A comparative study of class-D beta-lactamases.

A

comparative

study of class-D f-lactamases

Philippe

LEDENT,* Xavier

RAQUET,*

Bernard

JORIS,* Josef VAN

BEEUMENt

and Jean-Marie

_FRERE*j

*Laboratoire d'Enzymologie, Universite de Liege, Institut de Chimie, B6, B-4000 Sart-Tilman (Liege 1), Belgium,

and tRijksuniversiiteit, Gent Laboratorium voor Microbiologie, Ledeganckstraat, 35, B-9000 Gent, Belgium

Three class-D

_{fl-lactamases}

(OXA2, OXAl and PSE2)

were produced and purified to protein homogeneity. 6,-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same fl-lactamase with 6,-iodo[3H]penicillanate allowed theidentification of Ser-70 as

the active-site serineresidue.Inagreementwithpreviousreports,

the apparent Mr of the OXA2 enzyme as determined by molecular-sieve filtration, was

significantly

higher than that

INTRODUCTION

Two groupsof ,-lactamasescanbedistinguishedon the basisof

theircatalytic mechanisms.Class-B enzymes are metalloproteins

containing at least one Zn2+ ion per active subunit (Ambler,

1980).Incontrast,8-lactamases ofclasses A, Cand D are

active-site serineenzymes(JaurinandGrunstrom, 1981; Mossakowska etal., 1989). Thesethreeclassesdifferin theirprimary structures.

Membersof classesA andC have been widely studiedandsome

high-resolution three-dimensionalstructureshave been obtained

(Kelly etal., 1986; Samraoui etal., 1986; Oefner et al., 1990; Knox and Moews, 1991; Herzberg, 1991). At the present time,

five enzymesclearly belongto class D: OXAI, OXA2, OXA5, LCR1 and PSE2. The geneshave been sequenced (Dale et al.,

1985; Ouelletteetal.,1987;Huovinenet al.,1988; Couture et al.,

1992), and the deduced primary structures do not exhibit

statistically significant similarities to those of the enzymes of

classesAand C (Jorisetal.,1988).Nonetheless,a closeanalysis

(Jorisetal., 1991) shows thatsome of the motifsbordering the active sites of classes A and C enzymes and of the R61

DD-peptidase can be found in similar positions in class-D /1-lactamases. Inconsequence,itseems safe to assume that Ser-70

[numbering of Joris etal. (1991)] is theactive serine of class-D

enzymes. The class-Denzymes exhibitoriginal substrate profiles, oxacillin being themostsensitive tohydrolysis,in contrast with

the situation withmany class-A enzymes (Matagne et al., 1990) and,even moreso, withall class-Cenzymes(Galleni andFrere, 1988; Galleni et al., 1988). If these observations canbe

extra-polated, it is likely that the OXA3, OXA4, OXA6 and OXA7 enzymes also belong to class D (Medeiros et al., 1985). Immunologicalcross-reactionshave, for instance, been observed

with antibodies raised against the OXA2 and OXA3 proteins

(Hollandand Dale, 1985).

The presentinvestigationwasinitiated toobtaindetaileddata about the chemical and catalytic properties of class-D ,-lactamases. Three of the recognized members of the class have beenstudied.

deduced from the gene sequence, but this was not due to an

equilibriumbetween a monomer and a dimer. Theheterogeneity of the OXA2 ,-lactamase on ion-exchange chromatography contrasted with the similarity ofthecatalytic properties ofthe

variousforms. Afirst overview of the enzymic properties of the three 'oxacillinases' is presented. With the OXA2 enzyme,

'burst'kinetics,implying branched pathways, seemedtoprevail

with many substrates.

MATERIALS AND METHODS Bacterlal strains and plasmids

PlasmidR46(52kb)wasthesourceof the OXA2gene(Anderson

and Datta,1965), plasmid pMON234 (5.05kb)wasthesourceof the PSE2 gene(Huovinenetal., 1988) and plasmidpBGS18+ (4.4kb)wasusedasa vector(Sprattetal., 1986). Escherichiacoli

JM105 [endA sbcB15 hsdR4 rpsL thi A(lac-proAB) F' (traD36

proAB+ lacIP lacZAM15)] was used for the production of the

OXA2,f-lactamase,

E.coliHB101(supE44 hsdS20 recA13ara-14 proA2lacYlgalK2 rpsL20 xyl-5mtl-i)for theproductionof the PSE2

,-lactamase

and E. coliHBIOI (pMON301)(Levesque et

al., 1987) for the production of theOXAI,-lactamase.

Antibiotics

Ampicillin and oxacilinwerefrom Bristol Benelux S.A. (Brussels, Belgium), 6-aminopenicillanic acid, carbenicillin, cloxacillin, methicillin, flucloxacillin and dicloxacillin were from Beecham Research Laboratories (Brentford, Middx., U.K.),

benzyl-penicillinwasfromRhone-Poulenc(Paris, France), cefamandole,

cefazolin, cephaloglycin, cephaloridine and cephalosporin C

were from Eli Lilly and Co. (Indianapolis, IN, U.S.A),

6,i-iodopenicillanic acid was fromPfizer Central Research

(Sand-wich, Kent,U.K.), ceftazidimewasfrom GlaxoGroupResearch (Greenford, Middx, U.K.) and cefotaxime was from Hoechst-Roussel(Romainville,France).All thesecompoundswerekindly

given by the respective companies. Nitrocefin was _purchased

from Oxoid (Basingstoke, Hants, U.K.) and 7-amino-cephalosporanic acid from Janssen Pharmaceutica (Beerse,

Belgium).

6fl-Iodo[3H]penicillanic

acid(2.1mCi/mmol)wasthe sample describedpreviously (DeMeesteretal., 1985). Kanamycin waspurchased fromBoehringer (Mannheim, Germany). Proteins

Tos-Phe-CH2Cl ('TPCK')-treated trypsin was from Millipore

Corp. (Freehold, NJ, U.S.A.), BSA and ovalbumin were from

Abbreviations used: BH medium, brain/heart medium; TB, Terrific broth.

$

To whom correspondence should be addressed.

556 P. Ledent and others

Sigma Chemical Co. (St. Louis, Mo, U.S.A.)andlysozymewas

fromBelovo (Bastogne, Belgium).

Protein concentrations were estimated bymeasuring the A280 of the solutions.

Growth media

Brain/heart (BH) medium contained, per litre, 37 g of

brain/heart infusion (bioMerieux, Charbonnieres-les-Bains, France). 'Terrific broth' medium (TB) contained,perlitre, 12 g

of Bactotryptone (Difco), 24 g of Bacto yeast extract (bio-Merieux) 4ml of glycerol and 100 ml of 0.17 M KH2PO4,

0.72 MK2HP04buffer.

Chromatography

Amberlite CG50 was from Serva (Heidelberg, Germany), Sephadex G-25, Sephadex G-75, Sephadex 75 HR 10/30, Sephadex G-100, Mono S HR5/5,QSepharoseHiLoad 26/10 andPepRPCHR5/5 werefrom Pharmacia(Uppsala, Sweden). Theagarose-phenylboronic acid (type B) columnwasprepared

asdescribedby Cartwright and Waley (1984). Spectrophotometers

Allthe spectrophotometricmeasurementswereperformed with the help of a Uvikon 860 (Kontron instruments)

spectro-photometer interfacedtoa Copam+ PC 88C microcomputer.

Gas-phase sequenator

Theamino acidsequenceofthe active-sitepeptidewasdetermined withanApplied Biosystems 470-A gas-phase sequenator.

Genetic techniques

Standard DNArecombinant techniques wereused (Maniatiset

al., 1982).

Kinetic measurements

Usually,acomplete timecourseof thehydrolysis of the substrate was recorded at482nmfornitrocefin, at260nm forthe other cephalosporins, at 240nm for carbenicillin, at 260nm for oxacillin, cloxacillin, dicloxacillin and methicillin,at272nmfor flucloxacillin, and at 235nm for the other penicillins. The interaction between 6,-iodopenicillanic acid and the OXA2 ,J-lactamase was studied by using the reporter substrate method (De Meesteretal., 1987; Galleni and Frere, 1988). When biphasic hydrolysis time courseswereobserved, the values of the initial

rates(v0), the steady-staterates

_(v.,),

andthepartial inactivation

and re-activation rate constants

_(ki

and kr respectively) were determined withthe help of the microcomputeras describedby De Meesteretal. (1987).

Dilutions of theenzymesbelowaconcentration of 0.1 mg/ml were performed with buffer solutions containing 0.1mg of BSA/ml.

RESULTS

Subcloning ofthe OXA2 genefromthe R46 plasmid Inthe

pBGS18+ multicopy vector

In orderto increase the production of the OXA2 ,-lactamase,

the OXA2 gene was subcloned by inserting the 2.5 kb PstI

50860-BamHI 1160DNAfragment of R46 into the BamHI-PstI

site of the multiple-cloning region ofpBGS18+. The ligation mixture was used totransformE.coliJM105.Ampicillin-resistant and kanamycin-resistant transformants were selected. The recombinant plasmid pDML303 was isolated, purified and its restriction mapestablished (Figure 1).

The levels of /8-lactamase production by E. coli JM105 transformed by R46 or pDML303 are compared in Table 1. Fusion of the pMON234 plasmid harbouring the PSE2 gene with thepBGS18+vector

As forOXA2,varioussubcloning experimentswereattemptedin

pBGS18+.Noampicillin- andkanamycin-resistanttransformants wereobtainedwhen the transformation was made with aligation mixture in which thePSE2 gene was on ashortfragment (i.e.the 1350pb BamHI-SphI or the 1550pb BamHI-SstI fragments).

However,each ofthe twoplasmids contains onlyoneEcoRIsite.

Ligation of the plasmids after digestion by EcoRI yielded the

recombinant plasmid pBGS18+: :pMON234 (Figure 1). After

transformation with pBGS18+::pMON234, E. coli HBO1I

overproduced the PSE2 /3-lactamase (Table 1).

Production and purification of the OXA2

f,-lactamase

Twenty 1-litre conical flasks each containing 500ml of BH

mediumwereinoculated with 10ml ofa 16hpreculture in the

same medium and stirred overnight at 37'C. The cells were

separated by centrifugation and submitted to three rounds of

sonication (2x3min). The combined supernatants were

centrifuged for30 min at 39000 g. Atotal

,-lactamase

activity of

27600,umol/min was measured with benzylpenicillin as

substrate.

The isoelectricpH of theOXA2 enzyme being8.0 (±0.5), a

batch adsorption on a CG50 cation exchangerwas performed afteradjusting thepH to 6.0 with dilutedH3P04.Twosuccessive desorptions with 0.1 M Tris/H3P04 buffer, pH 8.6, containing

0.25 M K2SO4 allowed the recovery of 71 % and 10 % of the initialfl-lactamase activity respectively.

Thelaststepofthe purificationwas affinity chromatography

on anagarose-phenylboronic acid (type B) column. The OXA2

enzyme was only retarded on washing with the Tris/ H3P04/K2S04, buffer. Three successivepassages were necessary to obtain the pure enzyme. Table 2 summarizes the different

stepsof the purification.

Production and purffication of the PSE2

fi-lactamase

(Table 3) Aninitialtestof productionwasmade in 100 mlofTBmedium.

Alinear increasein

_,-lactamase

activitywasobserved after the end of the exponential phase.Theactivity after45 hof cultureat 37'C wasapproximatelytentimes higherthanafter 12h.

The bacteria were grown for 2 days at 37 'C in a 20-litre

fermenter containing 15 litres of TB medium. After centrifugation, the cellsweresuspendedin100 mMTris adjusted

to pH 9.4 with EDTA. Four rapid freeze-thaw cycles were

performedto extractthe PSE2

_,-lactamase

from theperiplasm.

Abatchadsorption-desorption on aCG50 cationexchanger

wasalso the firstpurificationstep for this enzyme. The isoelectric pH oftheenzyme is 6.1 and thusadsorptionwas completed by adjustingthe mixture topH4.6with 5MH3P04.Twosuccessive

desorptionsin 50 mM sodiumphosphate buffer, pH 7.6,allowed the recoveryof46 % and 14% of the initialf,-lactamase activity

respectively.

The samplewas filtered at 4'C through a Sephadex G-100 column (80cm x4.0cm) in 50mM sodium phosphate buffer,

Aval3700 SstI3550 SphI3350 XbaI 3000 PstI EcoRl N2670

Figure1 ConstruetonoftheplasmidspDML303andpBGS18+::pMON234 pH 7.6, at aflow rate of 30ml/h. The f6-lactamase was eluted

atavolumeof 320ml.Fractionscontainingfl-lactamaseactivity werepooled and concentrated.

A Q-Sepharose Hiload column coupled with an f.p.l.c.

ap-paratuswasthenused forthe last step of thepurification:buffers

A and B were 50mM sodium phosphate, pH 7.6, and 50 mM

sodiumphosphate, pH 7.6, containing 0.5MK2SO4 respectively;

thegradientwentfrom0to 100% ofbuffer B in500ml;the flow ratewas 5ml/min.The fl-lactamase was elutedatavolume of

100mlcorrespondingto20% of buffer B.

11500 111600

558 P. Ledent and others

Table 1 Levelsofproducton of theOXA2and PSE20I-lactamases

The values are expressed in umol of substrate hydrolysed/min per litre of culture. They

representthemeansof fivemeasurements+S.D.

Enzyme Activity (umol/

Strains produced Substrate min per 1) E.coli (R46) Ecoli (pDML303) Ecoli (pMON234) Ecoli (pBGS18+:: pMON234) OXA2 Benzylpenicillin (500 tsM) OXA2 Benzylpenicillin (500 uM) PSE2 Nitrocefin (1 00,sM) PSE2 Nitrocefin (100

_#M)

160(±15) 640(±40) 10(±1)

Table 4

Purfficatlon

oftheOXAI

fl-lactamase

For details, see the legend to Table 3.

Total Specific

activity Total activity Purifi- Recovered

(/umol/

proteins (4tmol/minper cation activity min) (mg) mg of protein) factor (%)

Crude extract 60 150 0.40 - 100

SephadexG-100 52 40 1.3 3.3 87

Mono S 36 2.7 13.3 33 60(69)

360(±20)

Table 2 PurIficaton of theOXA2 -lactamase

The values in parentheses give the yield (%) of each step. The activity is expressed as ,umol ofbenzylpenicillinhydrolysed/min per mg ofprotein.

Total Specific

activity Total activity Purifi- Recovered (umol/ proteins (,umol/min per cation activity min) (mg) mgof protein) factor (%) Crude extract CG50 Phenylboronate 27600 25000 1.1 - 100 22400 2000 11 10 81 1 19300 650 30 27 70(86) 2 15250 110 140 127 55(79) 3 14000 56 250 227 51(92)

Table 3 Purfflcaton ofthePSE2

fl-lactamase

Theactivity is expressed as,smolof oxacillinhydrolysed/min per mg of protein. The yield (%) of eachstepis_givenin_parentheses.

Total Specific

activity Total activity Purifi- Recovered

(4mol/ proteins (tmol/minper cation activity min) (mg) mg ofprotein) factor (%)

Crude 17000 48000 0.35 - 100

extract

CG50 10200 6700 1.5 4.3 60

Gl00 8300 2500 3.3 9.5 49(81)

QSepharoseHL 6200 150 41 117 36(75)

Production and purfflcation of theOXAI /i-lactamase (Table 4)

A 1-litre conical flask containing 500 ml of TB medium was inoculated with 10 ml ofa 16 hprecultureinthe samemedium and stirred overnight at 37'C. The cells were separated by centrifugation, resuspendedin 100mM Trisadjusted topH9.5

with EDTA and submitted to two freeze-thaw cycles. The solutionwasfilteredat4'C throughaSephadex G-100column (80cmx4.0cm)in 10 mM sodiumphosphate buffer, pH 7.0, at

aflowrateof 40ml/h.The fl-lactamasewaselutedatavolume

of 440ml. Active fractions were pooled, concentrated and dialysed against 50mMpotassiumacetate, pH 4.5.

K _k+2 k+3

E+CC± EC >- EC* - E+P

I _k+4

ECi

Scheme 1 General model for the interactionoffi-lactamase(E) with

6fl-lodopenicillanate (C)

EC* is the acylenzyme and EC' the rearranged adduct containing the dihydrothiazine chromophore.

The sample was then submitted to chromatography on a MonoS columncoupledto anf.p.l.c.apparatus. Buffers A and

B were 50 mM potassium acetate adjusted to pH 4.5 with concentrated acetic acid and the same buffer containing 0.5 M

K2SO4 respectively;theflowrate was 0.5ml/minand thegradient

wentfrom0 to100% of bufferB in 25ml. The/8-lactamasewas

eluted ata volumeof17 mlcorresponding to66 % of buffer B. Interaction ofthe

OXA2

f8-lactamases

with

6-fp-lodopenicillanic

acid

Titration of the OXA2

fi-lactamase

with

6fi-iodopenicillanic

acid

A 'branched-pathway' mechanism (Scheme 1)seems to prevail in the interaction of this

fi-lactam

with several class-A

/1-lactamases where the acylenzyme can rearrange into an

irreversibly inactivated speciesorundergo normal hydrolysis(De Meester etal., 1986). The hydrolysisproduct and the rearranged

inactivator moiety molecule bound to the active site of the enzyme both contain a dihydrothiazine chromophore. The

adsorption maxima are at305nm (e=_{8200 M-1 cm-') and} at 325 nm (e= ₁₂₅₀₀

M-'

_{cm-') for the former and the latter}

respectively (Frereetal., 1982).

Titration oftheOXA2

/J-lactamase

with

_{6,#-iodopenicillanate}

showed that this reagent inactivated the enzyme without apparent turnover

_(k+4

>

k+3).

Theinactivator/enzyme ratio necessary to obtain total inactivationwas 1:1(±5%). Indeed, at the end of thetitration,theabsorptionspectrumexhibitedaclear maximum at325 nm (Figure 2).

The enzyme concentration determined on the basis of the titration endpoint (27.6 ,uM)was ingood agreement with that deducedfromthe absorbance valueat325nm(27.2,uM). From

the known molecular weightand the tryptophan and tyrosine contentof the enzyme, thetheoretical A280(1%)value (Cantor and Schimmel, 1980) could be computed (21.9), yielding a

100 Zg 80 60 40 20 0 (b 2 A 1 0 230

Volumeof6f-iodopenicillanate(pi)

270 2 (c) - 0 ' 320 230 250 0.04 .,- _0.02 600 500 - 400 -5 300 O 200 100 300 350 A (nm) A(nm)

Figure2 Interaction of theOXA2fp-lactamasewith

6fi-iodopenicillanic

acid

(a) Titration of the OXA2 ,8-lactamase by 6,8-iodopenicillanic acid. Identical samples of inactivator(2.4 nmol in 4_,ll)wereaddedto500,ul of theenzymesolution until nofurther increase inA325wasobserved. The end point of the titration correspondedto23,1u (13.8 nmol) of6fl-iodopenicillanicacid.(AA=A325after each addition of inactivator-A325of the solution without inactivator; AA tot.=_A325atthetitration end point -_{A325 of the solution without}

inactivator.) (b) and (c) Absorptionspectraof theOXA2,-lactamase before(b)and after (c) inactivation by6,f-iodopenicillanicacid.

0

[C] (PM)

Figure 3 Inactivaton of theOXA2 f-lactamase with 6fl-iodopenicillanic

acid

(a) Pseudo-first-order inactivation rate constant (ks)as afunction of the inactivator concentration (C). Each point is themean+S.D. of five measurements. (b) Plot of_[C]/kias afunctionof[C]:

r=0.985.

iodopenicillanate]= 100lM; [OXA2]= 1-10,uM),

k,

wasequal

to

_k+2

for whicha value of 2.5 (±0.7)x10-2 s-I was found, in goodagreementwith that determined above.

25.4 uM concentration ofenzyme in the titration experiment,

thus ingoodagreementwith the valuesdetermined above. Kinetic measurements

The determination of constants

_k+2

and Kwas made with oxacillin (500,uM) as a reporter substrate. Pseudo-first-order

inactivation rate constants (k0) were measured for inactivator concentrations ranging from 0.7,M to 12.6,uM (Figure 3). After correction for the protection bythe reportersubstrate,the constants

_k+2

and Kwere calculated with the help ofeqn. (1)

(Frereetal., 1982):

gKms+[S]8

whereS ) kl [I

(1)

where Kms and [S] are respectively the _K,,, for and the

con-centration of the reporter substrate. The

_k+2,

K and

_k+2/K

values

were respectively 3.2 (+0.2)x10-2 s-1, 0.95 (±0.20) ,sM and

34000(±7000)M-1 s-1.Theerrorswerethose obtained for the

parameters after alinear regressionwith the Enzfitterprogram

(Leatherbarrow, 1987).

The value of

_k+2

was verified by directly following the absorbance at 325nm. Under these conditions

([6,8-Identification ofthe active-site serine ofthe OXA2,-Iactamase

Sequencealignments indicated Ser-70astheprobable active-site

serineresidue.Inordertoverifythisassumption,the enzyme was

labelled by adding 95nmol of 6,-iodo[3H]penicillanate to a

stoichiometric amount ofenzyme in a total volume of3.5ml. After 10 min at 30° C,thesamplewasfreeze-dried, and the solid residue redissolved in 500,1 of 8 M urea. The solution was

incubated for6 h at 37° Cand the urea concentrationdecreased

to 4 M by addition of500,1 ofwater. Trypsin digestion was realized in two steps: after addition of 250,g oftrypsin, the

sample was incubated for 12h at 37° C and a further 3 h

digestion was performed after a second addition ofthe same amountofthe same proteaseatthesame temperature.

The digest was filtered through a Sephadex G-25 column (120cmx1cm) in 10mMNH4HC03.Theelution profile showed four radioactive peaks; fractions corresponding to the smallest labelled peptides (18nmol) were pooled and freeze-dried. The

powderwas redissolved in 500 1u of 1% trifluoroacetic acidin water and further purified with the help off.p.l.c. apparatus

equippedwithaPEP-RPCHR(5/5) column. The solventswere

0.1% (v/v) trifluoroacetic acid inwater (A)and in acetonitrile

(B). Theflowrate was0.7ml/minand thegradientwentfrom 0

to60%(v/v) ofsolvent Bover42.5 ml.Thelabelledpeptidewas

elutedat avolumecorrespondingto 28% of solventB. Theresults ofgas-phase sequencing showed the presence of two peptides. Some residues were difficult to identify with

560 P.Ledent and others

Table 5 Active-sitepeptde sequencing

32 39 66 70 73

Sequence (Dale, 1985) F FS E F Q A K

Fraction 49: peptide 1 F FS E-Q A K peptide 2

* _{Residue not identified with certainty.}

Y S P A S*T F K Y-P A- -F K

Table 6 Apparent molecular masses of the OXA2, PSE2 and OXAI

pi-lactamases

as determined onSephadex75HR 10/30,and

_K.,

values

The linear-regression analysis [log (molecular mass) =_f(Ka)] was performed with BSA, ovalbumin and lysozyme (r=-0.988).

Proteins Molecular mass (kDa) Kav

BSA Ovalbumin Lysozyme OXA2 PSE2 OXAl 66 43 14.4 50.6+0.9 35.3+0.6 24.6 +1 0.118+0.001 0.183+0.001 0.602+0.002 0.171+0.002 0.293+0.003 0.415+0.015

the dihydrothiazine-labelled serine was generally poor on

gas-phase sequencing (Amicosante et al., 1988). Although the

sequences wereincomplete, they could bereplacedin theamino acid sequence deduced from the known OXA2 nucleotide

sequence(Daleetal., 1985), and,inparticular,the well-identified residues ofpeptide2allowedanunambiguous positioningin the

sequence. As predicted bythe sequence alignments, itcould be concluded that Ser-70wastheactive-siteserineresidue(Table 5). Behaviour of the

OXA1,

OXA2 and PSE2 f8-lactamases on Sephadex 75

The determination ofthe apparent molecularmassof thethree class-D fl-lactamases was performed on a Sephadex 75 HR

10/30column coupled to an f.p.l.c. apparatus. The

_Kav

values

and the corresponding molecular masses are given in Table 6.

The three class-D

fl-lactamases

were characterized by quite

differentapparentmolecularmasses.Those ofOXA2andPSE2 werehigher thanthose deduced from theirprimary structures;

that of OXAI waslower than 30 kDa.

Thehighapparent molecularmassofthe OXA2

fl-lactamase

on Sephadex 75 HR was not due to non-specific interactions between theproteinand the matrix since these wouldincreasethe

K,V,

leading to a lower apparent molecular mass. It has been

suggested that the OXA2 and OXA3 enzymes could exist as

dimeric proteins (Dale and Smith, 1976), whichwould explain thehighapparentmolecularmassof the former.Inconsequence,

weanalysedthebehaviour of theOXA2enzyme underdifferent

conditions whichmightinfluence theequilibriumbetweendimer and monomer.

The

_Kav

value on the Sephadex 75 HR column was not

influenced by pH variations (from 4 to 10), high ionic

strength (0.5M Na2SO4) or the presence of 200 mM borate (a

reversibleinhibitor), 1 M NaCl04 (a dissociating agent)or 1 M guanidinium chloride. Preincubation of the enzymes with

Table 7 Substrate profiles of OXA2, OXAl and PSE2

fl-lactamases:

penicillins

voand vssareexpressedaspercentages of thevssofbenzylpenicillin arbitrarilytakenas100%. Thekj value characterizes therateof transitiontothefinalsteadystate. Itcorrespondsto an

increase inactivityfor methicillin (lag) andto adecreaseinall other cases. Abbreviations: 6-APA, 6-aminopenicillanic acid; ND,notdetermined; +,burst; 0,noburst; +,very small burst;/, not relevant sincenoburstwasobserved.

Substrate OXA2 OXAl PSE2

6-APA(500 uM) + 0 0 VO 170 70 16 VSS 98 / / vo/vss 1.7 / / kj(s-1) 28x / / Benzylpenicillin (500 ,M) + 0 0 V0 140 100 100 VSS 100 / / vo/vss 1.4 kA(s-1) 35x10 / / Carbenicillin (5004M) + + + VO 80 110 120 VSS 2.3 63 28 vo/vss 35 1.7 4.3 kj(s-1) 77 x10-3 10x10-3 27x10-3

Methicillin (1 mM) + lag lag

VO 45 155 75 VSS 14 390 230 vo/vss 3.2 0.4 0.3 k, (s-1) 14x10-3 47x10-3 61x10-3 Oxacillin(1 mM) 0 + + VO 710 260 500 VSS / 180 430

vo/

vss / 1.4 1.2

ki

(s-1)

,

7.6x10-3 15x10-3 Cloxacillin (1 mM) + + + v0 220 250 1430 VSS 48 75 230 vo/vss 4.6 3.3 6.2

kA (s-1)

42x_10-3 6.9x10-3 22 x10-3 Dicloxacillin (1 mM) + + + VO 120 280 1460 VSS 21 55 155

vo/

vss 5.7 5.1 9.4 kj(s-1) 22x10-3 14 x10-3 24x10-3 Flucloxacillin(1 mM) 0 + + VO Competitive 110 1 VSS inhibitor 1.5 0.2 vo/vss 73 5 kj

(s-1)

20x10-3 ND

cephaloridine,

a substrate

exhibiting

burst kinetics

(see below),

also failedto

modify

the enzyme behaviour.

Affinity chromatography

on an

agarose-phenylboronate

column

of the

OXA2

f8-lactamase

partially

inactivated

by

618-lodopenicillanate

The dimericnatureof the OXA2

,-lactamase

didnotseemtobe

confirmed

by

the

gel-filtration

results. The existence ofa

rapid

equilibriumbetween monomeric and dimeric formsofthe enzyme

was further

investigated by

chemical derivatization

experiments

coupled with

affinity chromatography

on an

agarose-phenylboronate

column.

A

sample

oftheOXA2

/8-lactamase

was 50% inactivated

by

6,-iodopenicillanate

sothatthe ratioofadducttointact enzyme

was1.The mixturewas

applied

tothe

affinity

column whichwas elutedat 20

_{° C}

with 0.1 M

Tris/phosphate,

pH 8.6,

containing

Table 8 Substate profiles of OXA2, OXAl and PSE2

pl-lactamases:

cephalosporins

For the legend, seeTable 7. 7-ACA,7-aminocephalosporanicacid.voand

_vs.

areexpressedas

percentagesof the vssvalue ofbenzylpenicillin.

Substrate OXA2 OXAl PSE2

7-ACA(200

_IsM)

+ 0 0 V0 0.67 1.1 0.1 VSS 0.13 slow and / vo/vss 5.0 total / kA(s-1) 19xlO- inactivation / Cephaloglycin (200,M) + 0 0 VO 3.1 3.5 3.6 Vss 0.5 1 vo/vss 6.2 / / ki(s-1) 19xlO- / I CephalosporinC(300 ,M) + + 0 VO 46 2.2 3.0 vSS 2.4 1.7 / vo/vss 19 1.3 ki(s-1) 29x10-3 12x10-3 / Cephaloridine (150aM) + 0 + VO 45 34 8.2 VSS 1.4 / 6.8 vo/vss 32 / 1.2 kA (s-1) 42x10-3 / 19x1-3 Ceftazidime(100,M) + 0 0 V0 0.12 2.6 0.02 VSS 0.02 Slow and / v0/vss 6 total / ki(s-1) 41 x1-3 inactivation / Cefazolin (1501M) + 0 0 VO 16 13 3.1 vSS 3.6 / / vo/vss 4.4 / / k;(s-') 25x10-3 I / Cefamandole (200,M) + 0 0 VO 8.4 9.5 10 vSS 6.5 / I vo/ vss 1.3 I I

ki (s-1)

4.5x10-3 / / Nitrocefin(150 ,uM) + 0 0 V0 80 215 165 VSS Complex / / vo/vss kinetics / / kA(s-1) / /

0.25MK2SO4;the flowratewas2.5ml/min. The elution profile clearly showed thepresenceoftwopeaks. The firstone,centred

onthe deadvolume, correspondedto

_,8-lactamase

labelled by the inactivator; its absorbance spectrum exhibited a secondary maximum at 325nm. The secondpeak contained non-labelled

enzyme interacting with the boronate groups. The same result wasobtained withadduct/freeenzymeratios of 1: 3and 3:1; the

peak areas determined at 280nm were proportional to these ratios.

This experiment invalidated the hypothesis ofa rapid equi-libriumbetweenmonomeric and dimeric forms ofenzyme.Inthe

presenceof suchanequilibrium, onlyonepeak should have been observed,atanelutionvolume intermediate betweenthose of the non-retarded andretarded forms ofenzyme.

Heterogeneity of theOXA2 8-lactamase on Ion exchangers

Matthew and Hedges (1976) and Matthew et al. (1979) clearly showed the existence of at least three bands on isoelectric

focusing ofanOXA2 preparation. Inorder to determine if these

various forms of enzyme had similar kinetic properties, the following experiment was performed. A sample of OXA2

/3-lactamase (50mg)wasloadedon to aQ Sepharose Hiload 26/10

column with50 mMsodiumphosphate,pH7.0, at aflowrateof

5ml/min. During the washing of the column with the same

buffer, four distinct peakswereseparated. After330ml, thesame

buffercontaining 0.5 M K2S04was used and two other peaks appeared within thenext 100 ml.Thefractions centred oneach ofthepeakswerepooled, concentrated and their kinetic

proper-ties studied. The hydrolysis ofcephaloridine by the OXA2 ,-lactamasebeingbiphasic,aninitialrate/steady-state ratio(v0/v,8) and apartial inactivation rateconstant (k1) could be measured (see below). These differentparameters were the samefor all the

enzymeforms. Moreover, theproteinsinthe sixseparatefractions exhibited similar mobilitieson SDS/PAGE.

Substrate profile of OXA1, OXA2 and PSE2

f8-lactamases

Tables 7 and 8 give the relative rates of hydrolysis of several penicillins and cephalosporins respectively (benzylpenicillin being

thereference) and, whenabiphasic hydrolysiswasobserved, the

vo/v..

ratiosandthefirst-orderrateconstant

ki

characterizing the rateof transitionto thefinalsteady state. Ascouldbepredicted from the data available in the literature, the three enzymes

consistently hydrolysed oxacillin and cloxacillin faster than benzylpenicillin, justifying their appellation as 'oxacillinases'. However,surprisingly,in most cases,thehydrolysis time courses

didnotcorrespondtothesimple Henri-Michaelis model.'Bursts' wereoften observed and 'lags'inthehydrolysis of methicillin by

OXA1 and PSE2. In all these experiments, the enzyme

con-centration (0.04-0.1 ,M) was much lower than that of the substrate (0.1-1 mM) and the size of the burstwassignificantly larger than the enzymeconcentration, thus indicating that the

burstwasnotduetoacylenzyme accumulation butmoreprobably

tothe occurrenceofa branchedpathway.

The

_vo/vS,

ratios increased with the number of Clatoms on

the isoxazolyl side chain butthe replacement of the second Cl withFproduced specific results dependingon the enzyme.

Cephalosporins,with thepossible exception of nitrocefin,were generally poorer substrates than penicillins. With these substrates, the behaviour of OXA2 was clearly different from thoseofthe twootherenzymes.Indeed, OXA2did nothydrolyse

anyof the cephalosporins accordingtosimple Henri-Michaelis

kinetics whereas OXAI and PSE2generally obeyedthis simple model.

DISCUSSION

Duringthisstudy,wehavepurifiedthree class-D

,-lactamases

to

protein homogeneity. Our protocol of purification of the OXA2 and

OXAl

enzymes gave higher specific activities and better

yields than thosepreviously published (Yamagishiet al., 1969;

Dale andSmith,1971;Monaghanetal.,1982; Holland andDale, 1984). Philippon et al. (1983) purified the PSE2 ,-lactamase 56-foldwith a 26 %yieldbut didnotindicatethespecific activity ofthe pure enzyme.

Surprisingly, despite the apparent lack of affinity for

3-aminophenylboronate, the OXA2

_,/-lactamase

was retardedon

theaffinitycolumn. Thisphenomenon seemedtobespecificsince theinactivationofthe enzymeby

6,8-iodopenicillanate

appeared

to suppress theinteraction. Onemight arguethat the observed

results were due to anion-exchange phenomenon and that the additional negative charge supplied by the inactivator moiety

562 P. Ledent and others

seems unlikely since thechromatography wasperformed in the

presence of 0.5M

_K2SO4.

The interaction of the OXA2 ,-lactamase with 6,-iodopenicillanate followed a simple linear pathway and the

k+2/K

valuewasintherange of thoseobserved with class-A fi-lactamases. Moreover, theKvaluewas strikinglylow.

Isolation of an active-site peptide after labelling with

6,/-iodopenicillanate allowed the identification of Ser-70 as the

active-site serineresidue. Thiswasin agreementwiththe tentative identificationbased onthe gene sequence (Daleetal., 1985). It

couldbe argued thatpeptide 1 also contained a serine residue, but Ser-34 is not conserved in other class-D enzymes, which excludes it asthepotential active-site serine (Joriset al., 1991). Moreover, in the analogous class-A /-lactamases (Joris et al., 1991),the corresponding peptide ispartofana-helix inthea/fl

domain, faraway from the active site.

Inagreementwith the resultsof Dale (1971) andFoster(1983), the behaviour of the OXA2

_,f-lactamase

during molecular-sieve chromatography indicated anMr value twiceaslargeasthat of

OXAlandconsiderably largerthanthat deducedfrom thegene sequence. Since covalent linking oftwoidentical subunits could berejected onthe basisof the normal behaviour of theenzyme on SDS/PAGE and the absence of cysteine residues in the sequence, we explored the possibility of a rapid equilibrium betweena monomerand adimer.Noconditionswerefoundthat

could increase the elution volume of the enzyme. Moreover,

when

6fl-iodopenicillanate-inactivated

enzyme was mixed with

the intact protein, no subunit interchange leading to the

formation of hybrid dimers (or polymers) could be detected. In

conclusion, thecausesof the apparently larger Mr value observed for the OXA2 enzymeduringmolecular-sieve chromatography

remain amystery, since onthebasis of the structural analogies withthe class-A,3-lactamases,onewouldnot expecttheOXA2 proteinto present anespeciallyelongated shape.

As observed with many other

/8-lactamases

(Matagne et al., 1991), several different forms of the OXA2 enzyme could be separatedbyion-exchange chromatography. The Mr values of all theseforms, determined by SDS/PAGE,weresimilaranditwas

safe to conclude thatthey originated from slightly 'ragged'

N-termini or deamidation of particularly fragile asparagine residue(s). Moreover, the catalytic properties of the various forms appearedtobe identical.

Afirst overview of the catalytic properties ofthethreeenzymes

has been realized. As expected, the hydrolysis ofoxacillin was

catalysedmoreefficiently than that ofbenzylpenicillin. Moreover, themostunexpected resultwasthefinding that,atleast with the

OXA2 enzyme, 'burst' kinetics seemed to prevail with many

substrates.Infact,thisphenomenonperfectly explained the wide dispersion ofthe

_Vm,.

orinitial-rate values foundinthe literature. Indeed, we observed an apparent correlation between that dispersion and the size of the 'burst'. The two other enzymes more consistently produced 'bursts' with substrates that are

knowntoinduce thattypeofbehaviour, suchastheisoxazolyl penicillins. These phenomena, which imply the existence of a

branched pathway, are at present underdetailed investigation.

This work wassupported,inpart bytheBelgian GovernmentintheframeoftheP6le

d'Attraction Interuniversitaire (PAI no. 19), Actions concertees with the Belgian

Government(conventions 86/91-90 and89/94-130), the Fonds de la Recherche

Scientifique Medicale (contractno. 3.4537.88)and aconventiontripartite between the RegionWallone, Smith Kline Beecham, U.K. andthe University of Liege. P.L.

and X. R. are fellows of the Institut pour 1'encouragement de la Recherche

Scientifique dansl'Industrie etl'Agriculture(IRSIA, Brussels,Belgium)and B. J. is

'Chercheur Qualifie' of the Belgian National Foundation for Scientific Research

(FNRS). P. L. thanks everybody in the Laboratoire d'Enzymologie (Liege) for their help and encouragement and P. Cuvelier (Boehringer Mannheim, Belgium) for financial support.

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