the

thioester during condensation Perhaps in the mutants a

nearby hydroxyl group could restore this function (Fig 7)

or the stabilization is not absolutely necessary Moreover,

no covalently enzyme bound waiting position of the

activated acylsubstrate was found Thus, for the

C-domains of peptide synthetases, no such covalent

waiting position should be expected, either The conserved

serine in the crystal structure of chloramphenicol acetyl-

transferase was 4.3 A away from the reactive hydroxyl

group of chloramphenicol, ruling out its direct involvement

in catalysis Interestingly, two arginine residues were also

discussed as possible participants, but were found not

to be sufficiently near to the catalytic center of the

reaction Chloramphenicol acetyltransferase, dihydrolipoyl

transacetylase and TycBlp,.-CAT show no significant

sequence homology between each other, which is also true

for chloramphenicol acetyltransferase and dihydrolipoyl

transacetylase when compared separately They do

though, show significant structural homology, suggesting

a common structure-function motif for this type of

enzymes Future structure investigations will have to

determine, weather the C-domain also belongs to this

family

The prospect of using NRPSs to produce new bioactive

peptides has inevitably led to the demand for a more

detailed understanding of each process involved Although

it is possible to direct the biosynthesis to new derivatives

by reprogramming the substrate specificity of A-domains

and reorganizing the assembly of modules, we still have to

fight with loss of enzyme activity and specificity A

milestone in the study of peptide synthetases was the

determination of the A-domain structure of GrsAp,,-A [8]

Aspects of specificity and binding were superimposed on

other A-domains and with the help of eight residues in the

primary sequence the substrate can now be predicted and

even altered [9] Recent results in this field have now

demonstrated that substrate selectivity in the C-domain

can be another obstacle to be overcome on the way to

controlled and effective engineering of peptide synthetases

[6,7,33] In consequence a more detailed analysis of the

structurally related functions inside the C-domain still

demands our efforts for the future and probably requires

structural data from crystals Nevertheless, the data

presented in this study excludes the idea of a catalytic

triad involved in C-domain catalysis of NRPSs With the

finding of the additional residue R62 and confirming the

residues H147 and D151 abolishing condensation activity

of TycBlp,.-CAT, the idea of a similar mechanism to

chloramphenicol acetyltransferases, with the direct involve-

ment of only one residue (H147) in catalysis, is strongly

supported

His147

AA».‹s S—4'-Ppan

O

H

O-Á )—Tyr 66

His147

Nt \ Nya

backbone

—4'-Ppan

backbone

Fig 7 Proposed model for the catalytic mechanism at the C-domain This improved model is based on the proposed analogy to chloram- phenicol acetyltransferase, for which the mechanism has been estab- lished by the crystal structure and kinetic studies Residue H147 of TycBlp,o.-CAT plays the key role in the catalytic mechanism and is essential, whereas Y166 may be replaced by another nearby hydroxyl group, according to our data According to the model R62 and D151 play a more structurally important role rather than being involved with the catalytic mechanism (i.e displaying the backbone carbonyl in position).

ACKNOWLEDGEMENTS

We thank J6rg Tost and Robert Finking for their valuable contribution

during their undergraduate studies and Dr Torsten Stachelhaus for

critical reading of the manuscript This work was supported by the

Deutsche Forschungsgemeinschaft and the Fond der chemischen

Industrie For their reliable technical support we also thank Inge

Schiiler and Gabi Schimpff-Weiland

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