Appl Microbiol Biotechnol (1988) 28:144--147 Applied Microbiology

Biotechnology © Springer-Verlag 1988

Conformational stability of the penicillin G acylase from Kluyvera citrophila

Gabriel M~rquez, Jos~ M. Buesa, Jos~ L. Garcia, and Jos~ L. Barbero

Molecular Genetics Laboratory, Antibi6ticos S. A., Bravo Murillo 38, E-28015 Madrid, Spain

Summary The mature penicillin G acylase from Kluyvera citrophila was examined by circular di- chroism (CD). The far-UV CD spectrum at neu- tral pH revealed 11% alpha-helix, 44% beta-sheet, 11% beta-turn and 34% random coil. Far-UV and near-UV CD spectra showed that the enzyme pre- sented a high conformational stability under dif- ferent conditions of pH and salt concentration. The predictive model of Chou and Fasman indi- cated the presence of several beta-segments that could be arranged in antiparallel beta-sheets, which might explain the structural stability. The near-UV CD spectrum in the presence of penicil- lin G sulfoxide showed that the binding of this inhibitor to the enzyme resulted in modification of the dichroism of several aromatic residues.

Introduction

Penicillin G acylase (PA) (penicillin amidohydro- lase EC 3.5.1.11) is the enzyme used commercially to hydrolyse penicillin G to 6-amino penicillanic acid, the intermediate for production of semi-syn- thetic penicillins (Mahajan 1984). This enzyme has also been used to catalyse the reverse reac- tion, i.e. the synthesis of beta-lactam antibiotics (Takahasi et al. 1977). Although PAs have been found in different microorganisms, their physio- logical role still remains unknown (Mahajan 1984). In recent years, great interest has been focused on this enzyme, not only for its industrial applica- tions but also because PA is one the few proka- ryotic proteins that undergo a characteristic post-

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translational processing, i.e. the elimination of both a signal peptide and an internal polypeptide, to render the active mature form of the enzyme (BOck et al. 1983a, b).

Several PAs from Gram-positive and Gram- negative bacteria have been cloned (Bruns et al. 1985; Daumy et al. 1986; Meevootisson and Saunders 1987) and the pac gene coding for the PA from Escherichia coli has been sequenced (Schumacher et al. 1986). We have recently cloned (Garcia and Buesa 1986) and sequenced (Barbero et al. 1986) the pac gene from Kluyvera citrophila. As previously reported for the PAs from E. coli (B6ck et al. 1983a, b) and Proteus rettgeri (Daumy et al. 1985a), the enzyme from K. eitrophila con- sists of two dissimilar subunits which are gener- ated from a common precursor (Barbero et al. 1986). The PAs from K. citrophila and E. coli pres- ent a high sequence homology, suggesting that both enzymes have a common ancestral origin (Barbero et al. 1986). In spite of this information, very little is known about the other structural lev- els of the protein. This fact prompted us to initiate some conformational studies on the PA from K. eitrophila as an initial step before engineering the protein in order to improve its catalytic proper- ties.

We report here the analysis of the structure of the PA from K. eitrophila by circular dichroism (CD) compared with the predictions made using the method of Chou and Fasman (1978). The pro- tein showed high stability in different conditions of pH and salt concentration. These results are discussed according to the predicted secondary structure, which suggests a high content of beta- sheet segments. These are the first conformational studies on a penicillin acylase and we think they must contribute to knowledge on this industrially important enzyme.

G. Mfirquez et al.: Conformational studies of penicillin G acylases 145

Materials and methods

Isolation and purification of PA from K. citrophila. PA from K. citrophila was isolated from an E. eoli strain harbouring the recombinant plasmid pYKH5, containing the pac gene from K. citrophila (Garcia and Buesa 1986), and the enzyme was puri- fied as described elsewhere (Barbero et al. 1986).

Circular dichro&m spectra. CD spectra were recorded in a Mark III dichrograph (Jobin-Yvon). For spectra in the near- UV region (320--250 nm), cells of 1 cm optical path were used and the instrument was operated at a sensitivity of 2 - 10 -6 AA ram. Cells of 0.05 cm optical path were used and the sensitivity was set at 5 - 10 -6 AA mm to obtain spectra in the far-UV re- gion (250--200 nm). Results are expressed in molar elliptiei- ties with the dimensions of deg • cm 2 • (drool of protein)- 1 in the near-UV region or deg • cm 2 • (dmol of residue) -~ in the far-UV region. Results are the mean of at least three determi- nations. Calculations of the contributions of alpha-helix, beta- sheet, beta-turn and random coil to the secondary structure were carried out according to the method of Chang et al. (1978).

Results and discussion

Circular dichroism in the far- UV region

We have observed in the far-UV region that the secondary structure of PA from K. citrophila un- dergoes slight modifications as a consequence of varying the pH value from 2.7 to 9.4 (Fig. 1A, B). Within this pH range only a small increase of beta-sheet could be detected. At neutral pH the

Wavetength (nm) p H - -

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0.2 -0.~ 0.6 [Kd M

10 12

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C 80 --2 cn

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Fig. 1 A- -C . Circular dichroism spectra (far-UV) of the ma- ture penicillin G acylase (PA) from Kluyvera citrophila. A Spectra in 50 mM sodium phosphate at pH 2.9 (.__), pH 7.0 (--) and pH 12.0 (...). B Effect o f p H on the secondary struc- ture. C Effect of ionic strenght on the secondary structure. Contributions of alpha-helix (O) , beta-sheet (A) , beta-turn (A) and random coil ( l I) were determined according to Chang et al. (1978).

W a v e l e n g t h (nm)

260 280 300 250 280 300 r ~ r 50 5 0 ~ ~ J A B / \ \

\ \ \

1 5 '\ ~' 3O

'~ ~B

0 3

Fig. 2 A, B. Circular dichroism spectra (near-UV) of the ma- ture PA from K. citrophila. A Spectra in 50 mM sodium phos- phate at pH 7.0 ( - - ) and pH 12.0 (...__). B Spectrum at pH 7.0 in presence of 0.024 mg/ml penicillin G sulfoxide.

secondary structure of the protein was composed of 11% alpha-helix, 44% beta-sheet, 11% beta-turn and 34% random coil (Fig. 1B). On the other hand, at pH values above 9.4 the secondary struc- ture of the protein was clearly changed; the beta- turn and alpha-helix contributions increased while the beta-sheet diminished. It is worth noting that at pH 12, at which the highest structural modifications were observed, the enzymatic activ- ity was irreversible lost (data not shown). The ti- tration of basic amino acid residues, such as ly- sines and arginines, could account for the in- crease of the alpha-helix observed (Barbero et al. 1980). On the other hand, at high pH values the appearance of negative charged residues might be the cause of the change of a great portion of beta- sheet into beta-turn (Fig. 1B).

Virtually negligible changes were observed in the PA secondary structure when the ionic strength was increased up to 1M KF at pH 7.0 (Fig. 1C). The small variations detected might be due to the quenching of some charged residues and they had practically no effect in the overall secondary structure of the protein.

Circular dichroism in the near- UV region

Figure 2A shows the CD spectra in the near-UV region of PA from K. citrophila at two different pH values, i.e. 7.0 and 12.0. Both spectra show po- sitive dichroism in a range of wavelengths where the residues responsible for the absorption de- tected are mainly tyrosines and tryptophans. Be- tween pH 3.0 and pH 8.0, the spectra recorded were similar to that obtained at pH 7.0. These re- suits are in agreement with those registered in the

146 G. M~rquez et al.: Conformational studies of penicillin G acylases

far-UV region, suggesting that not only the sec- ondary but also the tertiary structure is modified to a minor extent throughout this pH range. Above pH 9.4 the spectra recorded showed an ef- fect corresponding to ionization of the phenolic group of the tyrosine residues. The mature PA from K. citrophila has 33 tyrosines and 25 trypto- phans as determined by its nucleotide sequence (Barbero et al. 1986), so the CD observations do not allow us to assign which residues contribute to the near-UV region spectrum. Nevertheless, the data obtained suggest that both the lLa and the lLb transitions of the the tyrosinate chromophore are optically active, as denoted by the increase of the ellipticity at 290 nm and 250 nm.

More interesting seems to be the spectrum of the enzyme registered at pH 7.0 in 50 mM sodium phosphate, in the presence of 0.024 mg/ml peni- cillin G sulfoxide, a competitive inhibitor of the enzyme (Fig. 2B). The binding of the inhibitor to the protein resulted in modification of the dich- roism of some aromatic residues. In consequence, the spectrum was altered both qualitatively and quantitatively. So, while the spectrum at pH 7.0 showed two local maxima located at approxi- mately 267 nm and 290 nm, and only a local min- imum at approximately 282 nm, the spectrum in the presence of the inhibitor showed only one maximum at approximately 272 nm. Besides, the maximum ellipticity reached in the latter case was about three times higher than that obtained at pH

7.0. Daumy et al. (1985b) have reported that the PA subunits from P. rettgeri play different roles; thus, the large subunit would contain the catalytic site, while the small one would confer the sub- strate side chain specificity. Furthermore, by com- paring the amino acid sequences of PA from E. coli with those of other penicillin-binding pro- teins, Oliver et al. (1985) suggested that the region between methionine-168 and lysine-191 could be involved in the binding of the penicillin G mole- cule. The amino acid sequence of this region is also highly conserved in the PA from K. citrophila (Barbero et al. 1986). However, this sequence only contains an aromatic residue, namely one pheny- lalanine, thus making it difficult to explain the high ellipticities registered in the presence of pen- icillin G sulfoxide by the interaction of this aro- matic residue with the inhibitor. A more likely ex- planation could be that the interaction with the inhibitor results in a conformational change af- fecting several tyrosine and tryptophan residues of both subunits.

Secondary structure predictions

Using the predictive model of Chou and Fasman (1978) we have determined the probable second- ary structure of the PA from K. citrophila (Fig. 3A). According to this prediction the alpha-helix, beta-sheet, beta-turn and random coil content of

I ~ 1 ~ t ~ 1 ~ - - ~ - r ~ t - - J - ~ t - ~ m ~ 1 ~ - ~ - ~ l ~ - ~ 1 ~ - - - L - u - ~ i 1 ~ _ ~ H ~ i ~ - - ~ 1 1 ~ - - a "-¢II

itlflfll ~ l l l ~ t t l t l I I I t t l l l l 1111 I l t l

~ ` - J ~ u ~ D H ~ ~ ~ - ~ ~ ~ m ~ N ~ ` ~ - ~ A V ~ y ~ i ~ t ~

~tt-1mt----'--ra-jmt-mmmut-muI~mtt-luu--a--~aaa~-~--r-uttmt~mu-j~tttN~v~rN~`t-r-tmta--r-numt-t---r~N~

HPi I 1 3

-I

-3

50 100 200 300 400 500 600 700 800

Fig. 3. Predicted secondary structure (A) and hydropathy profile (B) of the PA precursor protein from K, citrophila. / Residues in beta-sheet; 0 residues in alpha-helix; ~ four residues involved in a beta-turn. Numbers indicate amino acid sequence positions, while arrows show the cleavage sites which generate the mature PA. Probable secondary structure was predicted according to the method of Chou and Fasman (1978) and the hydropathy profile was determined according to Kyte and Doolittle (1982).

G. Mfirquez et al.: Conformational studies of penicillin G acylases 147

the mature PA is 20%, 34%, 20% and 26%, respec- tively. These results are in reasonable agreement with the values obtained by CD at pH 7.0. Al- though, the alpha-helix content predicted is higher than the CD estimate, Chou and Fasman (1974) pointed out that the percentages of helicity estimated by CD are usually lower than the X-ray results, probably due to the fact that helical seg- ments of globular proteins are short, whereas long polypeptides were mostly used for CD reference spectra. In addition, some regions in which alpha- helix conformation has been predicted (i.e. re- gions located within the residues 437--448, 455-- 459 and 696--701) also present a high probability for beta-sheet structure. The high content of beta- sheet, reaching 44% in the small subunit, as well as the high hydrophylicity of the PA (Fig. 3B), might be the reason for the high stability of the protein in water solutions. It should be noted that there are five regions, two in the small subunit and three in the large one, composed of numerous beta-segments which could be arranged in a re- peating antiparallel configuration by intervening beta-turn. This beta-sheet configuration could contribute to the stability of the PA. Interestingly enough, according to the predicted secondary structure, the postulated penicillin-binding site of PA (Oliver et al. 1985) should be located within one of the antiparallel beta-sheet domains of the small subunit.

The results presented here show that the PA from K. citrophila has a secondary structure with a great contribution of beta-sheet, possibly ex- plaining the high conformational stability of the enzyme in different conditions of pH and ionic strength. Given the similarities of PAs from K. ci- trophila, E. co6 and P. rettgeri, it is probable that our results reflect the conformation of a PA mole- cule, regardless of its source. Using the same con- ditions as those employed with the K. citrophila enzyme, we have recorded several CD spectra with the PA from E. coli and have obtained simi- lar structural results (data not shown). The most striking changes in ellipticity were observed in the presence of penicillin G sulfoxide, a molecule which is supposed to bind to the same structural environment as the substrate. More detailed struc- tural studies on the amino acid residues involved in the interaction with this PA activity inhibitor could shed light on how this enzyme carries out its catalytic activity.

Acknowledgements. We thank Dr. A. P6rez-Aranda for his en- couragement throughout the course of this work and Dr. J. Gavilanes for providing facilities for CD determinations.

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Received July 31, 1987/Accepted October 5, 1987