Streptomyces DD-carboxypeptidases as transpeptidases. The specificity for amino compounds acting as carboxyl acceptors

Biochem. J.(1973) 131, 707-718 Printed in Great Britain

Streptomyces

DD-Carboxypeptidases

as

Transpeptidases

THE SPECIFICITY FOR AMINO COMPOUNDS ACTING AS CARBOXYL ACCEPTORS By HAROLD R. PERKINS and MANUEL NIETO*

National Institute for Medical Research, Mill Hill, London NW7

IAA,

U.K.

and JEAN-MARIE

FRLRE,

MltLINA

LEYH-BOUILLE and JEAN-MARIE GHUYSEN Service deMicrobiologie,Institut deBotanique, Faculte de Medecine, Universite deLiege,

SartTilman,4000Lie'ge, Belgium (Received 23 October1972)

Theability of the water-solubleDD-carboxypeptidases of Streptomyces strains albus G, R61, Kll andR39 to perform transpeptidation wasstudied. The donor was diacetyl-L-lysyl-D-alanyl-D-alanine, and awhole range of amino acids, peptides and structurally

related amino compoundsweretestedfor acceptor function. No compound testedwas anacceptorfortheenzyme fromstrainalbus G whereas the enzymes from strains R61 and

Ki1

could utilize with varying efficiency a wide range of substances

including

peptides

with N-terminal

glycine

or

D-alanine,

co-amino acids, aminohexuronic

acids,

6-aminopenicillanic acid and D-cycloserine. Certain peptides, when present in higher

concentration, inhibited the transpeptidation observed at lower concentration. The enzyme fromstrainR39wouldnotuse anydipeptideas anacceptor, butafewcompounds

that were notglycine or a-aminoacidsoftheD-configuration didfunction thus. These wereD-cycloserine and the lactams ofmeso- orracemic-diaminoadipic acid.

Soluble DD-carboxypeptidases that hydrolyse the C-terminal peptide bond ofacyl-D-alanyl-D-alanine

and analogous peptides have been isolated from certain strains ofStreptomyces (Ghuysenetal., 1970; Leyh-Bouille etal., 1970b, 1971,1972), and it has been proposed that suchenzymesmightrepresentasoluble

form of the transpeptidases that perform the final peptide cross-linking in peptidoglycan synthesis (Leyh-Bouille etal., 1970b). This hypothesiswasgiven

considerable support when it was shown that the

penicillin-sensitive DD-carboxypeptidases from

Streptomyces strains R61 and R39 would transfer thepenultimate carboxylgroupofthe synthetic sub-stratediacetyl-L-lysyl-D-alanyl-D-alaninetothe amino

group of a suitable acceptor such as D-alanine,

glycineormeso-diaminopimelic acid (Pollocketal., 1972). Theenzymefromstrain R61 would also use

the dipeptide glycylglycine as an acceptor, thus giving a product that was not a substrate for the

enzymeand hencecouldnotpossibly be the result of

asimple reversal of hydrolysis. This peptide could be

consideredasananalogue of the

glycyl-LL-diamino-pimelic acid N-terminus that would be expectedtobe theacceptor forcross-linking during biosynthesis ofa peptidoglycan such as that found in Streptomyces

strainalbus G(Leyh-Bouilleetal., 1970a). However, theenzymefromstrainR39didnotuseglycylglycine asanacceptorandtheonefromstrainalbus Ggave notranspeptidation withanyoftheacceptorstried.

*_{Present address: Instituto de Biologla Celular,}

Velazquez 144, Madrid-6, Spain.

Vol. 131

These findings stimulated an investigation of the

specificity of theseenzymesforacceptorsinthe

trans-peptidation reaction, the results of which are

de-scribedinthepresentpaper. MaterialsandMethods

Chromatographyandelectrophoresis

The chromatographic solvent systems were as

follows: (1) butan-l-ol-acetic acid-water-pyridine (15:3:12:10, by vol.); (2) butan-l-ol-acetic

acid-water (3:1:1, by vol.). Paper electrophoresis was

performed in the following buffer solutions: A, aceticacid-collidine-water (2.65:9.1:1000, by vol.), pH7; B, acetic acid-pyridine-water (1:25:475, by vol.), pH6.5. Whatman no. 3 paper required for

preparative purposes was first exhaustively washed

by irrigation with 1M-ammonium acetate followed

bywater. Peptides

The peptide used as a substrate for

carboxy-peptidase activity and as the carboxyl donor for

transpeptidase activity, diacetyl-L-lysyl-D-alanyl-D-alanine,waspreparedasdescribed by Nieto&Perkins

(1971). The same peptide radioactively labelled in

the acetyl groups was prepared as follows.

[1-"4C]-Acetic anhydride

_(50pCi;

26.6,Ci/4mol)

was

con-densed at the bottom of the ampoule provided by thesuppliers (The Radiochemical Centre, Amersham, 707

Bucks., U.K.) bycoolingit at -80° C and warming up slightly therestofthe ampoule. The top ofthe ampoulewas brokenand

L-lysyl-D-alanyl-D-alanine

(3,mol)

and

triethylamine

(lul)

in water-dioxan

(1:1, v/v) (40,u) were added quickly. The frozen solution wasmelted, mixed thoroughly andkeptin an ice bath for 1h. Then 1 ,ul of acetic anhydride (non-radioactive) was added and the reaction was

allowedtoproceedfor afurther 1h intheicebath.

The reactionmixturewasheated for1minat 100° C, concentrated to dryness, redissolved in water and

finally purified bypaperelectrophoresis inbufferA

(tOV/cm, 3h). After radioautography, two main

bands were observedcorresponding to the

[14C]di-acetyl-peptide (17,uCi)

and the

mono[14C]acetylated

derivatives.

Thefollowing radioactive acceptorpeptides were

synthesized as described by Nieto etal. (1973) for D-alanyl-D- [U- 4C]alanine: D-alanyl-L- [U-

14C]-alanine,

glycyl-L-[U-14C]alanine,

glycyl-D-[U-14C] alanine,

D-alanyl-[U-14C]glycine

and

L-alanyl-L-[U-14C]alanine.

N-Glycyl-N'-[1- 4C]acetyl-LL-diaminopimelic

acid

was synthesized as follows.

Mono[1-14C]acetyl-LL-diaminopimelic acidwasobtained

by

acetylation with radioactive acetic anhydride

(50,uCi;

26.6,uCi/,tmol)

as described above but with a large excess of

LL-diaminopimelic acid

(1.4mg)

and omitting

acetyl-ation in non-radioactive acetic anhydride. By that

means 21

_,uCi

ofmonoacetyl derivativewasobtained after purification by paper electrophoresis as de-scribedabove.

Mono

[1

-

14C]acetyl-LL-diaminopimelic

acid (21

pCi)

inwater-acetonitrile(1:1,v/v)

(lOO,ul)

was

treated with 1.7mg of NaHCO3 and 1.5mg of

t-butoxycarbonylglycine

N-hydroxysuccinimide ester.

Thereactionwasallowedtoproceedfor 2h at room temperature. Thereaction mixturewasthenapplied

to Whatman 3MM paper for ascending paper

chromatographyinsolvent1andthe productlocated by radioautography (RF0.64,compared with RF0.27

forthe monoacetyl

derivative).

After elution ofthe radioactive band with

methanol-0.04M-NH3

(1:1,

v/v),

the dry product was dissolved in trifluoro-acetic acid-dichloromethane (1:1, v/v) (400,d) and

kept for 10min at 0° C followed by 10min at room temperature.The

resulting

N-glycyl-N'-[1-14C]acetyl-LL-diaminopimelic

acid trifluoroacetatewas

concen-tratedto

dryness,

redissolved in buffer Aandagain

concentrated. This step wasrepeated.Itwasfinally

appliedtowashedWhatman no. 3 paper for electro-phoreticpurificationinbufferA(10V/cm,3h). The

product moved towards the anode with a mobility

relative to glutamic acid of0.55. Its RF value by ascending paper chromatography in solvent 1 was

0.22. Theproduct elutedfrompaperelectrophoresis (14.8,iCi)should be of the samespecific radioactivity

asthe acetic anhydride used tolabel it.

The synthesis of the same peptide not

radio-actively labelledwascarried outsimilarlyon alarger

scale. Intermediatesandproducts were identifiedby

theirelectrophoreticproperties compared with those of the parent compounds, their amino acid

com-position and the Dnp-derivatives obtained after treatmentwithfluorodinitrobenzeneandsubsequent hydrolysis in 4M-HCI at 100° C for 6h. Also the

acetyldipeptide gave a grey colour withninhydrin,

compared with a normal purple colour for the

mono-acetyl derivativeofdiaminopimelic acid.

oc-Acetyl-L-lysine was preparedbytreatingL-lysine monohydrochloride with 1 mol.prop. of acetic an-hydride and 4mol.prop. ofNaHCO3. After 2h at

0° C,the mixture was adjusted to pH7 with acetic acid andsubjected toelectrophoresis onwashed paper in buffer A (1OV/cm, 3h). The neutral ninhydrin-positive band was eluted and re-submitted to paper

electrophoresis,this time in 0.25M-formic acid

(IOV/

cm,3 h). The main band wasa-acetyl-L-lysine(moved 16.4cm towards the cathode) and there was a minor band of E-acetyl-L-lysine (moved 11.5cm). The

identitiesofthese bands wereconfirmed by prepara-tion of the mono-Dnp derivatives, hydrolysis and

paper-chromatographic separation in phosphate

buffer (Levy, 1954). oc-Acetyl-L-[4,5-3H]lysine was prepared by a similar method. L-[4,5-3H]Lysine

monohydrochloride (0.2mCi) was added to

un-labelled compoundtogiveatotalof8nmol,andthe samplewasdried under vacuum.NaHCO3 (0.125M;

501l)

was added, followed by acetic anhydride (0.5,ul). After 30minat0° Cthe mixturewasacidified with formic acid and separated by paper electro-phoresis in 0.25M-formic acid as described above. Marker spots weredetectedwithninhydrinorbythe

procedure ofRydon &Smith (1952). Measurement of radioactivity in the eluted sample showed the

following approximate proportions of the total:

unchanged L-lysine, 3%; e-acetyl-L-lysine, 7%;

a-acetyl-L-lysine,40%;

diacetyl-L-lysine,

50%.

e-Glycyl-oc-acetyl-L-[4,5-3H]lysine

was then

syn-thesized in a manner similar to that used for the diaminopimelic acid peptide. Chromatographic

pro-perties(solvent 1, ascending, Whatman3MMpaper)

were asfollows:

oc-acetyl-L-lysine,

RF 0.35,RGIY 1.92, typical purple colour with

ninhydrin;

c-(t-butoxy-carbonylglycyl)-ac-acetyl-L-lysine,

RF 0.85, RGIY 4.6; e-glycyl-a-acetyl-L-lysine, RGIY 1.35, bright-yellow

colour whentreated with

ninhydrin

after chromato-graphyin thissolvent.

Other acceptors

2-Aminoglucuronic acidand2-aminogalacturonic

acid werekindly given by Dr. H. Paulsen, Institut fur Organische Chemie und Biochemie, Hamburg, Germany,andmuramic acid wasgivenbyDr.R.H.

STREPTOMYCES DD-CARBOXYPEPTIDASES/TRANSPEPTIDASES

were prepared as described by Nieto etal. (1973). Homologous w-aminocarboxylic acids and D-cyclo-serine(D-4-amino-3-isoxazolidone) werecommercial samples.

Enzymes

The solutions of Streptomyces

DD-carboxypep-tidases were those described earlier: S. albus G, approx. 20

units/,ul

(Ghuysen et al., 1970); strain R61,approx. 27 units/,ul (Leyh-Bouilleetal.,1971);

strain R39, approx. 100 units/,ul; strain Kit,

approx. 30

units/pl

(Leyh-Bouilleetal., 1972). One

unit of enzyme releases l nmol of D-alanine from

diacetyl-L-lysyl-D-alanyl-D-alanine in 1 h at 37° C, whenthe substrateis present atsaturating

concentra-tion.

Transpeptidation experiments

The buffer solutionused was sodium phosphate,

pH8, 17mM in phosphate ions, except for the S.

albus G enzyme, where it was Tris-HCI, pH7.5, 17mM in Tris, containing MgC12, 1.7mM. The

temperature was 37° C, the time ofincubation was 1h,the amount of enzyme solution usedwaseither

51t

(S.

albus

G,

strain

R61,

strain

Kll)

or

2,1t

(strain R39), and the donor peptide,

diacetyl-L-lysyl-D-alanyl-D-alanine (5Onmol) was present in a final

volumeof 30,u1. Theconcentrationofacceptor was varied as indicated in the text.

Afterincubation, samples were cooled' in ice and

then applied underastream ofhot airtoWhatman 3 MM paper forelectrophoresis either in bufferA (10 V/cm, 2h) or in a high-voltage apparatus (Electro-phorator model D; Gilson Medical Electronics,

Middleton, Wis., U.S.A.) with buffer B (80V/cm, lh). In experiments with "4C-labelled compounds the labelled components were located by

radioauto-graphy and appropriate areasof paper wereeluted

with water (0.3-0.4ml). The eluates were placed in

vials withscintillation fluid [lOml of a solution'in dioxan (final vol. 1 litre) of naphthalene (180g), 2,5-diphenyloxazole (4g) and

1,4-bis-(4-methyl-5-phenyloxazol-2-yl)benzene (1g)], and radioactivity

wasmeasured in aPackardTri-Carb liquid-scintilla-tioncounter. Theefficiency of countingwas70-73%

for 14C and 25%for 3H.

Inmostexperimentshigh-voltage electrophoresis in

buffer B was employed, since this allowed simul-taneous measurement of residual donor tripeptide, thedipeptide producedfrom itbycarboxypeptidase action and the transpeptidation product. Relative

mobilities aregiven in Table1.

Table 1.Electrophoretic mobilityatpH6.5ofdiacetyl-L-lysyl-D-alanyl-D-alanineand itstranspeptidationproducts

Electrophoresis was performed on Whatman 3MM paper in a high-voltage apparatus, with buffer B

(80

V/cm, 1h).

Compound Mobility relativeto

-meso-diaminoadipic acid -meso-diaminopimelic acid -Gly-Gly -Gly-Gly-Gly -Gly-Gly-Gly-Gly -Gly-L-Glu

-lactamofmeso-diaminoadipic acid -aminoglucuronic acid -3-aminopropionic acid -4-aminobutyric acid -5-aminovaleric acid -6-aminohexanoic acid -Gly-oc'-acetyl-LL-diaminopimelicacid -Gly-Gly-NH2 -Gly-Gly-L-Leu -Gly-L-Ala -6-aminopenicillanic acid -oc-acetyl-L-lysine -D-cycloserine

bis-(Ac2-L-Lys-D-Ala)-DD-diamninoadipic

acid Ac2-L-Lys-D-Ala-D-Ala 1.14 0.87 0.84 0.96 0.90 0.82 1.60 0.92 0.89 0.93 0.88 0.79 0.75 1.40 0 0.80 0.92 0.89 0.86 0.38 1.26 Ac2-L-Lys-D-Ala Ac2-L-Lys-D-Ala 709

Results

Water-soluble DD-carboxypeptidases from some Streptomyces strains, when incubated with diacetyl-L-lysyl-D-alanyl-D-alanine in buffer solution, liberate theC-terminal D-alanine residue. When these enzymes were incubated with the same substrate (donor) and

certain amino compounds (acceptors),

transpeptida-tionoccurred (Pollock et al., 1972) and their specific-ity for these acceptors has been studied. In initial experiments radioactive acceptors were used and thus only the proportion of acceptor thatwasconverted into transpeptidation product could be measured directly. The proportion of donor converted was then

calculated (Table 2).

Enzynes from strains R61 andKI1

These enzymes were able to use as acceptor glycine, D-alanine or the D-centre of meso-diaminopimelic acid, and in addition a whole range of dipeptides in which the free amino group was that of glycine or

D-alanine.Withacceptors of the general composition glycyl-R, the highest proportion of transpeptidation occurred when R was L-alanine, glycine or L-lysine linked by its E-amino group. The presence ofa D-asymmetric centre (R=D-alanine) considerably de-creased the effectiveness of the acceptor for

trans-peptidation. A similardecrease occurred when the

side chain of the L-amino acid forming the

C-terminusof thedipeptidewasfairly bulkyandcarried

Table 2. Transpeptidation by Streptomyces DD-carboxypeptidases toradioactively labelledacceptors

Theincubation mixture contained donor (diacetyl-L-lysyl-D-alanyl-D-alanine, 50nmol), buffersolution(sodium phosphate, pH8, finalconcentration 17mM,except forexperiments with enzymefrom S. albus G when it was

Tris-HCl, pH7.5, final concentrations 17mM, Mg2+ 1.7mM), enzyme solution 5,l (except for strain R39,

2,ul)

and acceptor, finalvolume

3Ou1.

Incubation wasfor 1 h, 37° C. Transpeptidation product was separated

fromacceptorbypaperelectrophoresisinbuffer A, and their radioactivities were measured. The radioactivity in the product, calculated as a proportion of the donor used, is shown as % transpeptidation. Withan acceptor R, thetranspeptidationproduct wouldbeAc2-L-Lys-D-Ala-R. Some of theresultswith amino acids and glycyl-glycine as acceptors were reported by Pollock et al. (1972) and are included here for comparison.

Transpeptidation (%) x-Amino acids [14C]Glycine

D-[14C]Alanine

L-[(4C]Alanine

meso-[3H]Diaminopimelic

acid* Mono[14C]acetyl-LL-diaminopimelic acid Dipeptides 1-[14C]Glycylglycine

Glycyl-D-['4Cjalanine

Glycyl-L-[14C]alanine

E-Glycyl-oc-acetyl-L-[3H]lysine

x-Glycyl-oc'-[14C]acetyl-LL-diaminopimelicacid D-Alanyl[l4C]glycine

D-Alanyl-L-['4C]alanine

D-Alanyl-D-[14C]alanine

L-Alanyl-L-['4C]alanine

*_{It was shown by Pollock et al.}

transpeptidation.

Acceptor/donor

molarratio Enzyme fromstrain . ..

1:1 10:1 1:1 10:1 10:1 1:1 10:1 1:1 R61 13.7 48 7.5 50 0 16.0 0 1:1 10:1 1:1 1:1 1:1 Kll R39 S.albus G 45 30 18.2 48 18.4 3.6 25.5 18.2 0.56:1 4.1:1 1:1 1:1 1:1 1:1 4.8 2.7 3.6 2.6 0.4 0.2 4.0 24.6 35 0 7.4 45 0 0 0 0 0 0 3.4 0 2.1 0 0 0.2 0 0 0 0 0 0 0 0 0

STREPTOMYCES DD-CARBOXYPEPTIDASES/TRANSPEPTIDASES

afree carboxylgroup(R=

monoacetyl-LL-diamino-pimelic acid). This peptide was synthesized as an

analogueoftheside chainthatwouldbe thenatural

acceptor for atranspeptidase cross-linking

peptido-glycan in thecell walls of strains S. albus G, R61

andKl1 (Leyh-Bouilleetal., 1970a). It isnoteworthy that with thisparticular peptideatenfoldincrease in

acceptor/donor ratio produced a decrease in the

amount of donor undergoing transpeptidation. This

observationwillbe discussed further below.

Dipeptides with D-alanineatthe N-terminus were muchpoorer acceptorsthanthe correspondingglycyl

peptides.

Indeed, D-alanyl-D-alanine was hardly an acceptor atall, whereas glycyl-D-alaninewas

reason-ably good. Since glycyl-L-alanine was such agood

acceptoritwastheoreticallypossiblethat L-alanyl-L-alaninemight beanacceptor, eventhough L-alanine itself was not. However, the L-alanine dipeptide hardly functioned as an acceptor, showing that an asymmetric centre ofthe L-configuration at the aminoendof thedipeptide effectivelyprevents

trans-peptidation.

Theenzyme fromstrainKl 1closelyresemblesthat

from strain R61 in many respects (Leyh-Bouille et al.,1972),and it wasthereforehardly surprising that

it behaved similarly in transpeptidation of the few acceptors examined.

Enzyme from strain R39

This enzyme, when functioning as a

carboxy-peptidase,

hasa

fairly

similarsubstrate

specificity

to that observed with the enzymes from strains albus

G, R61 andKl1 (Leyh-Bouille etal., 1970b, 1971, 1972).Thechief differenceis that apeptide withafree

c-amino group as in

ax-acetyl-L-lysyl-D-alanyl-D-ala-ninewas aparticularly good substrate,whereasitwas averypoor one forthe otherenzymes. However,in the transpeptidation reaction with diacetyl-L-lysyl-D-alanyl-D-alanine as donor, only glycine and the

D-centres of a-aminoacidswereaccepted (Table2). Itseemed possiblethat with this enzyme the nature of the donormight affect the efficiencyof transpeptida-tion,and soaparallel experimentwassetup inwhich

the donor was

x-acetyl-L-lysyl-D-alanyl-D-alanine

(a particularly good substrate for carboxypeptidase action)and theacceptors, at a molar ratioof10:1,

wereglycine, glycylglycine orglycyl-L-alanine.Once more only glycine served as acceptor (53% of the donor wasconvertedinto transpeptidationproduct). Hence, with either donor, none of the dipeptides studied served as acceptor, and this wasparticularly surprising for the c-glycyl-cx'-acetyl-LL-diamino-pimelic acid, which was known to be the 'natural' acceptor for thepeptidoglycan cross-linkingreaction in other strains of Streptomyces (Leyh-Bouille et

al.,1970a). Vol. 131

Enzyme fromS.albus G

This enzyme would not effect transpeptidation

with any of the acceptors tried. In other respects it is alsoexceptional(Pollocketal., 1972) and it may well be that the true

transpeptidase

of this

particular

organism is not the same as the isolated

carboxy-peptidase.

Simultaneous measurement of donor hydrolysis and

transpeptidation

Previous results showed that with D-alanine as acceptor, transpeptidation took place faster than hydrolysisofthedonorpeptide (Pollocketal.,1972),

andTable2 showedthat increased concentrationsof

a-glycyl-a.'-acetyl-LL-diaminopimelic acid decreased

the amount of

transpeptidation.

To facilitate the simultaneous study ofhydrolysis and transpeptida-tion andtheuseofagreater range of acceptors, we

prepared

di[14C]acetyl-L-lysyl-D-alanyl-D-alanine

for use as donor. This compound was incubated with

enzymes and acceptors and components of the

re-action mixture were separated by paper

electro-phoresis at high voltage (pH6.5, buffer B);

com-pounds

werelocated by radioautography and their radioactivitywasmeasured.

Theeffectofincreasingtheacceptorconcentration

wasstudied withglycine andwithglycyl-L-glutamic acid,acommercially available analogue (inthe sense that it has a free w-carboxyl group) of

o-glycyl-a'-acetyl-LL-diaminopimelic acid, and which is also closely relatedto

glycyl-L-alanine, previously

shown to be a very good acceptor for the enzyme from strain R61 (Table2).The enzymewasincubatedwith

radioactive donor and increasing

concentrations

of acceptors, and the amounts of transpeptidation productandresidual donorweremeasured(Table 3).

Theresultsshowthat, as might beexpected,increasing the concentration of glycine at fixed donor

con-centrationincreased the

proportion

of

transpeptida-tion relative to hydrolysis. The amount of donor

remaining unattacked after incubation for 1 h

decreased slightly as more acceptor was added.

However, with

glycyl-L-glutamic

acid as acceptor the

result was different. Although the ratio of

trans-peptidation to hydrolysis again increased with acceptor concentration, the proportion of residual donor rose steadily from 18.5% in the absence of acceptor to 55% in thepresence of

50mM-glycyl-L-glutamic acid. A somewhat similar result was

ob-served with oc-glycyl- oc'-acetyl-LL-diaminopimelic acid, although in this case overall breakdown of donor decreased even more in the presence of the

acceptor. An analogous peptide, identical save for the carboxyl groupato theamino group bearing the glycine residue, was

c-glycyl-oc-acetyl-L-lysine.

In-creasing the concentration of this peptide also

decreased the overall breakdownofthe donor (Table

4)00 .-o 00 00 4 S- 0 ~~~ V- 0~~~ P40 .,-4 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Cd~ ~ ~~~~~~~~~~~~~~~~~~~' P",

04

St r ek)"A. 8 0 . 0~ 0~Q - tn t D00 (Na t t0 0 [-4 000 t N 0 ' e 0l% 0 q Cd -0 Cd~~~~~~~~~~~~~~~~~~~~~~~~~~~4 0~~~~~~~~~~~~~~~~~. 00 (L 0~~~~~~~~~~~~~ > c 0 0 0: C;4 (~0 4 ci o 60 ; ~~ ~ ~ ~ 4)0I0l nelC4)~ 87~~-4 Z _0, U l 04 d -4r4 s-40~ C:'0 0 0 Cd0 C4 C) 0C4 a o U,o ~~~~j 0~~~~ene W5 2! t) 0~~~~~~~~~ Cd 4)a 0 .00

STREPTOMYCES nD-CARBOXYPEPTIDASES/TRANSPEPTIDASES 3). Atthe highestconcentration used, however, the

proportionofdonorhydrolysed todipeptide hardly changed, whereas the amount of transpeptidation

product diminishedgreatly. This contrasts withthe

results obtained with glycyl-L-glutamic acid, where

thehydrolysiswas much themoreaffected, orwith

cx-glycyl-a'-acetyl-LL-diaminopimelic acid, where the twoprocesses wereaffectedmorenearlytothesame extent.

Toseewhether theside-chain carboxylgroupwas

essential for the

inhibitory

effect, therelative

pro-portionoftranspeptidationandhydrolysiswas

com-pared with glycyl-L-alanine and glycyl-L-glutamic acidasacceptors, eachat aconcentrationof16.7mM

(acceptor/donor ratio 10:1). Withincreasing timeof

incubation the decrease in residual donor remained exactly parallel for both acceptors, each of which decreased overall donor breakdown (Fig. 1). At any time the proportion oftranspeptidation relative to hydrolysis was higher for glycyl-L-alanine than for glycyl-L-glutamic acid.

Theeffect of theconcentration ofglycyl-L-alanine

on transpeptidation was further examined under initial velocity conditions (a maximum of 17% of available donorwasconsumed). Acceptor

concentra-tionsranged from 2.5 to 20mM. Aplotof 1/vagainst l/[acceptor] (Fig. 2) shows that, starting at a con-centration below 10mM, glycyl-L-alanine exercised

considerable substrate inhibitiononthe transpeptida-tion reactranspeptida-tion.

Acceptors other than simple amino acids and dipeptides Although the enzyme from strain R39appearedto require glycine or a free D-aminoacid asacceptor,

the enzyme from strain R61 was evidently far less

specific. We therefore studied a range of amino

compoundsasacceptorstodiscovertheeffectofthe

length ofthe peptidechain, whether amino groups

0

so

* 0 0 0 50 100 150 Time (min)

Fig. 1. Relative transpeptidation and hydrolysis of

donorbyenzymefromstrain R61 inthe presence of glycyl-L-alanine orglycyl-L-glutamic acid

The incubation mixture

(90,ul)

contained

di[14C]-acetyl-L-lysyl-D-alanyl-D-alanine

(150

nmol),

acceptor

(1.5

p,mol),

phosphatebuffer, pH8 (17mM), and en-zyme fromstrainR61

(15,ul).

The temperaturewas 37° C.Atintervalssamplesweretaken and the com-ponents separated by high-voltage paper electro-phoresisasdescribedin the Methods section.

Trans-peptidation product, ;hydrolysis product,---;

residual donor,...; o, acceptor, glycyl-L-alanine; A, acceptor, glycyl-L-glutamic acid. Under thesameconditions in the absence of acceptor, the

residual donor after 60min had decreased to only

18

%

oftheinitial

value.

Vol. 131 1.5r '-. 04 S-0

-E

1.0 ,-- 1--Q

0.5.1

₂

10-2/[Accptor]

(litre/mol) 4 Fig. 2. Kinetics of transpeptidation with

glycyl-L-alanine as acceptor

The incubation mixture

_(35/4)

contained

di[14C]-acetyl-L-lysyl-D-alanyl-D-alanine(87.5nmol),enzyme from strain R61 (25ng), phosphate buffer, pH7.0 (5mM),andvarious concentrationsofglycyl-L-alanine.

Incubationwas at37° C forperiodsof upto80min. The product was separated and measured as de-scribed in the Methods section. VT iS thevelocity of

the

transpeptidation reaction.

02 '0 '.4 0 i 0 ',.4 0 C) .02 02

'o

n -£oo b eN c'oo r o X 4orn o ooo o- o 0

'

o ----o - ei CI CI - o ,F N t vo t N o

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other than a-amino groups could undergo trans-peptidation and if a terminal carboxyl group was also

required. The effectiveness ofvarious peptides and amides as acceptors is shown in Table 4. Increase in

chain length ofpolyglycines beyond the dipeptide

caused a decrease intranspeptidation, butintroducing abulkyside chain on the C-terminal residue of the

tripeptide (L-leucine replacing glycine) improved acceptor capacity somewhat. Amidation of the

C-terminal carboxyl group ofglycine orglycylglycine

greatly decreased their function as acceptors. In each case theintroduction of anamidegroup resultedin apoorer acceptor than ifanotherglycine residuehad

beenintroduced. Thustoachievethe same decrease

in acceptor function produced by amidation of glycylglycine, it was necessary to add another two glycineresidues. Therewas adistinction herebetween

the enzymes from strains R61 and R39. Although the enzyme from strain R39 would not use any of

the dipeptides tried as acceptors (Table 2), it gave

weak transpeptidation with glycineamide (0.5% at anacceptor/donor ratio of 1:1 and2.7%at aratio

of 10:1). Thus for acceptor function by this en-zyme, amidationofthe a-carboxyl group was not so

damagingastheintroductionof a secondamino acid residue.

D-Alanyl-L-alaninewas an acceptor fortheenzyme fromstrain R61 (Table 2). For otherpurposes we

synthesized ananalogue of this peptide, namely the lactam ofmeso-diaminoadipic acid, in whichthetwo

methyl side chainswere

joined

byacovalent bond, thus forcingacisconformationonthe peptide link (Nietoetal.,1973),and wetherefore testedthis

com-poundas atranspeptidation acceptor. Itfunctioned quite well for the enzyme from strain R61 (Table 5),

being probably

abetteracceptor than D-alanyl-L-alanine(Table 2). Thus the changeinconformation enforcedby cyclizationmayfavouracceptorfunction.

Surprisingly,

theenzyme fromstrainR39could also

usethe meso-diaminoadipic acid lactamas a

trans-peptidation acceptor.Apartfromglycine amide,this was the only substance so far found, other than

simple ac-amino acids, that wouldserve thisfunction.

Forcomparison, Table5 also shows the proportion oftranspeptidation thatoccurredwiththelactam of

racemicdiaminoadipic acid,andthe uncyclized meso-and DD-isomers. The values for the straight-chain

meso-diaminoadipic acidmay besomewhat low, as

thiscompound is ratherinsolubleand gave a slightly turbid solution at the concentration used. The DD-isomer ofdiaminoadipicacid was a particularly good acceptor for both enzymes, and it is noteworthy that both were able to perform transpeptidation at each end of the same acceptor molecule, leading to a

di-substituted acceptor. A similar result was also observed with the enzyme from strain R61 when

DD-diaminopimelicacid was theacceptor.

Since the lactams ofdiaminoadipic acid are

six-membered rings with an amino and a carboxyl

groupattacheddiametrically, it seemed possible that other molecules of the same general pattern might also function as acceptors. The 2-amino-2-deoxy-hexuronic acids are such molecules, and Table 5 shows that the two tested, with the D-gluco and D-galacto configurations, were both quite good accep-tors forthe enzyme from strain R61, but not at all for theone from strain R39. Thus the arrangement ofcarboxyl and amino groups was clearly acceptable totheformer enzyme, even with the large number of hydroxyl groups present, but the latter could no

longer perform transpeptidation, either because the relative distance or arrangement of charged groups was incorrect, or because of steric hindrance by the hydroxyl groups.

Tocheck that theaminosugarconfigurationalone was notenough to make acompoundanacceptor for the enzyme from strain R61, glucosamineand mur-amicacid were examined. Like

2-amino-2-deoxy-D-glucuronic acid, both have the D-glucose

configura-tion, but the former has no carboxyl group and the

latter has a carboxyl group in adifferent situation. Neither compound was an acceptor for either enzyme

(Table5).

Thelactamsof thediaminoadipicacid isomers and

the aminohexuronic acids each have a carboxyl

groupseparatedfrom an amino groupbyan

approxi-mately similar distance. Atomic models suggested thatthis distance would be aboutthe sameasin the

extended form of4-aminobutyric acid or

5-amino-valeric acid. These co-amino acids, and their next lowerand higherhomologues, werethereforetested as transpeptidation acceptors (Table 5). None was an acceptor for the enzyme from strain R39, and contrary to the above suggestion, the four-carbon andfive-carbon acidswere poorer acceptors forthe

enzyme from strain R61 than their three- and six-carbonhomologues. Themoststrikingdifferencewas betweenglycine,with its a-amino group, for which

thepercentagetranspeptidationatanacceptor/donor ratioof 5:1 was calculatedtobe about 33% (inter-polation on a semi-logarithmic plot of the results

given in Table 3), and 3-aminopropionic acid, with avalue ofonly 6%. Similarly, 5-aminovaleric acidwas amuch poorer acceptor thanglycylglycine, which has about the same separation between its free carboxyl and amino groups. Clearly this dis-tance alone is not the major factor governing the

effectiveness of a compound as a transpeptidation

acceptor.

The introduction of anacetamidogroup atothe

carboxyl group of6-aminohexanoic acid (a-acetyl-L-lysine)decreased its acceptor functionconsiderably (Table 5). This contrasts with the effect of a bulky side chain at the C-terminus of triglycine (glycyl-glycyl-L-leucine),whichsomewhat improved acceptor

STREPTOMYCES DD-CARBOXYPEPTIDASES/TRANSPEPTIDASES

some extent resemble the lactams ofdiaminoadipic acid isomers are 6-aminopenicillanic acid and

D-cycloserine. The former is rather a poor antibiotic ofthe penicillintype and the second isregardedasan analogue ofD-alanine,normally supposedtofunction

byinterfering with alanineracemase and D-alanyl-D-alanine synthetase(Stromingeretal.,1960;Neuhaus

&Lynch, 1964).

6-Aminopenicillanic

acidfunctioned as a poor acceptoronly for theenzyme from strain R61 (Table 5). In the same experiment

carboxy-peptidase

actionwas

greatly

inhibited. Thus

6-amino-penicillanic acidwasable to occupytheacceptor site

of the enzyme from strain R61, but this does not

necessarily mean that this site was the seat of

inhibitory action. It could mean, however, that in some organisms the action of 6-aminopenicillanic acid,asdistinctfromitsacyl derivatives the penicillins, might becomplicated byitsabilityto functionas a

transpeptidaseacceptor.

D-Cycloserine, however,

was anexcellentacceptor fortheenzyme fromstrainR61

(better than glycine, for instance: compare Tables 5 and3) andquiteagoodone evenfor theenzyme from strain R39. It had no effect on the overall

breakdown ofthe donor. Thusinthis respect,asin

others,D-cycloserine serves as anexcellentanalogue

ofD-alanine, which is alsoagoodacceptor(Pollock

etal.,

1972).

Some ofthe

transpeptidation product,

di[14C]acetyl-L-lysyl-D-alanyl-D-cycloserine,

was

iso-latedafter paperelectrophoresis and testedas a

sub-strate andinhibitorfor the

carboxypeptidases

from

strains albus G, R61 andR39. It functioned as an

inhibitor fortheformertwoenzymes, butwasalsoa

substrate(Nietoetal.,1973).

Discussion

The observation that the soluble DD-carboxy-peptidasesfromstrainsR61 andR39, butnotstrain albus G, will function in vitro as transpeptidases (Pollocketal.,1972)hasbeengreatly extended.The enzyme fromstrain R61, whichas acarboxypeptidase has high Km values for awholerange ofsubstrates

(Leyh-Bouille

et al., 1971), would transpeptidatea

large number of acceptors, including glycine,

D-amino acids, peptides withD-alanine orglycine

N-terminal,andcertain cyclic compoundswithsuitably arranged aminoandcarboxylgroups. Ingeneral,the

presenceofafree carboxylgroupseemedtobehighly

desirable for acceptorfunction,andamidation greatly decreasedit.Theonlyeffectiveacceptor studied that had no free carboxyl orsimple amidegroupwas

D-cycloserine. However, this antibiotic behaves as a zwitterion withpKa valuesof 4.4 and7.4,and ithas beenproposedthat thecarbonyloxygen atomatothe amino group bears an effective negative charge

(Kuehl etal., 1955). Similarly, at pH6.5 the

trans-peptidation product

diacetyl-L-lysyl-D-alanyl-D-cycloserine behaved as an anion on paper

electro-phoresis (Table 1). ThusatpH8, the value used for the transpeptidase assay, the charge properties

would be similar to those of an a-amino acid, andits functionas an acceptor would be

explicable.

The results show that certain acceptors such as

glycyl-L-alanine, glycyl-L-glutamicacid and c-glycyl-a'-acetyl-LL-diaminopimelic acid, when present at higher concentrations, caused a decrease in overall

breakdownof the donor to an extent independent of their function as acceptors. Thus glycyl-L-alanine

inhibited overall breakdown of donor to exactly the same extent as glycyl-L-glutamic acid, and yet at acceptor/donor ratios of 10:1

transpeptidation/

hydrolysisratios were 1.80 and 1.08 respectively(Fig.

1). Similarly o-glycyl-a'-acetyl-LL-diaminopimelic acid was arelatively poor acceptor and yetgreatly decreased the overall breakdown of donor. Never-theless, the effect of this peptide on the relative proportion of transpeptidation and hydrolysis was different from that of the analogous E-glycyl-oc-acetyl-L-lysine (Table 3).

One mechanism by which an acceptor might de-crease the overall breakdown of the donor is as follows. Alanine may be released by two pathways, either by direct hydrolysis or, in the presence of acceptor, by the competing process of transpeptida-tion. Ifbinding ofthe acceptor to the enzymewere good, but the transpeptidation reaction were slow relative to hydrolysis, then the presence of acceptor would lead to a decrease in the rate of release of alanine. If, however, excess of acceptor led to a decrease in the amount oftranspeptidation,then this

inhibitoryeffect might well be exercised separately from acceptor function, perhaps by binding at a different site on the

enzyme.

Further workwith the enzyme from strain R39 and acceptorsderivedfrom

peptidoglycans

has produced results strongly sup-porting this suggestion (Ghuysenet al., 1973).

The limited range of compounds that would function as acceptors for the enzyme from strain R39 was surprising, particularly since

oc-glycyl-oc'-acetyl-LL-diaminopimelic

acid, an analogue of the

transpeptidase acceptor site in some Streptomyces

peptidoglycans,

was not an acceptor. However,

further work has shown that strain R39 has

meso-diaminopimelic acid andnobridging amino acid in

its cross-linked peptidoglycan, and that 'natural' acceptors will function perfectly well in vitro with the enzyme from the same strain (Ghuysen et al.,

1973).

Wethank Mr. C. S. Gilbert and Mr. I. D. Bird for technicalassistance. This work was supported in part by grants (to J.-M. G.) from the Fonds de la Recherche Fondamentale Collective, Brussels (no. 1000) and the Institut pourl'Encouragementde la Recherche Scientifique dans l'Industrie etl'Agriculture, Brussels (nos. 1699 and 2013).

References

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