1 Department of Biochemistry and 3 Laboratory of Biophysical Chemistry, BIOSON Research Institute, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen and 4 DSM Gist, PO Box 1, 2600 MA Delft, The Netherlands
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Abstract |
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Keywords: ß-lactam binding site/penicillin acylase/site-directed mutagenesis/substrate-induced conformational change/X-ray structure
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Introduction |
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Binding of the ß-lactam nucleus is important for the application of PA in the production of semi-synthetic antibiotics. In order to obtain structural information on the nucleophile binding site, we constructed the inactive ßN241A mutant and soaked crystals of this mutant with penicillin G. The structural data indicated an important role for the residues R145 and
F146 in binding of the ß-lactam moiety of penicillin G. The function of
F146 was further investigated by site-directed mutagenesis and kinetic characterization of mutants. The results showed that substituting the
F146 residue can lead to mutants which are improved with respect to their use in the synthesis of ß-lactam antibiotics.
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Materials and methods |
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For site-directed mutagenesis and expression of the recombinant enzymes, the plasmid pEC was used, which was provided by DSM Gist (Delft, The Netherlands). This plasmid carries the penicillin acylase gene of E.coli ATCC 11105, but has a ßV148L mutation compared with the GenBank sequence 42247. E.coli HB101 was used as a host.
PCR mutagenesis
Mutants were made by PCR. For introducing the F146 mutations, a fragment of 305 bp was amplified from plasmid pEC using the oligonucleotide 5'-GAAGTGCTTGGCAAA-3' as the forward primer. This primer anneals 21 bp upstream of an EcoRV site in the PA gene. As the reverse primer 5'-GCCAGATTATCGATTTCGCTAGTACTATCAGAXXXGCGGTTT-3' was used. In this primer a ClaI site is shown in italics and the codon for amino acid
F146 is underlined. The codons GTA, GGC and GAG were used to change
F146 to Tyr, Ala and Leu, respectively. After amplification using Pwo polymerase (Boehringer Mannheim), the fragment was digested with ClaI and EcoRV and ligated into pEC that was digested with the same enzymes.
The reverse primer used to construct the ßN241A mutant was 5'-AAAGGCAAACAGATCTGAAGCGGGATAATCTTTTTGGGGAGAATTCGCCCAGTTAGC-3' in which a BglII site is shown in italics. Introduction of the underlined codon for the ßN241A mutation simultaneously creates an EcoRI restriction site. As a forward primer, 5'-CTGACCAGACGACACAAACGGCTTA-3' was used which binds 30 bp upstream of an NheI site in the PA gene. The 382 bp fragment was amplified using PCR and after digestion with NheI and BglII ligated into pEC that was digested with the same enzymes.
Competent E.coli cells were transformed with ligation mixtures using standard protocols (Sambrook et al., 1989). Transformants containing the desired fragment were sequenced in order to confirm the mutation and the absence of second site mutations.
Purification
Wild-type and mutant PAs were obtained as follows. Recombinant E.coli was grown at 17°C in LB medium (Sambrook, et al., 1989) at 150 r.p.m. and induced with 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). Cells were harvested in the late exponential phase by centrifugation at 5000 g for 10 min. The pellet was resuspended in 1/10 volume of ice-cold osmotic shock buffer A (20% sucrose, 100 mM TrisHCl, pH 8.0, 10 mM EDTA) and centrifuged for 10 min at 5000 g. The pellet was then resuspended in 1/10 volume of ice-cold osmotic shock solution B (1 mM EDTA) and centrifuged for 10 min at 5000 g. Potassium phosphate buffer (1 M, pH 7.0) was added to the supernatant (periplasmic extract) to a final concentration of 50 mM, then (NH4)2SO4 was added to a final concentration of 1.5 M. The sample was loaded on a Resource-Phe column (Amersham Pharmacia Biotech) and eluted with a linear gradient of 1.50 M (NH4)2SO4 in 50 mM phosphate buffer, pH 7.0. The PA of E.coli eluted at a concentration of 1 M (NH4)2SO4.
This preparation was used for kinetic experiments but was further purified for crystallization experiments. Enzyme eluted from the Resource Phenyl column was pooled and desalted by dialysis against 2x1 l of 50 mM TrisHCl, pH 8.5. The desalted protein was then applied to a HiTrap-Q column (Amersham Pharmacia Biotech) mounted on an Äkta explorer (Amersham Pharmacia Biotech). A gradient from 0 to 250 mM NaCl in 50 mM TrisHCl, pH 8.5, was used to elute the enzyme. PA eluted from the column at 37 mM NaCl.
Crystallization and data collection
Purified PA was crystallized as described earlier, with small modifications (McVey et al., 1997). After purification, the buffer was exchanged for 100 mM MOPS, pH 7.5, and the sample was concentrated using a Microsep 30 filter (Filtron) to a final concentration of 10 mg/ml. Crystals were obtained with the hanging drop vapour diffusion technique at 4°C. Enzyme solution (2.5 µl, 8 mg/ml) was mixed with 2.5 µl of precipitant containing 1215% PEG MME 2000 and 50 mM MOPS, pH 7.2, and crystals with a bad morphology (two-dimensional intergrown plates) typically appeared within 12 weeks. These crystals were used for streak seeding as described by McVey et al. (1997). Crystals from streak seeding showed better morphology and were used for data collection.
Crystals with bound penicillin G and crystals with bound phenylacetic acid were obtained by soaking crystals of ßN241A PA with 10 and 1 mM penicillin G in mother liquor [20% (w/v) PEG MME 2000, 4.4% (v/v) glycerol and 50 mM MOPS, pH 7.2] for 22 min and 5.5 h, respectively.
Data were collected from single flash-frozen crystals (120 K). For this, the crystals were transferred from the drop to a mother liquor containing 20% (w/v) PEG MME 2000, 4.4% (v/v) glycerol and 50 mM MOPS, pH 7.2. Prior to freezing, the crystals were dipped in a cryo solution, containing 35% (w/v) PEG MME 2000, 6.5% (v/v) glycerol and 50 mM MOPS, pH 7.2. The crystals showed a large variation in size but only small crystals with typical dimensions of 0.2x0.1x0.04 mm could successfully be frozen. Larger crystals tended to give double spots and smeared diffraction images.
Diffraction data were collected using Cu K radiation from a rotating anode source and a MacScience DIP2030 area detector. The 5.5 h penicillin G soak of ßN241A was measured at beamline X31 of the EMBL outstation at DESY, Hamburg. Data were processed using Denzo and Scalepack (Otwinowsky, 1993
).
Molecular replacement and model refinement
The unit cell of PA crystals in this study deviates slightly from previously published unit cells (Hunt et al., 1990; Duggleby et al., 1995
) (Table I
). Therefore, molecular replacement was used to obtain initial phases. The wild-type structure (1PNK.PDB) was used as a search model in the program AMORE (CCP4, 1994
). Electron density of phenylacetic acid and penicillin G was already clearly visible after rigid body refinement. The obtained solution was used for further refinement in X-PLOR (Brünger, 1992
), including Powell energy minimization and overall and individual B-factor refinement. After four rounds of refinement the ligands were modelled with O in the improved electron density of phenylacetic acid and penicillin G, respectively (Jones et al., 1991
). Final refinement cycles were done in CNS (Brünger et al., 1998
) using the maximum likelihood function of this program. Data collection and refinement statistics are presented in Table I
. Penicillin G was present with both the thiazolidine and the ß-lactam ring intact as has been observed in the electron density of isopenicillin N (Burzlaff et al., 1999
).
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Determination of kinetic parameters
Steady-state kinetic parameters were determined by measuring initial velocities at different substrate concentrations and fitting the data using the program Enzfit (Elsevier Biosoft, 1987). The hydrolysis of 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB) and D-2-nitro-5-[(phenylglycyl)amino]benzoic acid (NIPGB) was followed by measuring the increase in absorbance at 405 nm in a Perkin-Elmer spectrometer. Rates were calculated using a of 9.09 mM1·cm1 for the conversion of NIPAB and NIPGB. The inhibition constants of the ß-lactam nuclei were determined by measuring the Km for the hydrolysis of NIPGB in the presence of various concentrations of inhibitor. 6-APA was found to be a competitive inhibitor and the inhibition constant was calculated using
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where Km,app is the Km in the presence of inhibitor, [I] the inhibitor concentration and Ki the inhibition constant. Conversion of phenylacetamide and penicillin G was followed by reversed-phase HPLC using a Chrompack C18 column with Jasco PU-980 pumps and a Jasco MD-910 detector set at 214 nm. All compounds were eluted isocratically using a solution containing 340 mg/l SDS, 5 mM phosphate, 30% acetonitrile, which was adjusted to pH 3.0 with dilute phosphoric acid. All enzymatic conversions were carried out in a 50 mM phosphate buffer at pH 7.0 at 30°C.
Chemicals
NIPAB was purchased from Sigma Chemical and NIPGB from Syncom (Groningen, The Netherlands). Penicillin G was a gift of DSM Gist (Delft, The Netherlands). PEG MME 2000 was purchased from Hampton Research.
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Results |
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Binding constants of wild-type penicillin acylase for ß-lactam nuclei are of the order of 10100 mM (Kutzbach and Rauenbusch, 1974; Kasche et al., 1984
; Ospina et al., 1992
). Since the solubility of these compounds is approximately the same, co-crystallization or soaking crystals with ß-lactam nuclei was expected to be difficult. Since co-crystallization or soaking with the natural substrate penicillin G results in hydrolysis, due to catalytic activity in the crystals (Duggleby et al., 1995
), we studied the interaction of the enzyme with the ß-lactam moiety by soaking crystals of an inactive mutant with penicillin G. An inactive mutant was made by replacing asparagine ß241 by an alanine, thereby removing the side chain amide that stabilizes the oxyanion that is formed in the transition state. The ßN241A mutant was about 10 000-fold less active than the wild type using NIPAB as the substrate. When periplasmic extracts of clones overexpressing this mutant protein were analysed on SDSPAGE gels, a prominent band with a molecular weight of ~90 kDa was seen, indicating the presence of large amounts of precursor protein. This suggests that export of the precursor protein to the periplasmic space takes place, but that processing is severely impaired in this mutant, although not completely blocked. After incubation for 48 h at room temperature, the precursor protein precipitated, whereas the processed mutant protein that was present remained soluble and could be purified as described. After crystallization, the ßN241A mutant was soaked with penicillin G. Soaking the crystals for 22 min with penicillin G revealed the intact substrate in the active site, although not fully occupied as indicated by higher B-factors of the penicillin G (Figure 3A
). Soaking for 5.5 h resulted in crystals with an intermediate structure, where the active site was occupied with phenylacetic acid, showing that slow hydrolysis of the amide bond still can take place without the stabilizing side chain of the asparagine in the oxyanion hole.
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Upon binding of the penicillin G to the enzyme, the position of the phenyl ring of the phenylacetic acid moiety changes as compared with the enzymephenylacetic acid complex (Figure 3B). Because of this shift, the carbonyl carbon of the bond that is to be hydrolysed in penicillin G is at a distance of 3.3 Å from the OG of the nucleophilic serine, whereas in the structure with phenylacetic acid this carbon atom is located at 2.9 Å from the OG.
A significant difference between the structures of the enzymepenicillin G and the enzymephenylacetic acid complex is the position of the side chains of R145 and
F146 (Figure 3B
). The side chain of
R145 is protruding into the solvent and is at a distance of 8 Å from the carboxyl group of the ligand. Two water molecules are positioned in such a way that hydrogen bonding between the NH1 atom of
R145 and one of the oxygen atoms of the carboxylate group can take place. The side chain of
F146 has moved away from the binding pocket towards ßY31, thereby opening the active site in order to bind penicillin G. The CD1 and CG atoms of the side chain of
F146 in this open conformation are located at 3.1 and 3.5 Å from the 2ß-methyl group of the thiazolidine ring of penicillin G. If penicillin G were bound to the enzyme without this conformational change taking place, the CD1 atom would be at a distance of 1.8 Å from this methyl group, creating steric hindrance. This suggests that the
F146 has to shift position in order to accommodate the large ß-lactam nucleus in the active site. The short distance between the CD1 and the 2ß-methyl group of the thiazolidine ring of penicillin G indicates that in this conformation
F146 assists in binding of the substrate by van der Waals interactions with the ß-lactam moiety of the substrate.
The side chain of ßF71 moves slightly upon binding of penicillin G towards a staggered stacked conformation with the thiazolidine moiety of the substrate. In this conformation the CZ atom of ßF71 is located at 3.2 Å from the sulfur atom and the C5 atom of the thiazolidine moiety. The backbone carbonyl oxygen of the ßQ23 residue is hydrogen bonded to the carbonyl oxygen of the phenylacetic acid moiety via an H2O molecule in the active site. This H2O molecule is only observed in the structure with penicillin G and might be the nucleophilic water that deacylates the acyl enzyme. Van der Waals interactions of the main chain oxygen of ßQ23 with the carbonyl oxygen of the ß-lactam ring, which is at 3.4 Å, may also take place (Figure 3C).
The results indicate that both van der Waals interactions and hydrogen bonding play a role in binding of the ß-lactam moiety of penicillin G. The fact that the distances between the enzyme and penicillin G are shorter than expected for van der Waals interactions may be caused by the low occupancy of penicillin G in the active site.
Activity of the site-directed mutants
The structure of the penicillin Genzyme complex showed a positional shift of F146. This phenylalanine closes the active site, shielding the hydrophobic binding site from the solvent (Figure 3B
) and upon binding of the substrate the phenyl ring assists in binding of the ß-lactam moiety (Figure 3C
). The phenylalanine on this position is conserved in penicillin acylases of E.coli, Alcaligenes faecalis and Kluyvera citrophila (Verhaert et al., 1997
). In order to study the role of this residue in more detail, it was mutated to an alanine, leucine or tyrosine. The effect of the mutations was studied by measuring the steady-state kinetic parameters for the chromogenic substrate NIPAB as well as for penicillin G and phenylacetamide (Table II
).
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Interaction of F146 with the ß-lactam nucleus
The activity measurements with the mutant enzyme suggested that the interaction of the mutant enzyme with substrates was dependent on the type of leaving group. To study the interaction of ß-lactam substrates with the acyl enzyme of these mutants, we measured the kinetics of the synthesis of penicillin G, using 6-APA as the nucleophile and phenylacetamide as the acyl donor. The acylated enzyme can be deacylated by H2O or 6-APA, yielding either phenylacetic acid or penicillin G.
All mutant enzymes differed markedly from the wild-type enzyme in their ability to synthesize penicillin G (Figure 4). It appeared that 6-APA was no longer able to deacylate efficiently the covalent intermediate of
F146Y and small amounts of penicillin G were synthesized. The rapid production of phenylacetic acid is in agreement with the observed steady-state parameters for the hydrolysis of phenylacetamide and indicate that H2O is still able to deacylate the
F146Y enzyme efficiently. Since the wild-type and the
F146Y mutant enzyme have a similar affinity for 6-APA (Table II
), this difference in synthesizing capacities seems to be caused by a difference in reactivity of bound 6-APA rather than by differences in the degree of saturation of the acyl enzyme with 6-APA. Using the
F146L and
F146A mutants, much more penicillin G than phenylacetic acid was produced, indicating that 6-APA very efficiently deacylates the
F146L and
F146A mutant enzymes, even though binding of 6-APA is weaker. The results indicate that the synthesizing capacity of penicillin acylase, i.e. the ratio of aminolysis by a ß-lactam nucleophile vs hydrolysis by H2O, can be improved by mutations that influence the interaction of the enzyme with the substrate in the ß-lactam binding site.
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Discussion |
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Three amino acid residues assist in binding of the ß-lactam moiety of the substrate. The side chain of R145 is 8 Å away from the carboxylate group of the ß-lactam nucleus but has an interaction with an oxygen of this carboxylate group via two bridging water molecules. This is in line with observations made by Svedas et al. (1996), who concluded that a positively charged residue in or near the active site might play a role in binding nucleophiles by interacting with the negatively charged carboxylate group. For this interaction to take place, the enzyme has to be in the open conformation, since in the closed form the side chain of this
R145 is pointing towards the active site and forms a hydrogen bond with the carbonyl oxygen of ßF24.
Upon binding of penicillin G, the phenylalanine on position ß71 moves slightly towards a stacked conformation with the thiazolidine ring of penicillin G and is probably involved, by means of hydrophobic interactions, in binding of the hydrophobic ß-lactam nucleus. Roa et al. (1994) identified an F360V mutant of penicillin acylase of K.citrophila, which was obtained after random mutagenesis and showed that it had altered substrate specificity. The mutant was able to hydrolyse glutaryl-L-leucine and NIPAB, but could not cleave the corresponding ß-lactam substrates with the same acyl group, glutaryl-7-ACA and penicillin G. Since F360 is equivalent to ßF71 in the E.coli enzyme, this indicates that also in PA of K.citrophila this phenylalanine is involved in binding of the ß-lactam moiety.
The most important residue for interaction with the leaving group of the substrate is the phenylalanine on position 146. Kinetic data indicate that removing the aromatic function on position
146 decreases the affinity for the phenylacetylated substrates. Svedas et al. (1996) have already proposed that hydrophobic interactions are the most important factor governing substrate binding. Moreover, it seems that an aromatic function is needed for correct positioning of the substrate relative to the catalytic serine in the active site, since also the kcat for penicillin G in the
F146L and
F146A mutants has decreased significantly.
Mutating F146 to a tyrosine could influence the binding of ß-lactam substrates in the following way: when the phenylalanine moves to the open conformation, the CZ position is located 3.2 Å away from the hydroxyl group of ßY31, whereas in the closed conformation the distance is 4.6 Å. A tyrosine on position
F146 would place the hydroxyl groups of both tyrosine residues at 4.0 Å from each other in the closed structure, but this distance would decrease to 2.1 Å in the open conformation of the enzyme. Therefore, steric hindrance of the phenyl ring of residue
146 of the
F146Y mutant may occur when the enzyme switches to the open conformation, which may explain the reduced kcat value of the
F146Y mutant for hydrolysis of the ß-lactam substrate penicillin G. In contrast to the
F146Y mutant, a conformational change in the
F146L mutant would place the side chain of the leucine at a distance of 4.6 Å from ßY31. This may explain the fact that the
F146L mutant is still able to hydrolyse and synthesize penicillin G efficiently.
The observation that soaking the ßN241A mutant with penicillin G yielded a structure with intact substrate in the active site confirms the crucial role of the asparagine residue in catalysis by means of stabilizing the oxyanion (Duggleby et al., 1995). However, a low but significant activity for NIPAB was observed and also prolonged incubation of the crystals with penicillin G resulted in a structure with phenylacetic acid in the active site. Probably also the backbone amide of ßA69 plays a role in stabilizing the negative charge of the oxyanion, as suggested by Duggleby et al. (1995), allowing catalysis to take place, albeit at a much lower rate.
The ßN241A mutant is also impaired in processing, as indicated by the large amount of precursor protein formed. This indicates that an intact oxyanion hole is necessary for maturation of the enzyme, suggesting that this is an autocatalytic process as proposed for the family of Ntn-hydrolases (Brannigan et al., 1995). The structure of the precursor protein might give more insight into the precise details of the processing events (Hewitt et al., 1999
).
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Notes |
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5 To whom correspondence should be addressed. E-mail: d.b.janssen{at}chem.rug.nl.
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Acknowledgments |
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References |
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Brannigan,J.A., Dodson,G., Duggleby,H.J., Moody,P.C., Smith,J.L., Tomchick,D.R. and Murzin,A.G. (1995) Nature, 378, 416419.[ISI][Medline]
Bruggink, A., Roos,E.R. and De Vroom,E. (1998) Org. Process Res. Dev., 2, 128133.
Brünger,A.T. (1992) XPLOR Version 3.0: a System for X-ray Crystallography NMR. Yale University Press, New Haven, CT.
Brünger,A.T. et al. (1998) Acta Crystallogr., D54, 905921.[ISI]
Burzlaff,N.I., Rutledge,P.J., Clifton,I.J., Hensgens,C.M.H., Pickford,M., Adlington,R.M., Roach,P.L. and Baldwin,J.E. (1999) Nature 401, 721724.[ISI][Medline]
CCP4 Collaborative Computational Project, No. 4 (1994) Acta Crystallogr., D50, 760763.[ISI]
Cole,M. (1969) Biochem. J., 115, 733739.[ISI][Medline]
Done,S.H., Brannigan,J.A., Moody,P.C. and Hubbard,R.E. (1998) J. Mol. Biol., 284, 463475.[ISI][Medline]
Duggleby,H.J., Tolley,S.P., Hill,C.P., Dodson,E.J., Dodson,G. and Moody,P.C. (1995) Nature, 373, 264268.[ISI][Medline]
Esnouf,R.M. (1997) J. Mol. Graphics, 15, 133138.
Hewitt.L, Kasche,V., Lummer,K., Rieks,A. and Wilson,K.S. (1999) Acta Crystallogr., D55, 10521054.
Huang,H.T., Seto,T.A. and Shull,G.M. (1963) Appl. Microbiol., 11, 16.[ISI][Medline]
Hunt,P.D., Tolley,S.P., Ward,R.J., Hill,C.P. and Dodson,G.G. (1990) Protein Eng., 3, 635639.[Abstract]
Jones,T.A., Zou,J.Y., Cowan,S.W. and Kjelgaard,M. (1991) Acta Crystallogr., A47, 110119.[ISI]
Kasche,V., Haufler,U. and Zollner,R. (1984) Hoppe Seylers Z. Physiol. Chem., 365, 14351443.[ISI][Medline]
Kutzbach,C. and Rauenbusch,E. (1974) Hoppe Seylers Z. Physiol. Chem., 355, 4553.[ISI][Medline]
McVey,C.E., Tolley,S.P., Done,S.H., Brannigan,J.A. and Wilson,K.S. (1997) Acta Crystallogr., D53, 777779.
Morillas,M., Goble,M.L and Virden,R. (1999) Biochem. J., 338, 235239.[ISI][Medline]
Ospina,S.S., Lopez-Munguia,A., Gonzalez,R.L. and Quintero,R. (1992) J. Chem. Technol. Biotechnol., 53, 205214.[ISI][Medline]
Otwinowsky,Z. (1993) In Sawyer,L., Isaacs,N. and Bailey,S. (eds), Proceedings of the CCP4 Study Weekend: Data Collection and Processing. SERC Daresbury Laboratory, Warrington, UK, pp. 5662.
Roa,A., Garcia,J.L., Salto,F. and Cortes,E. (1994) Biochem. J., 303, 869875.[ISI][Medline]
Roa,A., Goble,M.L., Garcia,J.L., Acebal,C. and Virden,R. (1996) Biochem. J., 316, 409412.[ISI][Medline]
Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning. A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Stambolieva,N., Mincheva,Z. and Galunsky,B. (1998) Biocatal. Biotransform., 16, 225232.[ISI]
Svedas,V.K., Savchenko,M.V., Beltser,A.I. and Guranda,D.F. (1996) Ann. N. Y. Acad. Sci., 799, 659669.[Medline]
Verhaert,R.M., Riemens,A.M., Van der Laan,J.M., Van Duin,J. and Quax,W.J. (1997) Appl. Environ. Microbiol., 63, 34123418.[Abstract]
Received June 28, 2000; revised October 10, 2000; accepted October 16, 2000.