expression of recombinant diaminopimelate epimerase in escherichia coli : isolation and inhibition...

Eur. J. Biochein. 186, 137-143 (1989) c; FEBS 1989

Expression of recombinant diaminopimelate epimerase in Escherichia coli Isolation and inhibition with an irreversible inhibitor

William HIGGINS ’, Chantal T.4RDIF1, Catherine RICFIAUD’, Michkle A KRIVANEK and Alan C4RDIN’

’ Institut de Microbiologie, UniversitE de Paris-Sud, Orsay, Francc Merrell Dow Research Institute, Strasbourg, France

Merrell Dow Research Institutc, Cincinnati, USA

(Receivcd May 10/August 8, 1989) - EJB 89 0584

Recombinant diaminopimelate epimerase is overproduced to give 1 YO of soluble protein when grown under the appropriate conditions in Escherichia coli. This compares with 0.02% of the constitutive level of wild-type enzyme. A new purification procedure now yields milligram quantities of homogeneous enzyme of high specific activity (192 U/mg). This has enabled sufficient amounts of enzyme both to compare with wild-type enzyme and to enable active site modification studies to be performed. Incubation of the enzyme with 2-(4-amino-4- carboxybutyl)-2-aziridine-carboxylic acid (AZIDAP), results in time-dependent irreversible inhibition. Tryptic digestion of the inactivated enzyme and peptide-mapping show that AZIDAP is specifically and covalently bound to the enzyme at a unique peptide. Determination of the amino acid sequence of this peptide and comparison with the sequence deduced from the DNA sequencc of the dapF gene shows that Cys73 is labelled. Finally based on limited sequence similarities around this cysteine and active-site cysteines of proline racemase and 1- hydroxyproline 2-epimerase, together with mechanistic considerations, we propose that all three non-pyridoxal- phosphate-containing racemases/epimerases derive from a common evolutionary origin.

The peptidoglycan layer of the bacterial cell wall is essen- tial to protect against osmotic shock and lysis. Several unusual amino acids, including D-alaninc and n,L-diaminopimelic acid (A,pm), are incorporated into UDP- N-acetylmuramyl- pentapeptide, the cytoplasmic precursor to peptidoglycan [l]. These unusual amino acids are unique to prokaryotes and should therefore be good targets for the rational design of new antibacterial agents. u-Alanine is formed from L-alanine by alanine racemase and this enzyme is both the target of known antibactcrial agents and the object of extensive studies for the design of new ones [2-51. Although apriorian attract- ive target, the enzyme diaminopimelate epimerase, which con- verts L,L-A,pm into L,u-A,pm or meso A2pm, has been little studied, mainly because it is present in low abundance [6]. The enzyme is also interesting per se bccause it is a non-pyridoxal- phosphate-containing enzyme and kinetic studies [6] have suggested that it operates via a two-base mechanism anal- ogous to proline racemase [7].

Only recently has the location of the dapF gene, which codes for Azpm epimerase, been determined [8]. We isolated the gene and expressed it from a high-copy-number plasmid. Thus we are now able to produce large amounts of the enzyme, making further work possible. Recombinant Azpm epimerase is essentially equivalent to enzyme previously isolated from E. coli strain W [6] (see Results). In addition, we show that Azpm epimerase is irreversibly inactivated by AZIDAP (F. Cierhart et al., unpublished work), a ncw spccific inhibitor of the enzyme that will be described in full elsewhere. We also

Correspondence fo W. Higgins. Merrcll Dow Rcsearch Institutc, 16. rue d’hkara, F-67009 Strasbourg Cedex, France

Ahhreviaiions. AZIDAP, 2-(4-amino-4-carboxybutyl)-2-aziridine carboxylic acid ; A2pm. 2,6-diamiiiopiinelic acid; Cin, carboxymcthyl ; Pth, phcnylthiohydantoin.

A’nzymps. Diaminopimelic-acid epimcrase (EC 5.1 .I .7): meso- diaminopinielic-acid dehydrogenase (EC 1.4.1 .-).

show that this irreversible inactivation results in the active site modification of a cysteine residue. Detcrmination of thc sequence of this cysteine-containing peptide showed it is in perfect agreement with the amino acid sequence deduced from the DNA sequence that we have recently determined [9]. Thus we confirm that this active site cysteine is Cys73 in the ovcrall sequence. Finally, based on limited active-site-scquencc similarity and mechanistic considerations, we propose that Azpm epimerase, proline racemase [lo] and 1 -hydroxyproline 2- epimerase [12] are homologous, and derive from a common evolutionary origin [ll].

MATERIALS AND METHODS Escherichiu coli strains and plasmids used have been de-

scribed [XI. Trypsin and soybean trypsin inhibitor were from Roehringer-Mannheim. 2-(4-Amino-4-carboxybutyl-2-aziridine carboxylic acid was provided by the chemistry laboratory

A p pm Ho2cw co2H NH, NH,

PROPOSED AZIDAP- CYSTEINE ADDUCT

H2N- CH-CO, H

Fig. 1 . Strirctirres ofA2pm, AZIDAP and AZIDAP-cysteini.

138

of the Merrell Dow Research Institute, Strasbourg. AG50W- X4 ion-cxchange resin and all electrophoresis chemicals were obtained from Bio-Rad. DEAE-cellulose was from Whatman, and matrix green A and blue A from Amicon. [G-3H]A2pm and ['4C2]iodoacetic acid (56 Ci/mol) were from Amersham, and Aquasol-2 scintillation fluid was from Du Pont. FPLC apparatus and all columns (pepRPC, Mono Q) were from Pharmacia.

Growth of plusmid-containing and host strainfor expression of A g m epimerase

The plasmid-containing strain (pdF3/JM109) and the host strain (JMI09) were maintained on N-broth agar slants (sup- plcmcnted with 50 pg/ml ampicillin in the case of pdF3/ JM109). Strains were inoculated into nutrient broth (5 ml) and grown at 37°C for 9 h. Precultures, consisting of either 50ml minimal medium (M63) [13] with amino acid sup- plement or 0.1 % acid hydrolysed casein, or rich medium [14], werc inoculated with 1 ml of the 9-h cultures and, after growth overnight, 10 ml of these cultures was diluted into 250 ml of the appropriate fresh medium contained in 1-1 flasks. Growth curves were determined according to Miller [13]. For dctermi- nation of Azpm epimerase levels, samples (1 3 ml culture) were removed. centrifuged, washed with buffer A (20 mM Tris/ HCl, pH 7.0, 1 mM dithiothreitol, 1 mM EDTA) and stored overnight at - 20 "C. Analysis for A,pm epimerase activity is as describcd below. In all cases ampicillin was added to all pdF3/JM109 culture media to SO pg/ml.

Large batches of pdF3/JMlO9 were grown in an 80-1 Biolaffittc fermentor containing 50 1 minimal medium (M63), 0.1 % acid-hydrolysed casein and ampicillin (50 pg,/ml). Over- night precultures (40 i d ) were inoculated into 2 1 and again grown overnight. This preculture was used to inoculate the fermentor and growth was continued for 5-6 h. Cells were harvested. when absorbance at 540 nm exceeded 1.5, by means of an ultrafiltration system (Amicon), centrifugation and washing with buffer A before freezing at -20°C. From 50 1 culture a yield of 110 g (wet mass) was obtained.

Azpm epimerase assays

A single time point assay was used based on the epimerase- catalysed release of 3H20, as described [6] during 40 min at 25°C [8]. In this assay activity is defined as 'H,O radioactivity released in 40 min (see Results). Jn addition, A,pm epimerase activity was also measured via a coupled assay system [6] in which L,L-A,pm is transformed by the epimerase into meso- A,pm in the presence of excess rneso-A2pm dehydrogenase which reinoves the me.sn-A2pm, producing NADPH, which can be continually monitored at 340 nm. In this assay, one unit of epimerase activity is 1 pmol NADPH formed/min under these conditions [6].

Airification qf Azpm epimerase

Frozen cell paste (55 g wet mass) from pdF3/JM109 was suspended in 125 ml buffer A in an Ultra-turrax apparatus (10 min). Passage through a French press (136 MPa), dilution with 125 ml buffer A and centrifugation (30000 x g for 20min) gave the S30 crude extract. The supernatant was adjusted to 30% saturation by adding solid ammonium sulphate. Following centrifugation and further addition or ammonium sulphate (to 45% saturation), the precipitated protein was recovered and dissolved in buffer A (final volume

2

0 Q N

a

(

1.0

z D z!

3 5

5

0

I I I I I I 0 10 20 30 40 50

RETENTION VOLUME

Fig. 2. Final.ceparation of A,pm epimeruse on Mono Q HR.515. Sample: 500 pi A,pm epimerase partially purified up to the matrix-green-A step. Flow rate: I ml/min. Eluant: 20 mM potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol. Gradient: NaCl as shown. Detec- tion: 280 nm. Shaded area: enzyme activity

10 ml) which was dialysed overnight against 2 1 buffer A. The dialysed crude enzyme preparation was applied to a DEAE- cellulose column (2 .3 x 49 cm), equilibrated with buffer A. The column was washed with 250 ml buffer A and eluted with a linear gradient formed by adding 250 1111 buffer A containing 0.25 M KC1 to 250 ml buffer A. The flow rate was 25 ml/h and 10-ml rractions were collected. A,pm epimerase eluted near the end of the gradient at 0.22 M KCI. Pooled fractions containing Azpm epimerase activity were dialysed overnight against 2 1 buffer A. EnLyme was concentrated by applying the dialysate to a small DEAE-cellulose column (0.9 x 10 cm), rinsing with 50 ml buffer A and eluting with buffer A containing 0.50 M KCl. The flow-rate was 20 mlih and 2.5-ml fractions were collected. Enzyme activity cluted immediately and pooled fractions were dialysed against 1 1 buffer A.

Enzyme, concentrated in this fashion, was stable at 4°C for six months but lost activity upon freezing.

Further purification was obtained by applying the concentrated and dialysed enzyme to a matrix-green-A column (1.4 x 5 cm). rinsing with 50 ml buffer A then eluting with buffer A containing 1.2 M KCI and collecting 2.5-ml fractions. Fractions containing A,pm epimerase activity were dialysed against bufkr A and cencentrated on a small DEAE- cellulose column as described above. A final purification step was obtained by injecting 500-pl aliquots of enzyme onto a Mono Q HR 5/5 column (see Fig. 2).

SDJ'IPAG E

SDSjPAGE was performed essentially as described [15] using 10% or 15% acrylamide. Silver staining of protein was performed according to [16] and protein was fixed in the gel by the method described in [17].

A Z I D A P-labelled enzyme

Azpm epimerase was treated with SO pM AZlDAP for 30 min at 37'C in 20 mM potassium phosphate buffer, pH 7.0. An aliquot was diluted 100-fold and tested for activity remaining to confirm that the enzyme was > 99% inactivated. The inactivated enAyme was dialyzed against 2 1 NH4HC03

139

Tablc 1. Purification of A p n epimerase from pdF3IJM109 Frozcn cell pastc (55 g) grown as described in Materials and Methods. was purified as described in Materials and Methods

Fraction Volume Total protein Specific activity Recovery Purification

ml mg S30 extract 281 4430 Ammonium sulphate 38 1250 DEAE-cellulose 9.3 173

Mono Q 2.7 1.6 Matrix green A 16.9 77

dPm/Pg %

89 800 100 148 000 47 468 000 20 934000 18

9 200000 10

-fold I 1.6 5.2

10.4 100

and lyophilized. AZIDAP-labelled enzyme was then carboxymethylated with either [ ''C]iodoacetic acid or iodoacetic acid as described below.

Curboxymethylution of A2pm epimerase

Enzyme ( z 200 pg) was taken up in 0.5 ml Tris/HCl/urea buffer (0.4 M TrislHCl, pH 8.3, containing 0.8 mM EDTA and 6.4 M urea) in a reactivial (Pierce) sealed with a rubber septum and kept under nitrogen for 10 min. An aliquot of 100 mM dithiothreitol was added through the septum to give a final concentration of 5 mM and the reactivial was incubated at 45°C for 180 min. The reactivial was enclosed in foil and 250 pCi of [14C,]iodoacetic acid, dissolved in 50 pl H,O was addcd. After 16 min at 22 C, 50 pl 14 M 2-mcrcaptoethanol was addcd to quench the rcaction and limit carboxymethylation to cysteine residues [l8]. The reaction mixture was dialyzed overnight against 250 ml 50 mM NH4HC03, followed by two further changes of 1 I NH4HC03 then ly ophilized.

Tryptic digestion

Lyophilized carboxymethylatcd A,pm epimerase (Cm-A,pm), AZIDAP-labelled carboxymethylated-A2pm epimerase or [14C]Cm-A,pm epimerase was dissolved in 250 p10.2 M N-ethylmorpholine acetate buffer, pH 7.8. A 5-pl aliquot of trypsin (1 mgjml in 1 mM HC1) was added and the mixture was incubated at 37°C for 90 min. Digestion was terminated with 5 p1 soybean trypsin inhibitor (5 mg/ml in N - ethylmorpholine acetate, pH 7.8).

FPLC peptide mapping

Peptide mapping was performed using a FPLC apparatus equipped with a pepRPC column. Gradient elution was performed with buffer A (0.1 "% trifluoroacetic acid in H20) and buffer B (0.1"A trifluoroacetic acid in H20/CH3CN, 50: 50) according to the program described in Fig. 5. Peptides werc detected at 214 nm and peptides for sequencing were collected manually. In certain experiments fractions were collected in I-ml volumes using a FRAC 100 fraction collector (Pharmacia) with mini-vials and the [' 4C]carboxymethylated peptides were detected by scintillation counting after adding 4 ml Aquasol-2 (Du Pont).

Sequencing of active-site peptides

Automated Edman dcgradation was performed on a model 470 A protein peptide sequencer (Applied Biosystems, Inc.) with reagents, instructions and standard programs sup- plied by the manufacturer. The phenylthiohydantoin deriva-

tives of amino acids were analysed at each cycle on a model 120 phenylthiohydantoin (Pth) analyzer (Applied Biosystems, Inc.) directly on-line with the sequencer.

RESULTS

Expression o j the dapF gene product and purification qf A2pm epimerase

In wild-type strains of E. coli (W. Higgins and C. Tardif, unpublished results) and host strain JM109, Azpm epimerase is expressed at a constant and low level throughout the growth phases. Following cloning of the dupF gene [8], expression of A,pm epimerase from plasmid pdF3 varied twofold or threefold during the growth cycle and with growth media, but total levels of A,pm epimerase increase from an estimated 0.02% to around 1 % (results not shown).

Purification Of'Azpm epimerase

Table 1 summarizes the purification scheme. Early steps include ammonium sulphate fractionation and DEAE-cellulose chromatography. However, dye-ligand chromatography and ion-exchange chromatography, using FPLC methodology, proved particularly useful for final purification steps. It had previously been shown [6] that reactive-blue agarose (Sigma) bound Aapm epimerase. We screened the enzyme against several dye ligands and found that it was retained by both blue A and green A. Green A was chosen as having superior purification properties to blue A and gave good recovery, but dialysis to remove 1.2 M KCI, which inhibits Azpm epimerase activity, was required. Mono Q ion-exchange chromatography, although using a N + R 3 group slightly different from DEAE-cellulose, follows the same principle but gives increased resolution. Surprisingly, the separation shown (Fig.2) could not be obtained with Tris/HCI buffer but required phosphate to gain the optimal resolution. Analysis of the enzyme at various stages of purification by SDSjPAGE is shown in Fig. 3. Only after the dye-ligand affinity column does Azpm epimerase represent a significant band on SDS/PAGE. In Fig. 3, lane G, which conlains 2 pg Aapm epimerase, silver staining shows that impurities represent < 0.01 % of the applied protein. The apparent subunit molecular mass (32 kDa) for A,pm epimerase is in agreement with previous results [6]. Using the coupled assay procedure [6] our purest preparation of A2pm epimerase had a specific activity of 192 U/mg, compared with 109 U/mg obtained previously [6]. A more detailed comparison (Table 2) shows differences in both K, and k,,,. Since strain differences between E. coli W and K37 (the origin of pdF3; see [19]) are unlikely to be the cause, these different kinetic paramcters are probably a reflection of thc higher specific activity of the current enzyme. The specificity constant

I40

z W

a Value obtained from plots according to Lineweavcr-Burk [25]. I, Value obtained from plots according to Lineweaver-Burk [25]

for V,,, and assuming pure active monomer 32 kDa.

kDa

4 10-7

f ix

A B

92 - 66- 45- 31 -

A2 Pm --. c

epimerose

92 - 66- 45- 31 -

A2 Pm --. c

epimerose

C D E F kDa

-92 -66 - 4 5

-3 1

-2 1 21 -

- 1 4 14-

Fig.3. SDSIPAGE of Azpm epimerase at various stages ofpurijicalion. (A and B) 15% gel of S30 crude extracts of pdt'3,/JMIU9 and JM109, respectively, after 7 h of growth in L-broth [13]. (C-G) 10% gel; C, S30 crude extract; D, ammonium sulphate fraction; B1 pooled DEAE- cellulose fraction: F, matrix-green-A fraction; G, final purification step after Mono Q column

Table 2. A comparison of' wild-type and rwomhinant Azpm epirnerase Values for strain W are &ken from [6]. Values for the recombinant

Materials and Methods. (A) The enzyme was obtained from wild- type E. coG strain W (chromosomal). (B) The enzyme was obtained

T I M E ( m i n )

0 enzyme were obtained using identical couplcd assay procedure. see 100

- from recombinant pdT3iJM109 - s Source of Molecular Ampli- Puri- Spccific K,,, k,,, + 50

k enzyme mass fication fication activity ?

~ I\\'?* \ I- 0 kDa -fold U/mg pM s Q

A 34 k 2 1 6000 108 160 84 2 W

B 3 2 + 2 53 102 192 238" 132b ; 2 0 \

k,,,/K, is generally considered a truer measure of kinetic parameters and these are virtually identical for the two preparations; 525000 s- ' . M-' for strain W and 555000 s-l . M-' for pdF3.

Wiseman and Nichols [6] have suggested a second form of Azpm epimerase. We suggest that this may result from aggregation of subunits during their DEAE-cellulose chromatography step. We have reproduced this effect only by grossly overloading a DE-52 DEAE-cellulose column. It is unlikely that a second form of the enzyme cxists since mutant JC7623 dapF [8] shows no activity by either of our assay procedures (unpublished result).

As previously reported [6, 201, the en7yme is most stable when stored at 4°C.

Enzjwze inactivation

A,pm epimerase shows time-dependent inhibition with the Azpm analog AZIDAP (for structure see Fig.1). The enzyme inactivation followed pseudo-first-order kinetics (Fig.4). A full kinetic analysis with the enzyme from E. coli and the rate of formation of AZIDAP from halomethyl-A2pm derivatives will be described elsewhere (Gerhart ct al., unpublished work).

When larger amounts of enzyme (100-200 pg) were inactivated in the presence of 50 ~ I M AZIDAP, 100-fold dilutions confirmed that all enzyme activity was lost. Furthermore, denaturation with 6 M urea in the presence of dithiothreitol, a necessary step prior to carboxymethylation, did not remove the label, as judged by mapping of tryptic peptidcs. We therefore concluded that inactivation was irreversible.

Fig.4. Inactivation of E. coli Azpm eppirnerase from plasmid pdF3. Aliquots of a 10 mM stock solution of AZIDAP wcrc diluted to give freshly prepared 100 pM% 10 pM and 5 pM working solutions. Ali- quots of' purified enzyme from E. coli were treatcd with the indicated concentrations (M) and remaining activity determined as describcd in Materials and Methods

Peptides mups und sequence of the active-site peptide

A comparison of peptide maps of the carboxymethylated enzyme (Fig. 5A) and carboxymethylated enzymc which had previously been inactivated by AZIDAP (Fig. 5 B), showed that peptide T10 was displaced to yield a unique peptide T10'. Both amino acid analysis (result not shown) and sequence analysis (see Table 3) confirmed that these two peptides were virtually identical. The only difference was at cycle 12 of the seventeen-residue peptide(s). Sequence analysis of TI 0' (from the AZIDAP-labcllcd enzyme) yielded a blank at this position (the Cys adduct had not yet been synthesised. see Fig. 1). In the case of peptide T10 (from the carboxyinethylated enzyme), the residue was assigned as Pth-Ch-Cys, eluting between Pth- Gln and Pth-Thr (see T d b k 3). To further substantiate the assignment at cycle 12 we prepared ['4C2]Cm-A,pm epimerase under conditions limiting carboxymethylation to Cys (see Materials and Methods and [18]) and identified all ''C-labclled peptides (Fig. 5C). T10 was indeed labelled, and since T10 contains no other residues which are likely ctindi- dates for carboxymethylatioii, this supports the assignment of Cys at cycle 12.

Following AZIDAP labelling, about 35% carboxymethylated T10 remains (see Fig. 5B). This may be explained

141

1; To

'0 n C

1 1 4 0 10 20 30 40 50 60 70

RETENTION VOLUME ( m i )

Fig.5. Peptide maps of Azpm epimerase. Flow rate: 0.7 ml/min. Eludnts: buffcrs A and B as described In Materials and Methods. Gradient: 0-5 ml, 0% buffer B; 5-60 ml. gradient 0-80% buf-fer B; 60-70 ml, gradient 80-0% buffer B. Detection: 214 nm. (A) Peptide map of 200 pg carboxymethylated A,pm epimerase incubated with trypsin (2.5 : 100, by mass) in 0.2 M N-ethylmorpholine acetate buffer, pH 7.8, for 90 min. (B) Peptide map of 200 pg carboxymethylated, AZIDAP-labelled A,pm epimerase treated as indicatcd in A. (C) ''C-Labelled-pcptide profile from [14C]Cm-A2pm epimerase. Peptides not shown for clarity but identical to A above

as follows: only active enzyme is labelled to give AZIDAP- labelled cysteine at the active site. Any inactive enzyme remaining is thus carboxymethylated under the reducing conditions used. It is important to note that more than half of T10 is labelled with AZIDAP. This eliminates any possible explanation of half-site reactivity. We would predict that if 100% active A,pm epimerase were labelled no T10 would remain. This final proof of stoichiometry must await even more active enzyme preparations.

Table 3. Amino m i d sequence y f the active site tryptic peptide of A g r n epimerose Thc sequence runs were performed at least twice using an Applied Hiosystems model 470A sequencer. The phenylthiohydantoin derivatives were analyzed at each cycle directly on-linc, using a model 120 phcnylthiohydantoin analyzer (see Materials and Methods)

Cycle Amino acid Yield of

T10' TI 0

pmol

1 2 3 4 5 6 I 8 9

10 1 1 12 13 14 15 16 17

Ilc Phe Asn Ala Asp GlY SerIdSer Glu Val Ala Gln CYS Gly

GlY

Arg

Asn

Ala

182 I59 138 160 53

107 35/96 61 66 73 63

47 33 33 20

1 -

3 -

563 560 359 352 237 1 24 97

120 117 105 81 952 43 4s 30 32 15

' This residue assigned as AZIDAP-labelled cysteine in peptide T10' as no discernible residue was obtained at this cycle for AZIDAP- labelled tryptic peptide.

Assigned as Pth-Cm-Cys in carboxymethylated pcptide T10, identified as a peak eluting between Gln and Thr. Yield is estimated using the Gln response factor.

Pth-Arg identificd but not quantified.

The protein-sequencing results were substantiated by later data on the DNA sequence of the dapF gene. The total DNA sequence has now been published [9] and a clone containing the active site region gave the sequencc shown in Fig.6, confirming cysteine at position 12 of the peptide (Table 3). Further work enabled this cysteine residue to be positioned in the overall structure as Cys73 of the dapF gene.

DISCUSSION The dupF gene coding for Azpm epimerase [8] has been

expressed from pdF3 and under the appropriate conditions it produces 3 of total soluble protein whereas A,pmepimerase represents only 0.02% of soluble protein in wild-type cells. This results in our ability to isolate 1.6 mg pure enzyme from 4.43 g soluble protein extract. This corresponds to a 72-fold higher yield than obtained previously [6].

The release of 3 H 2 0 lends itself to a very useful and simple assay for the purification of A,pm epimerase. However, the strong icotope effect for the removal of 'H instead of 3H [6] means that specific activity of the enzyme (expressed as dpm/ pg) is a function of the specific activity of [G-3H]A2pm. Fur- thermorc, specific activity cannot be simply related from one batch of [G3H]A2prn to another because k,,, the conversion of meso-A2pm to L , L - A ~ ~ ~ and k,,, of L,L-Azpm to meso- Azpni are different. For this reason, and in order to compare our enzyme with that obtained previously, we used the coupled assay described by Wiseman and Nichols [6]. L,L-A2pm is

142

TAT CGC ATT TTC AAT GCT GAT GGC AGT GAA GTG GCG CAG TGC GGC AAC GGT GCG CGC TGC TTT Tyr Arg Ile Phe Asn Ala Asp Gly Ser Glu V a l Ala Gln Cys Gly Asn Gly Ala Arg Cys Phe

Fig. 6. surnmarji of amino ncid widDNA sequence dutn. DNA sequence data was obtained from dideoxy sequencing methodology [26] on M 13 clones of dapf: fragments and is described in full elsewhere [9]. underlining indicates the amino acid sequence of peptide ’1’10 (TlO‘) as determined in Table 3

Table 4. Compari.von of active-site cystcine regions Sequence of Azpm cpimerase is from this paper. Sequence of hydroxyproline (Hyp) 2-epimerase is from [12]. Sequence of prolinc racemase is from [lo]. The sequence corresponding to the active-site cysteine region of Azpm cpimerase is shown [9, 1 I]. The DNA sequencc of the region shown of hydroxyproline 2-epimerasc and proline racemase are postulated and use the most frequently used codons [22]. The box shows scqucnccs which are identical in all three enzymes. The bases undcrlincd are those which, ifchanged, would give sequcnccs identical to A,pm epimerase. Notc that 5 out of 21 base changes in hydroxyproline 2-epimerase and 3 out of 15 base changes in proline racemasc would result in sequences identical to that of Azpm epimerasc

Enzyme Amino acid sequence DNA sequence

GCG CAG TGC GGC AAC GGT GCG ACC GGT TCG

A,pm epimerase Ala-Gln-Cys-Gly-Asn-Gly-Ala Hyp 2-epimerase Ser-Thr-Cys-Gly-Thr-Gly-Ser TCG S G TGC Proline racemase Ser-Pro-Cys-Gly-Thr T C G CCG TGC GGC ACC

transformed into meso-A2pm and then excess meso-A2pm dehydrogenase, partially purified from Corl;nehacterium glutanzicum [6,21] specifically transforms the rneso-A2pm into L-6-amino-2-oxopimelic acid, producing NADPH, which can be continually monitored at 340 nm.

Gel filtration experiments on Sephacryl S-300 in the prcs- ence of 1 mg/ml bovine serum albumin (Higgins and ‘Tardif, unpublishcd results) yields a single peak of enzyme activity corresponding to molecular mass 45 kDa, versus 32 kDa determined by SDS/PAGE. A similar value was reported by Wiseman and Nichols [6] following gel filtration on Sephadex G-200. The value obtained by gel filtration is proportional to the Stokes radius, and does not in all cases relate to molecular mass. In this case we know that A2pm epimerase contains an unusually high number of acidic amino acids since its PI, measured by chromatofocusing, is around 5.0. Therefore, wc suggest that the ratio of hydrophobic to hydrophilic residues, found in the ‘normal’ globular protein, does not apply in this case. It has previously been shown [ I l l that proline racemase was a diiner of identical subunits, 38.6 kDa. They showed that this dimer bound 1 mol of a competitive inhibitor, pyrrole-2-carboxylate, but that the enzyme was inactivated by iodoacetate with a stoichiometry if 1 mol/subunit. 1-Hydroxy- proline 2-epimerase [12] was shown to be a dimer of identical subunits of 32 kDa, but the enzyme was 80-85% inhibited by the incorporation of 1 mol iodoacetate/dimer. Clearly then the A,pm epimerase specics at 45 kDa might be a dimer and experiments are under way to determine the role of the active- site cysteine in monomer/dimer equilibrium.

In addition to the similarity in subunit molecular mass, these non-pyridoxal-phosphate-containing racemases/epimerases have some sequence similarity around the active- site eysteinc. Ramaswamy [I 21 showed previously sequence similarly between 1-hydroxyproline 2-epimerase and proline racemase, and these are compared with A2pm epimerase in Table 4. Although superficially this similarity is not striking, changes in 5 out of 21 bases in 1-hydroxyproline 2-epimerase and 3 out of 15 bases in proline racemase would give identical DNA sequences as shown in Table 4. It must be emphasized that the basc sequences deduced Tor proline racemase and 1 -hydroxyproline 2-epimerase have not been confirmed by DNA sequencing and therefore the argument is somewhat circular. Nevertheless the codon usage is that most frequently

A

Fig. I . Schematic representation of composite uctive-site of A2pm epipbnerase. (A) ‘Transition-state’ postulated for A2pm epirnerase (based o n [23]). (B) Proposed concerted mechanism with AZIDAP as inhibitor

found in say the ~ r p operon [22]. Based on kinetic evidence, Wiseman and Nichols [6] postulated a incchanistic rclation- ship bctwcen A2pm epimerase [6] and proline racemase [11]. They proposed a two-base mechanism and predicted that one of these bases be a monoproton base; probably the active-site thiol residue. Studies on proline racemase [l 11 suggested that these two cysteines came from two separate subunits making a composite activc sitc. Thus if A2pm epimerase involved similar cysteine residues on separate subunits, then only one

143

labelled peptide would be expected. We feel that our isolation of only one major cysteine-containing peptide labelled by A Z I D A P is further support for this thesis.

More recently, a series of kinetic studies on proline racemase (see [23] and references therein) showed that in certain situations of high substrate levels, exchange of thiol pro- tons may become ratc limiting. They concluded that proline racemase undergoes an enzymic route which involves a transition state or unstable intermediate in which the prolinc carbanion is flanked by the two catalytic thiol groups (see Fig.3 in [23]). Were Azpm epimerase to follow exactly this mechanism, then the postulated ‘transition state’ shown (Fig. 7) would be expected. However, in the case of AZIDAP inhibition, it is more likely that a concerted mechanism in- volving the two SH groups and the aziridine would occur (see Fig. 7). Such a mechanism has been previously proposed for proline racemase inactivation by aziridine 2-cdrboxylate [24]. One consequence of th is mechanism is that, instead of a chirai enzyme distinguishing prochiral substrates, a chiral inhibitor (e. g. the geometric isomer shown in Fig. 7) would selectively inhibit one of two essentially equivalent subunits. The demon- stration of this phenomenon remains to be determined.

If one considers all of the evidence for subunit structure, mechanistic considerations and sequence similarity around the active-site cysteine, it is very tempting to propose a strong relationship between all three enzymes. That is, all non- pyridoxal-phosphate-containing racemases/epimerases may be derived from a common evolutionary origin. Thus our active-site-directed inhibitor (AZIDAP), which was originally designed as an antibacterial agent, may have proved extremely useful in delineating a previously unsuspected homology.

We would like lo express our appreciation to Miss Raymonde Girardot and Dr Bertrand Rihn, Institut de Bacteriologie, Strasbourg for the large scale culturing of pdF3/JM109. We thank Dr Fritz Gerhart for synthesis of AZIDAP.

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expression of recombinant diaminopimelate epimerase in escherichia coli : isolation and inhibition...

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