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The Streptomyces K15 DD-peptidase/penicillin-binding protein. Active site and sequence of the N-terminal region.

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Biochem. J.(1989)260,601-604 (Printed in GreatBritain)

The

Streptomyces

K15

DD-peptidase/penicillin-binding

protein

Active

site and

sequence of

the

N-terminal region

Melina LEYH-BOUILLE,* Jozef VAN

BEEUMEN,t

Suzanne RENIER-PIRLOT,* Bernard JORIS,* Martine NGUYEN-DISTECHE* and Jean-Marie

GHUYSEN*T

*Service deMicrobiologie, Universite de Liege, InstitutdeChimie, B6, B-4000 Sart Tilman

(Liege

1), Belgium, and

tLaboratorium voor Microbiologie, Rijksuniversiteit-Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium

The N-terminal region of the Streptomyces K 15 DD-peptidase/penicillin-binding protein shows high homology with that of otherpenicillin-interactive proteins ordomains. The active-site serine residue of the conserved tetradSer-Xaa-Xaa-Lysoccurs atposition 35. There isno indication for the presence of a signal peptide or anN-terminal hydrophobicsequence, suggesting that the StreptomycesK15enzyme is probably anchored to the membrane by a C-terminal peptide segment.

INTRODUCTION

The membrane-bound 26000-Mr DD-peptidase of

Streptomyces K15 catalyses acyl-transfer reactions with

cyclic

,-lactam

and non-cyclicamide andestercarbonyl donorsviaformation ofanacyl-enzymethatinvolvesan

essential serine residue [1,2]. The acyl-enzymes formed

byreaction with,-lactamantibiotics havelonghalf-lives

[3]. Consequently, the DD-peptidase behaves as a

penicillin-binding protein (PBP). The peculiarity ofthe

low-Mr Streptomyces K15 DD-peptidase/PBP is that whenactingon anamidesubstratesuchas

Ac2-L-LyS-D-Ala-D-Ala it functions almost exclusively as a strict

transpeptidase [1]. In water, hydrolysis is negligible

because the D-alanine that is released during enzyme

acylation is effectively re-utilized in a transfer reaction

that maintains the concentration oftheamide carbonyl

donorataconstantlevel. In thepresenceofanexogenous

amino compound suchasGly-Gly, theacceptor activity

of which overcomes that of water and D-alanine, the

substrate is quantitatively converted into the

trans-peptidatedproduct

Ac2-L-Lys-D-Ala-Gly-Gly.

Asabasis

for the design of nucleotide probes that would help

identifying the gene that encodes the Streptomyces K15

DD-peptidase, the N-terminal region of the purified

enzyme was sequenced and the essential serine residue

was identified by active-site peptide mapping.

MATERIALS AND METHODS

SequenceoftheN-terminal region of theDD-peptidase

The enzyme (4.9nmol in 130

ll),

purified in the

presence of cetyltrimethylammonium bromide as

de-scribed previously [4], was supplemented with 5 vol. of

cold (-20 °C) acetone and maintained at -20°C for

30min, after which time the precipitated enzyme was

collected by centrifugation at 20000g for 30min. The

pelletwas washed with 4 vol. of cold acetone, dissolved

in trifluoroacetic acid and the solution supplemented

with an equal volume of water. Automated sequence

analysis of 4.9 nmol of protein was carried out on a

470 A gas-phase Sequenator. The amino acid

phenyl-thiohydantoin derivatives were analysed off-line the

sequencer on a 4.6mm x250mm IBM

cyanopropyl

column(Biotech Instruments, Hertford, Herts.,U.K.)as

described in ref. [5]. The h.p.l.c. set-up consisted ofan

Intelink System connected to a 440

fixed-wavelength

detector(Waters, Milford, MA, U.S.A.) set at254 and 313nm. Automated sequence analysis of 1.4nmol of protein was also carried out on a 477A pulsed-liquid

Sequenator (Applied Biosystems, Foster City,

CA,

U.S.A.). The amino acid phenylthiohydantoin deriva-tives wereanalysed on-line thesequencer ona21 mm x

220mm C18 MPLC Cartridge column (Applied

Bio-systems) as described by the manufacturer.

Preparation of the acyl-(I35Slbenzylpenicilloyl-)enzyme The enzyme (20 nmol) was incubated for 15min at

37 °C with [35S]benzylpenicillin (230nmol; 250nCi/ nmol; New England Nuclear) in 1100,l of 17.5 mm-potassium phosphate buffer, pH 7.5, containing0.016% cetyltrimethylammonium bromide. Protein acylation

was complete. The acyl-enzyme was precipitated with

acetoneasdescribed above. The pellet waswashed with

4.8 ml of cold acetone, dried in vacuo and dissolved

in 280 1l of 88% (v/v) formic acid. The solution was

supplemented with

32#1

of a Triton X-100 solution

(3%, w/v) and 2 ml ofwater, and freeze-dried.

Pronasedigestion of the

acyl-(I35SIbenzylpenicilioyl-)enzyme

Trypsin,subtilisin,thermolysin, Staphylococcusaureus

proteinase V8 and Pronase were tested as proteolytic

agents. Pronase, previously used for similar purposes

[6,7], caused the rapid disappearance of the acyl-([35S]benzylpenicilloyl-)enzyme (as monitored by SDS/ polyacrylamide-gel electrophoresis) and the appearance

offour major radioactive peptide fragments (as moni-tored by paper electrophoresis at pH6.5, 60 V/cm for

1 h). One of these fragments (I) migrated 2cmtowards

the cathode, another (II) remained at the origin ofthe electrophoretograms and the two others migrated 9cm

Abbreviationused:PBP,penicillin-binding protein. $ To whomcorrespondence shouldbeaddressed.

Vol. 260

(2)

M.

Leyh-Bouille

and others 100 4 3 0

~0

C) x o 1 (a) 80-60 -c) m

40i

co 20 -0 40 Fraction no. (b) E 0. C) 0 ._5

13

*1 I I / I I I I I I 20 30 Fraction no. *0

Fig. 1. Purification of the 35S-labelled active-site-containing fragment (III) of the Streptomyces K15

chromatography on Sephadex LH20(a)andbyf.p.l.c. on a PepRPC column(b)

DD-peptidase/PBP by

For details see thetext. (a)- -,Radioactivity. (b)----,--- Radioactivity; , A24;--,buffer Bgradient. Hatched areas

indicate the fragment-(III)-containing fractions.

(III) and 28 cm (IV) towards the anode. Compound (IV)

was [35S]benzylpenicilloate. Maximal yield of fragment (III) (31

%0

of total radioactivity) was obtained by incubating 20 nmol of acyl- ([35S]benzylpenicilloyl-)en-zymefor 2 h at 37 °C with 58 ,tg of Pronase in 1.6 ml of

0.4%

NH4HCO3. The solution was freeze-dried. Isolation of the 35S-labelled active-site fragment (III)

Step 1. The Pronase digest was dissolved in 100 ,ul of 8800 (v/v) formic acid. After addition of 250,ul of 9500

(v/v) ethanol, thesolutionwasloadedon a35mlcolumn of Sephadex LH20 equilibrated against an 88%

(v/v)

formic acid/95 00 (v/v) ethanol (1:4, v/v) mixture

[8].

Elution with the same mixture yielded three major fractions (Fig. la). Fractions 25-30 contained fragment (II), fractions 38-47 contained fragments (I) and (III) and fractions 53-60 contained

[35S]benzylpenicilloate

(IV).

Step2. Fractions 38-47 were pooled and freeze-dried and the pellet was dissolved in 1 ml of 0.1%

(v/v)

trifluoroacetic acid. Purification of

fragment

(III)

was

achieved on a f.p.l.c. apparatus (Pharmacia,

Uppsala,

Sweden)equippedwitha0.5cmx5cm

PepRPC column,

with various mixtures of buffer A(0.1 % trifluoroacetic

acid in water)and bufferB

[0.1

% trifluoroacetic acid in

water/acetonitrile (3:7,v/v)]. The

gradient

started with

1 ml of buffer A, and then went on from 0% to

200%

buffer B over 8

ml,

from

200%

to 4000 buffer B over

12mland from 40% to 100% buffer B over 5ml

(flow

rate 0.5ml/min; volume of the collected fractions

460,l). Fractions 24-26 (Fig. lb) were pooled and freeze-dried. The final preparation contained 0.72 nmol of purified fragment (III). Paper electrophoresis at

pH 5.6revealed the presence ofasingleradioactive

com-pound. As shown below, it was, however, contaminated by free glycine.

RESULTS AND DISCUSSION

The sequence of the N-terminal region of the protein is shown in Fig. 2. Ambiguity was observed at some cycles following cycle 30 withthe gas-phase Sequenator andfollowingcycle41with thepulsed-liquid Sequenator. The enzymepreparation was very pure,valine being the only N-terminal residue detected.

Although the first cycle of the Edman degradation was made difficult because of the presence of large amounts of glycine, analysis of the active-site peptide fragment (III) yielded the sequence

(Ser)-Thr-Gly-Ser-Thr-Xaa-(Lys), thus demonstrating that the active-site serine residue is at position 35 in the native protein. (The

amount of glycine phenylthiohydantoin derivative at

cycle 1 was solarge that,onthebasis of theradioactivity of the peptide, it was obviously a contamination. As a result of the wash-out effect, the amount of the glycine phenylthiohydantoin derivative gradually decreased during the subsequent cycles exceptat cycle 3.)

Ithasbeen proposedthat all the penicillin-interactive active-site-serine proteins and domains, i.e. the

/-lactamases of classesA,C and D, thelow-MrPBPs and the penicillin-binding domains of the

high-Mr

PBPs, 1989 602

(3)

Streptomyces K1 5 DD-peptidase ;4 H- H > U) Z Hq U) 4 H H z 4 U- U 0 P4 0 0 S1 :> :> C U) Cl) C' u) -14 44 4C4 ¢ H ) EH EH 4 > X >4 >4 -e > 1- E-4 > > PL4 P4 P4 r-4 ¢> >X > P X4 4 4CS 4CS 4CS 4CS H ¢ > -Z) 4 CH *EH 4C) CU) 4CC/4CU)* U- HP4 p4 0h -) 0 C C' C' C' H EH U) H H *44 *C4 *4 E-4 0P4 0;P4 O 00: z V Cz :t Z C) Z rJ P- 06 P4 p w p E-4 rX4 U *Z V > > *.Cc Z U) U U) Z) X A: En Z *> *:> >4 OU) -X p4 -D (DIrX p4 C V) C H z uz cH Pn z > U)s PX C ¢ ZCU 4CU > H cn E-4 : C C 4C44 E-4 :4 z z 0 OH 4 E4H * 0*4C' *CH CU) C0 P4 E4CU H E-4 H 1-4 z C' U) 4 V 4 p

~4

*CH 0 H4 U) 00 p4 z E-4 H ~4C 0 p4 H U-4 -4C *r) *0 P14 ¢ :U H ¢ 4ec-p H E4 04 z P4 CY ¢ P-4 z ¢4 Hq H U- 4 ¢ p4 ¢

E-4 - H'IO >Ll) %4CO) -4 ' P4 ID

--U-> c' r-*>as-Ir--4 r'-H r-4_ It -,t N CN C C V U) eC' 0 4CU H CU) H -C4 4CH 4 p-4 z *;4 *E-4 14C U-9 P4C OU) C' *44 *-9 a 4C ocn z OU) U) H >4 *H U) *PL4 0 opL4 :> o4 *14 U C FQ > O C4R 0c4 *4 04 >CU o; *P9 *H E-4 H U) P4 P sC E4 Om n 4CU) U)V ¢) U) scn Es OU) ¢-H-4 p-4 ~4 O44 (D ¢4 P z U) ¢ C ¢ :e U) H 4CH Z 4C H C ) tP 0 C' -4 rX4 U C U C' 04 0 0 0> o C 0 H C; C ¢ 4 04 0 4 U-;4 U) H 4 P4 U4 0> 4H H 4C 4C OCU) *E4 E4

>U

CU) U) u4) -) c0 U)4 p4 44 p4 (DU OU) PL4 P4H OU m C' 4 U) C' OU) pC4 p4 U)4 CY C OU -X CE w P4 E-4 UV) > 4 C Hy C Cy 0C 4n C' U CU) ¢) H H U) ¢U H C Hx 0C H C' 4 0# H n ¢ uo-4CU-(L ~~~~~~~~~N

~~~~~~~~~~0-

co ~~~~~ ~~~~~~~~~~~~~~~~~LO) LO CN a)a)4) CL~~ ~ ~ ~ ~ -L c

in)

00

0N.

v. CN Cl) a. ~ ~ a--o a-CU 0-z ci CL 0- ci co) co Ca c -.J co ~~~~F ~~~~0-J (O 603 :U 4 z '-4 Ur) z U) C' 4 C' EH Cl) E-4 U) H C Ev ~4 U) r: U) Hv '-0 H Hq p4 0 U) CU -Cd o U) 1 Cd Cd 0 C13 CJ I) Vol. 260

(4)

M. Leyh-Bouilleand others form a super-family of evolutionarily related proteins

[20]. One of theircommon features is the occurrence of the conserved tetrad Ser*-Xaa-Xaa-Lys, where Ser* is the active-site serine residue, close to the N-terminus of the proteinsordomains. The alignments of Fig. 2reveal

anespecially high similarity between the 20-amino-acid-residue stretcharound the active-site serine residue of the

Streptomyces K15 DD-peptidase/PBP (from Thr-21 to

Met-40) and the corresponding regions of the penicillin-binding domain of the

high-M,

PBP1B of Escherichia

coli (400 identity), the low-Mr PBP5 of E. coli (45% identity) and the low-Mr PBP ofBacillus subtilis (50

%

identity). The amino acid sequence failed to reveal the

presence of a signal peptide or an N-terminal

hydro-phobic sequence, thus suggesting that the Streptomyces

K15 enzyme, like the other known low-Mr PBPs, is

probably anchored to the membrane by a C-terminal

segment [21,22].

Thework inLiegewassupportedin partbythe Fonds de la

Recherche Scientifique Medicale (Contract no. 3.4507.83), an

Action Concertee with the Belgian Government (Convention

86/91-90), the Fonds de Recherche de laFacultedeMedecine

U.Lg. andacontractwith theE.E.C.(BAP-0197-B).The work in Gent was supported by the Belgian National Incentive ProgramofFundamental Research in Life Sciences, initiated by the Belgian State-Prime Minister's Office-Science Policy

Programming Department. REFERENCES

1. Nguyen-Disteche, M., Leyh-Bouille, M., Pirlot, S., Frere, J. M. &Ghuysen, J. M. (1986) Biochem. J. 235, 167-176

2. Leyh-Bouille, M., Nguyen-Disteche, M., Bellefroid-Bourguignon, C. &Ghuysen, J. M. (1987) Biochem. J. 241, 893-897

3. Leyh-Bouille, M., Nguyen-Disteche, M., Pirlot, S., Veithen, A., Bourguignon, C. & Ghuysen, J. M. (1986) Biochem. J. 235, 177-182

4. Nguyen-Disteche, M., Leyh-Bouille, M. & Ghuysen, J. M. (1982) Biochem. J. 207, 109-115

5. Hunkapiller, M. W. & Hood, L. E. (1983) Methods Enzymol. 91, 486-493

6. Frere, J. M.,Duez, C. & Ghuysen, J. M. (1976) FEBS Lett. 70, 257-260

7. Duez,C., Joris, B., Frere, J. M. &Ghuysen, J. M. (1981) Biochem. J. 193, 83-86

8. Khorana, H. G., Gerber, G. E., Herlihy, W. C., Gray, C. P., Anderegg, R.J., Nihei, K. & Biemann, K. (1979)

Proc. Natl. Acad. Sci. U.S.A. 76, 5046-5050

9. Broome-Smith, J. K., Edelman, A., Youssif, S. & Spratt,

B.G. (1985)EMBO J. 4, 231-235

10. Asoh, S., Matsuzawa, H., Ishino, F., Strominger, J.,

Matsuhashi, M. &Ohta, T.(1986) Eur.J. Biochem. 160,

231-238

11. Nakamura, M., Maruyama, I. N., Soma, M., Kato, J., Suzuki,H.&Hirota,Y.(1983) Mol. Gen. Genet. 191, 1-9 12. Song, M. D.,Wachi, M., Doi, M., Ishino,F.&Matsuhashi,

M. (1987) FEBS Lett. 221, 167-171

13. Spratt, B.G. (1988) Nature (London) 332, 173-176 14. Broome-Smith,J., Edelman, A. & Spratt, B. G. (1983) in

The Target of Penicillin (Hakenbeck, R., H6ltje, J. V. & Labischinski, H., eds.), pp. 403-498, W. de Gruyter, Berlin 15. Todd, J. A., Roberts, A. N., Johnstone, K., Piggot, P. J., Wintr, G. & Ellar, D. (1986) J. Bacteriol. 167, 257-264 16. Duez,C., Piron-Fraipont, C., Joris, B., Dusart, J., Urdea,

M. S., Martial, J. A.,Fr&re,J. M.&Ghuysen, J.M.(1987) Eur. J. Biochem. 162, 509-518

17. Sutcliffe, J. G. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3737-3741

18. Dale, J. W., Godwin, D., Mossakonska, D., Stephenson, P. &Wall, S. (1985) FEBS Lett. 191, 39-42

19. Jaurin,B.&Griindstr6m, T. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4897-4901

20. Joris, B., Ghuysen, J. M., Dive, G., Renard, A., Dideberg, O.,Charlier, P., Frere,J. M.,Kelly,J.A., Boyington,J. C., Moews, P. C. & Knox, J. R. (1988) Biochem. J. 250, 313-324

21. Ferreira, L.C.S., Schwarz, U., Keck, W., Charlier, P.,

Dideberg, 0. & Ghuysen, J. M. (1988) Eur. J. Biochem. 171, 11-16

22. Pratt, J. M.,Jackson,M. E.&Holland, I. B. (1986) EMBO J. 5, 2399-2405

Received 16January 1989/14March 1989; accepted28March 1989

1989 604

Figure

Fig. 1. Purification of the 35S-labelled active-site-containing fragment (III) of the Streptomyces K15 chromatography on Sephadex LH20 (a) and by f.p.l.c

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