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1 Department of Biophysics, Max-Planck Institute for Medical Research, Jahnstr. 29, D-69120 Heidelberg, Germany
2 Aventis Pharma Deutschland GMBH, D-65926 Frankfurt am Main, Germany
Reprint requests to: Karin Fritz-Wolf, Department of Biophysics, Max-Planck Institute for Medical Research, Jahnstr. 29, D-69120 Heidelberg, Germany; e-mail: fritz{at}mpimf-heidelberg.mpg.de; fax: 49-6221-486-437.
(RECEIVED July 11, 2001; FINAL REVISION October 8, 2001; ACCEPTED October 10, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.27502.
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Abstract |
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 TOP Abstract IntroductionResults and DiscussionMaterials and methodsReferences |
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-amino group of the catalytic serine acts as a base. A subtle difference is the presence of two catalytic dyads, His B23/Glu B455 and His B23/Ser B1, that are not seen in penicillin G acylase. In contrast to classical serine proteases, the central histidine of these dyads interacts indirectly with the O
through a hydrogen bond relay network involving the -amino group of the serine and a bound water molecule. A plausible model of the enzyme–substrate complex is proposed that leads to the prediction of mutants of glutarylamidase that should enable the enzyme to deacylate cephalosporin C into 7-aminocephalosporanic acid. Keywords: Cephalosporin acylase; glutaryl acylase; cephalosporin C; catalytic triad; Ntn-hydrolase; X-ray structure
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Introduction |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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The recently determined structure of GA (Kim et al. 2000) shows extensive similarity with the structure of penicillin G acylase (PGA) (Duggleby et al. 1995). PGA is a founding member of the Ntn (N-terminal nucleophile)-hydrolase superfamily (Brannigan et al. 1995). Like GA, members of this superfamily consist of two different amino acid chains generated by post-translational processing from inactive precursors (Brannigan et al. 1995). Ntn-hydrolases share a common fold in which the N-terminal nucleophile and other catalytic groups occupy equivalent sites. Serine proteases differ in details of proton abstraction from the catalytic Ser-hydroxyl group, involving either the classical catalytic triad, consisting of Ser, His, and Asp, or hydroxyl/
-amine or hydroxyl/-amine catalytic dyads (Paetzel and Dalbey 1997). It has been suggested that the catalytic triad should be regarded as two dyads, Ser/His and His/Asp dyad (Liao et al. 1992). PGA employs the -amino group of the catalytic serine for proton abstraction from the Ser-hydroxyl (Duggleby et al. 1995).
The two serine proteases PGA and GA share a common fold, but they convert different substrates (Scheme 1
). PGA catalyzes the hydrolysis of the antibiotic penicillin G into phenylacetic acid and 6-aminopenicillanic acid (6-APA), whereas GA deacylates Gl-7-ACA to 7-ACA. Another GA from the Pseudomonas sp. strain N176 (N176) converts in addition cephalosporin C (CPC), although at low efficiency (Ishii et al. 1995). CPC is the natural form of the antibiotic cephalosporin, which is produced by the fungus Cephalosporin acremonium. The substrates shown in Scheme 1 are all cleaved at the amide bond linking the ß-lactam core with the side chain. In penicillin G, the side chain consists of a hydrophobic phenyl moiety, while the side chains of Gl-7-ACA and CPC include charged groups (Scheme 1). Substrate specificity of the enzymes is associated with recognition of the side chain and not of the ß-lactam core, as shown for the case of PGA (Huang et al. 1963).
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We describe here a structure-based approach to achieve this goal that is guided by the structure of GA and comparison with the related enzymes PGA and N176. We have solved the structure of GA with a phosphate bound near the catalytic serine and with glycerol in the side-chain binding pocket, and derive the putative structure of Gl-7-ACA in complex with GA. This allowed us to pinpoint the required modifications that should enable the new enzyme to convert CPC to 7-ACA.
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Results and Discussion |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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In the active center the GA structure contains a glycerol and a water, whereas an ethylene glycol and a phosphate are found in the Se–GA structure. Otherwise, both structures are very similar.
Overall structure
The A2B2 heterotetramer of GA (Fig. 1A) consists of two A1B1 heterodimers, each built from two polypeptide chains (A: residues Ala A1–Glu A160; B: residues Ser B1–Pro B522). The two chains fold into a compact structure as one might expect for a protein derived from a single-chain precursor. The two heterodimers, related by a crystallographic twofold axis, form a tight contact (buried surface area 1862 Å2) that involves only residues of the B-chains (Fig. 1A). The symmetry axis is located between Tyr B380 (heterodimer 1) and Tyr B380 (heterodimer 2). The tyrosine rings are oriented antiparallel, and their hydroxyl groups interact, mediated by a bridging water, with the carbonyl group of Ser B377 of the other heterodimer (Fig. 1B).
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) appears to be very similar to the structure of cephalosporin acylase reported by Kim et al. (Fig. 1c
of Kim et al. 2000); the atomic coordinates of the latter structure were not released at the time of this writing. The heterodimer forms one large and two small domains. One of the small domains (B301–B452) contains mainly ß-sheets, while the other (A61–A86, A151–A160, B75–B142, B199–B221) is helical, and involved in A2B2 heterotetramer contacts (Fig. 1A). The central core of the large domain comprises four layers of ßß ( Fig. 2A) that display the characteristic structural motif of the Ntn-hydrolase superfamily (Brannigan et al. 1995).
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coordinates can be superimposed with a r.m.s.d. of 1.9 Å (Fig. 2B), which is similar to the results of Kim et al. (2000).
Structure of the active center
According to the above alignment the structural similarity between GA and PGA includes the active site centered at the catalytic residue Ser B1 (Fig. 2B). As shown in Figure 2A, Ser B1 is located at the top of the flat ß-sheet, while the substrate binding pocket extends into the more twisted ß-sheet. In the neighborhood of the catalytic residue Ser B1 we found significant electron density (4–5 
) in both of our structures that could not be explained by protein atoms.
In the GA structure this density can be explained by a glycerol and a water molecule (W1) (Fig. 3A). In both crystallographically independent copies, W1 occupies a well-defined position, as indicated by its low temperature factor of 21 Å2. Glycerol is somewhat more mobile, with an average temperature factor of 49 Å2, but shows clear density in the annealed omit map. Glycerol interacts with Ser B1, Tyr A150, Tyr B33, Gln B50, Arg B57, Val B70, and W1, which bridges the hydroxyl group and the amino group of Ser B1 (Fig. 4A).
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). Refined temperature factors of the ligand atoms are 30 Å2 and 40 Å2, respectively. Ethylene glycol interacts with Tyr A150, Tyr B33, and Arg B57. Phosphate oxygen O2 interacts with ethylene glycol and the O3 atom forms hydrogen bonds with the Ser B1 hydroxyl and backbone-amino group, while O4 interacts with the hydroxyl-group of Ser B1 and with the amino groups of residues Val B70 and Asn B244 (Fig. 4B).
In both structures the catalytic residue Ser B1 is fixed by numerous interactions with the neighboring residues His B23, Asn B244, and Arg B274 (Figs. 4A,B). Hydrogen bonds are formed between Ser B1 O and the main-chain amino group of His B23, between the -amino-group of Ser B1 with OD1 of Asn B244 and NE2 of His B23, and between the main-chain carbonyl group of Ser B1 and the side chain of Arg B274. The Asn B244 side chain is fixed by the guanidino group of Arg B274.
Mechanism
In our structures Glu B455 is buried in the protein, and there is no other charged residue to compensate for this negative charge. Glu B455 interacts with the imidazole of His B23 so that the three residues, Ser B1, His B23, and Glu B455, form a cascade of two dyads (Figs. 4A,B), reminiscent of the catalytic dyads of serine proteases (Liao et al. 1992). However, the hydrogen bond between Ser B1 and His B23 involves the amino group of Ser and not the hydroxyl group, as found in serine proteases. This points to the importance of the backbone amino group of Ser B1, indicating that the catalytic mechanism of GA could be similar to PGA (Duggleby et al. 1995). Indeed, we observed a close structural similarity between Se–GA and the PMSF–PGA complex that includes the active site residues around the catalytic Ser B1. If we superimpose equivalent main-chain atoms and Cß-atoms (Kabsch 1978), we obtain a r.m.s.d. of 0.5 Å for 69 atoms (GA: B1–B3, B22–B24, B68–B71; B242–B245; PGA: B1–B3, B22–B24, B67–B70, B239–B242).
The mechanism proposed by Duggleby et al. (1995) for PGA assigns the Ser B1 -amino group as a general base. This amino group subtracts a proton from water W1, which bridges the amino and hydroxyl groups of Ser B1. The resulting OH- attracts the proton of the Ser B1 hydroxyl group. The negatively charged Ser B1 O attacks the substrate's carbonyl carbon, thereby forming a negatively charged intermediate stabilized by the oxyanion hole. Collapse of this tetrahedral intermediate leads to a seryl acyl enzyme and release of free 6-aminopenicillanic acid (6-APA). Attack of the acyl enzyme by water W2 leads to the formation of a second tetrahedral intermediate that is stabilized by the same interactions as the first one. Finally, this intermediate collapses, releasing free phenylacetic acid.
The interactions made by the tetrahedral intermediates developing during the catalytic cycle have been deduced from the structure of the PMSF–PGA complex in which the sulphonyl group is thought to model the hemiacetal (Duggleby et al. 1995). The same interactions occur in our Se–GA structure if we identify O3, O4 of the phosphate group and water W2 with O1S, O2S of PMSF and W2 in the PMSF–PGA complex, respectively (Figs. 4B,C). In Se–GA, the O4 atom interacts with nitrogens of the main chain of Val B70 and the side chain of Asn B244. These residues form together with His B23 the oxyanion hole, a structural motif that is characteristic of serine proteases (Stroud 1974). The corresponding residues in PGA are Ala B69, Asn B241, and Gln B23, respectively.
We conclude that the network of hydrogen bonds seen in our structures closely matches the network in the active site of the PGA structures, which is a strong indication that both enzymes adopt the same catalytic mechanism. The Ser B1 -amino group in PGA and GA can only work as a base if this group is uncharged. This is likely to be true for both enzymes at their pH optimum (PGA: 8.2 [Cole 1969], GA: 7.8) if the pKa of the free -amino group assumes a value about 8, as is usually found in proteins (Stryer 1988). In GA, the uncharged state of the -amino group is maintained by His B23 and Glu B455.
One structural difference remains, however. In PGA, the side-chain oxygen of Gln B23 forms a hydrogen bond with the -amino group of Ser B1 (Fig. 4C), whereas the role of the oxygen is assumed by the nitrogen NE1 of His B23 in GA (Fig. 4B). As mentioned above, this interaction is stabilized by Glu B455. At the pH optimum of GA the His and Glu side chains (pKa 6; pKa 4.25, Stryer) are unprotonated. The unprotonated side of the histidine residue (NE1) is oriented towards the -amino group, while the histidine NE2 forms a hydrogen bond with the acidic residue. The strength of a hydrogen bond depends on the pK values of the heteroatoms sharing the proton: two similar pK result in a short, strong hydrogen bond with almost no barrier between the two energy minima of the shared proton (Cleland 2000). Transient positive charges develop during the catalytic cycle at the -amino group or at the His side chain. It is then likely that in GA the hydrogen bond between His B23 and Glu B455 (distance 
2.7 Å) changes to a low-barrier hydrogen bond (distance 2.5 Å), because the pK of the now charged histidine matches that of the Glu side chains. The proton could then readily move between the heteroatoms forming the low-barrier hydrogen bond. The energy released upon formation of the low barrier hydrogen bond is used to ease proton transfer during the catalytic cycle by lowering the activation energies.
Taken together, it appears that GA has efficiently combined reaction principles from the PGA mechanism and from the classical catalytic triad.
Side-chain binding pocket
GA deacylates Gl-7-ACA to 7-ACA, whereas PGA catalyzes the hydrolysis of penicillin G into phenylacetic acid and 6-APA. PGA specifically recognizes the side-chain phenyl moiety and not the ß-lactam core of the substrate Penicillin G (Huang et al. 1963). In our GA structure glycerol is located at a position in the active center that corresponds to phenyl acetic acid in the structure of the complex with PGA (1pnl; Duggleby et al. 1995).
The side-chain pocket in PGA consists of 20 residues (A142–A146, B48, B49, B52–B57, B153–B155, B175–B178), which could be aligned with residues (A146–A150, B49, B50, B53–B58, B152–B154, B175–B178) in our GA structures (r.m.s.d. = 1.4 Å for 99 equivalent pairs of main-chain and Cß atoms). Most of these residues reside on the twisted ß-sheet, except for the GA residues Tyr A150 and Met A146 located on the C-terminal helix of chain A, and TyrB33, which is at a branching point where the polypeptide chains follow different courses in the two enzymes (Fig. 2C).
Only hydrophobic amino acids line the side-chain pocket in PGA, while GA possesses Arg B57, which binds glycerol as observed in our GA structure (Figs. 4A, 5A,B). The hydroxyl group at the other end of glycerol can only marginally interact with the oxyanion hole because the molecule is too short. The glutaryl side chain of the Gl-7-ACA substrate is just long enough to allow simultaneous interactions with Arg B57 and the oxyanion hole (Fig. 5C).
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shows a model of the first tetrahedral intermediate. The carbonyl carbon is bound to Ser B1 O, and the carbonyl oxygen fits into the oxyanion hole. The carboxylate group of the glutaryl side-chain interacts with Arg B57, Tyr B33, and Tyr A150, similar to the interactions seen for glycerol. The glutaryl side chain of Gl-7-ACA in GA and the side-chain phenyl moiety in PGA are then orientated in the same manner, while the ß-lactam core makes no interactions with the protein.
Comparison with glutarylamidase from the Pseudomonas strain N176
Glutarylamidase from the Pseudomonas sp. strain N176 (N176) hydrolyzes Gl-7-ACA and also CPC, although the latter only at low efficiency (Ishii et al. 1995). GA and N176 share 23% sequence identity and 50% sequence similarity. To predict the structure of N176 we have aligned its sequence with the GA and PGA structures as shown in Figure 6. The alignment suggests that the three enzymes adopt the same catalytic mechanism, all using Ser B1 as catalytic residue and three NH groups forming the oxyanion hole. One of these NH groups is provided by the side chain of a conserved asparagine residue, which is fixed by a conserved arginine. It is possible that the catalytic dyads seen in GA are also present in N176. Both proteins use a histidine that is either linked to Glu B455 in GA or to Asp B21 in N176 (Fig. 5D,E).
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). At the C-terminal end of this loop we have aligned N176 Tyr B32 with Tyr B33 in GA and Tyr B31 in PGA. The side chain of Tyr B32 would then point towards Ser B1, consistent with mutational studies of N176 (Ishii et al. 1995) that show that Tyr B32 could interact with Ser B1, and that the mutation Tyr B32 Glu abolishes enzymatic activity (Figs 5D, 7). Our alignment nicely explains the loss of catalytic activity when this residue is mutated to the negatively charged Glu residue. The two neighboring residues of Tyr B32 in N176, namely Ala B33 and Met B31, correspond to Tyr B34 and Phe B31 in GA. Studies of the mutants Ala B33 Tyr and Met B31 Phe show moderately increased activities for catalysis of CPC with respect to the wild type, and essentially no change of activity for the substrate Gl-7-ACA (Ishii et al. 1995).
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). The carboxylate group of CPC interacts with Arg B24, whereas the carboxylate group of Gl-7-ACA is bound by Arg B57 in GA, as mentioned above (Fig. 7). In PGA, the side chain of Penicillin G is purely hydrophobic, and as a consequence, there is no positively charged residue in the pocket. In N176, His B178 interacts with the -amino adipyl moiety of CPC. This interaction is stabilized through the negatively charged side chain of Asp B177 that points towards the histidine side chain. The -amino adipyl moiety, together with the His and Asp residues, structurally resembles the catalytic dyads described above. For the formation of the dyads it is necessary that the side chains of the adjacent Asp and His residues point into the same direction, which leads to backbone torsion angles outside of the allowed region of the Ramachandran plot. It has been noted that steric strain in the polypeptide backbone, when it occurs, is often found in functional regions of the protein (Herzberg and Moult 1991).
Implications for CPC converting mutants of GA
An enzyme that is catalytically competent to produce 7-ACA directly from cephalosporin C (CPC) would be of great interest for the industrial production of semisynthetic cephalosporin antibiotics (Ishii et al. 1995; McDonough et al. 1999; Kim et al. 2000). Our approach to achieving this goal is based on the putative structures of Gl-7-ACA in complex with GA (Fig. 5C) and of CPC in complex with N176 (Fig. 5D). We attempt to pinpoint the modifications required in GA if its substrate is to be CPC.
Instead of the carboxylate moiety at the end of the Gl-7-ACA side chain, CPC carries a carboxylate and an amino group (Scheme 1, Fig. 5D,E). Replacing Gl-7-ACA in our model structure by CPC would lead to no interactions of the -amino adipyl moiety. This is consistent with the finding that GA does not accept CPC as a substrate.
For binding of the -amino adipyl moiety, Figures 5D–F and 7 suggest that the GA mutant Tyr A150 Glu could lead to CPC binding. The carboxylate group of CPC could interact with Arg B57 (Fig. 5F). Considering the putative structure of CPC in complex with N176 (Fig. 5D), binding of the -amino adipyl moiety would require the mutation Phe B177 His and perhaps Thr B176 Asp to match the triad seen in N176. In addition, we expect that the mutants Leu B24 Arg or Gln B50 Arg would lead to more favorable interactions with the carboxylate group.
Summarizing our analysis, a modified GA that accepts CPC as a substrate should include a histidine or a glutamate for binding the -amino adipyl moiety and exactly one positively charged residue for binding the carboxylate group. This leads to the specific suggestions for modifying the sequence of GA that are summarized in Table 1. The additional mutations Y A150 A and Y B153 L, which enlarge the side-chain binding pocket, may be needed to accommodate the larger substrate CPC. Preparation and biochemical characterization of these GA mutants are underway.
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Materials and methods |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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Gel chromatography of GA using superose 12 in the presence of a 50-mM potassium phosphate buffer at pH 7.0 revealed a molecular weight of 140,000.
The pH profile of GA activity was determined at 37°C with 25 mM Glutaryl-7-ACA in the presence of 100-mM sodium phosphate buffers (pH 6.0 to 8.0). Glutarylamidase was added in appropriate dilution to get a linear velocity. The reaction was stopped after 10 min by addition of acid and aldehyde according to Shibuja et al. (1981). The resulting Schiff base was determined photometrically. Conversion to 7-ACA was about 8%. The pH profile displayed a broad optimum between pH 7 to 8 and a significant increase of specific activity between pH 6 and 7.
Crystallization
For bulk industrial production the enzyme was crystallized in the last purification step as described in the patent (Bayer 1996). To obtain X-ray grade crystals, small crude crystals were dissolved at 20–30 mg/mL in 0.5 M potassium phosphate buffer at pH 7.5 in the presence of 5 mM DTT. Crystals of GA were grown at 18°C by the hanging drop method using potassium phosphate (1.5–2.0 M, pH 7.0–9.0) as the precipitating agent. Diamond-shaped crystals of the size 400 x 300 x 40 µm appeared after 2–3 days. The crystals were monoclinic and contained two A1B1 heterodimers in the asymmetric unit, leaving a solvent content of 58%. Preparation of selenomethionyl-GA (Se–GA) was carried out as described (Budisa et al. 1995), applying the same continuous expression system in 10 L lab-scale fermentation (Aretz et al. 1998). Se–GA crystals were obtained under the same conditions as above. For cryoprotection, the GA and Se–GA crystals were transfered to crystallization buffer containing 10% (w/v) glycerol and 10% ethylene glycol, respectively.
Data collection
All diffraction data were recorded by the rotation method and processed by XDS (Kabsch 1988), which includes routines for space group determination, scaling, and conversion to reduced structure factor amplitudes (Kabsch 1993). Data set GA (Table 2) was collected from a single crystal at 100 K in the laboratory (X-rays: Cu K; generator: Nonius Fr571, operated at 45 kV/80 mA; detector: Mar300 image plate, xrayresearch; rotation/image: 0.5°). Multiwavelength anomalous dispersion (MAD) data were collected from the same selenomethionyl crystal (Se–GA) at 100 K using synchrotron radiation (ESRF beamline BM-14). Data sets Se–GApk, Se–GAip, and Se–GArm (Table 2) were obtained at wavelengths at the peak anomalous signal, at the inflection point, and remote from the absorption edge, respectively (detector: MAR345, xrayresearch; rotation/image: 1°).
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Model building and refinement
An initial atomic model of Se–GA was fitted to this map using the interactive graphics program O (Jones et al. 1991). Refinement of the model was completed after a few rounds of model correction, thereby imposing restraints on the two independent A1B1 heterodimers to obey noncrystallographic symmetry (Table 3). The final model of the A2B2 heterotetramer comprises residues Gln 8 to Glu 160 of the A chains, residues Ser 1 to Pro 522 of the B chains, one phosphate, and one ethylene glycol in each of the two active sites, and 710 water molecules in the asymmetric unit. The model displays good stereochemistry, as verified by Procheck (Laskowski et al. 1993). Except for residue Phe B 177, all backbone torsion angles are within the allowed region of the Ramachandran plot, and four prolines (P A132, P B253, P B379, P B466) assume a cis-peptide conformation, as has been noted previously by Kim et al. (2000). The GA structure was solved by molecular replacement using the Se–GA structure as a starting model. The final model contains the same number of protein atoms as the Se–GA structure, one glycerol molecule in each of the two active sites, and 706 water molecules (Table 3).
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Acknowledgments |
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 L. G. Otten, C. F. Sio, J. Vrielink, R. H. Cool, and W. J. Quax Altering the Substrate Specificity of Cephalosporin Acylase by Directed Evolution of the beta -Subunit J. Biol. Chem., October 25, 2002; 277(44): 42121 - 42127. [Abstract] [Full Text] [PDF]
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