Insight into autoproteolytic activation from the structure of cephalosporin acylase: A protein with two proteolytic chemistries

Abstract

Cephalosporin acylase (CA), a member of the N-terminal nucleophile hydrolase family, is activated through sequential primary and secondary autoproteolytic reactions with the release of a pro segment. We have determined crystal structures of four CA mutants. Two mutants are trapped after the primary cleavage, and the other two undergo secondary cleavage slowly. These structures provide a look at pro-segment conformation during activation in N-terminal nucleophile hydrolases. The highly strained helical pro segment of precursor is transformed into a relaxed loop in the intermediates, suggesting that the relaxation of structural constraints drives the primary cleavage reaction. The secondary autoproteolytic step has been proposed to be intermolecular. However, our analysis provides evidence that CA is processed in two sequential steps of intramolecular autoproteolysis involving two distinct residues in the active site, the first a serine and the second a glutamate.

Posttranslational autoproteolysis is a mechanism used to activate many proteins via self-catalyzed peptide bond rearrangements, which play an essential role in a wide variety of biological processes. They include activation cascades such as blood coagulation and fibrinolysis, cell death, embryonic development, protein targeting and degradation, viral protein processing, and zymogen activation (1–6). Increasing evidence suggests that autoproteolysis may be involved in more biological activation processes than previously appreciated. These processes are all mediated by intramolecular or intermolecular autocleavage reactions. Four types of intramolecular protein modifications have been studied extensively: hedgehog proteins, inteins, N-terminal nucleophile (Ntn) hydrolases, and pyruvoyl enzymes (4, 7–9).

Members of the Ntn hydrolase family have divergent sequences, but share an αββα sandwich structural motif and a common enzyme mechanism (7, 8). In addition, they catalyze internal peptide bond rearrangement through an N → O or N → S acyl shift by a nucleophilic Ntn amino acid created by autoproteolytic processing (4, 7). Structures of mature Ntn hydrolases, including cephalosporin acylase (CA), penicillin G acylase (PA), penicillin V acylase, glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase, 20S proteasome β subunit (PRO), glycosylasparaginase (GA), and l-aminopeptidase, have been determined (10–17). Structures of the precursors of CA, PA, PRO, and GA have been reported, providing some insight into the activation mechanism (11, 18–22). However, the understanding of mechanism cannot be complete without structural information on the first cleavage intermediates.

Ntn hydrolases are of particular interest because they include acylase enzymes used in the biosynthesis of the β-lactam antibiotics cephalosporin and penicillin. We have focused on CA. This enzyme is expressed as a 76-kDa precursor that is activated by two sequential autocleavage steps (11, 23) (Fig. 1A). A primary cleavage between Gly-169 and Ser-170 generates the α′ subunit (the α subunit with a 9-aa pro segment) and the β subunit. A subsequent secondary autocleavage between Gly-160 and Asp-161 releases the pro segment from the α′ subunit, resulting in an active heterotetramer (αβ)₂. The release of the pro segment may be required for optimal CA activity. Ser-170, which becomes the N-terminal residue (Ser-1)^†† of the β subunit of CA is invariant and known to play a critical role in both autoproteolytic activation and enzyme catalysis (23–25).

Fig. 1.

Autoproteolytic cleavage of glutaryl 7-aminocephalosporanic acid acylase. (A) Schematic diagram of two-step autoproteolytic cleavage. The precursor is activated through a primary cleavage to generate the α subunit containing the pro segment (α′ subunit) and the β subunit. A subsequent secondary autocleavage releases the pro segment. (B) SDS/PAGE analyses of the autoproteolytic cleavage of CA. Lane M, molecular weight markers; S170A is a precursor form. Y202L, L was obtained by incubating Y202L at 37°C for 24 h. (C) Time-course experiments of secondary autocleavage. Y202L or S170C was incubated with mature enzyme (WT) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or at different pH values. Incubation and reconstitution experiments (Inc/Rec). Lane 1, freshly prepared Y202L; lane 2, crystals of fresh Y202L; lane 3, crystals of Y202L incubated at 37°C; lane 4, reconstitution with the combination of α_mature and β_mature subunits; lane 5, reconstitution with α′_S170C and β_mature subunits; and lane 6, reconstitution with α′_E159Q and β_mature subunits.

In an effort to gain a better understanding of the autoproteolyic mechanism, we have designed constructs containing mutations that partially prevent or retard autocleavage and carried out the structural determination of these mutants. Here, we report structural evidence that CA is processed in two sequential steps of intramolecular autoproteolysis involving two distinct proteolytic mechanisms, the first mediated by a serine residue and the second by a glutamate.

Results

Choice of Mutant Proteins.

A single amino acid mutation of Ser-170 to alanine (S170A) prevents primary cleavage, producing an enzymatically inactive single-polypeptide precursor (Fig. 1B). The crystal structures of precursor (S170A) and mature CA have been reported (11). In this study, we have focused mainly on four mutants. S170C and E159Q underwent primary autocleavage but no secondary autocleavage. Y202L and R226K, slow processing mutants, showed a delayed release of the pro segment in comparison with the mature enzyme. Tyr-202 and Arg-226 are located in the substrate binding site of CA. The Y202L and R226K mutants were initially analyzed as altered-activity mutants and found to be capable of sequentially producing α′ and α subunits during activation. In the case of Y202L, it took ≈24 h at 37°C to make a complete transition from the uncleaved to the cleaved form (Fig. 1C, Y202L control). Thus, early (Y202L) and late (Y202L, L) stages of Y202L during the time course of autoproteolysis could be captured for crystallization (Fig. 1B, lanes Y202L and Y202L, L). The presence or absence of the pro segment linked to the α subunit of the mutant (α′β)₂ or (αβ)₂ was confirmed by mass spectrometric and crystallographic results (see Fig. 6, which is published as supporting information on the PNAS web site). The electron density of the pro segment in Y202L, L revealed a clear discontinuity after Gly-160, indicating a complete release of the pro segment, which was consistent with the SDS/PAGE result (Fig. 1B, lane Y202L, L).

Comparison of Mutant Structures and Pro Segments.

The refined overall structures of the mutants, Y202L, R226K, S170C, and E159Q (see Table 1, which is published as supporting information on the PNAS web site), are similar to the precursor and mature structures reported earlier (11) (Fig. 2A). In brief, they adopt the αββα motif formed by two β-sheets packed against each other, sandwiched by two layers of α-helices. After superposition of the C_α atoms, the rms deviations of mutants against the precursor S170A range from 0.37 to 0.53 Å. The structural αββα core is highly conserved in the Ntn hydrolase superfamily (8), as in structures of glutarylamidase from Pseudomonas sp. and CA from Pseudomonas diminuta (10, 24). However, there are significant differences at the entryway to the active site, which is a deep cleft at the tip of the αββα sandwich motif, where the pro segment is located.

Fig. 2.

Structural comparison of CA proteins. (A) Superposition of the overall structures of S170A, S170C, E159Q, Y202L, R226K, and the mature CA. Each structure is shown in yellow, cyan, dark green, green, slate blue, and marine, respectively. To emphasize the differences of the pro segments, the pro segment of Y202L is shown in red. Residues Gly-160, Ser-170, Tyr-202, and Arg-226 are shown in a ball-and-stick representation in magenta. (B) Stereoview of the pro-segment conformation of Y202L. The electron density map corresponds to the 2 F_o − F_c map calculated at 0.5 σ.

In precursor CA the pro segment is α-helical from Pro-163 to Ala-166 (11). Proline is not favored in helices, and significant deviations from helical geometry suggest that this is a high-energy conformation, generating a peptide flip for initiation of primary autoproteolysis. A similar strained conformation, which was suggested to regulate the activation of glycosylasparaginase (21) and a pyruvoyl enzyme (22), is believed to be the driving force for autocleavage to break the scissile peptide bond (26). In the intermediate mutants of CA, which have undergone the initial but not the secondary cleavage, the pro segments adopt strikingly different conformations from that of the precursor (Fig. 2A). First, the pro segments show a loop conformation without regular secondary structure. While their N termini are covalently attached to the α subunit, the C termini upon primary cleavage are moved from residue 170 and may be partly exposed to solvent. In addition, there are no significant geometric deviations from ideal geometry in the pro segments, indicating unstrained conformations. Second, they are highly mobile with high B factors (68–83 vs. 18–35 Ų in precursor). The electron densities for the pro segments are visible only at a low contour level, as shown in Fig. 2B (and see Fig. 7A, which is published as supporting information on the PNAS web site). The pro segment may become highly mobile upon primary cleavage, a characteristic of a terminal end, which would probably facilitate secondary autocatalysis and subsequent dissociation of the pro segment. Thus the highly strained helical conformation of the precursor pro segment is transformed into a highly flexible and relaxed loop conformation in the intermediates, supporting the notion that the relaxation of structural constraints drives the primary autocleavage reaction.

A comparative analysis of the mutant structures reveals that the pro segments exist in a variety of conformations (see Fig. 7B). Although Y202L, R226K, and E159Q follow similar backbone paths from Glu-159 to Pro-162, moving down to the deep cleft at Glu-159, they extend away from each other at Pro-163. By contrast, residues Glu-159 and Gly-160 in S170C have ϕ conformational angles opposite to the signs of other mutants and the pro segment moves downward at Gly-160 to the deep cleft. The differences in the backbone path place Pro-163 of S170C and Y202L at positions ≈10 Å apart from each other. Moreover, S170A precursor structure reveals extensive contacts between the pro segment and protein residues (see Tables 2 and 3, which are published as supporting information on the PNAS web site), but there are significantly fewer contacts in the intermediate mutants. Although only partial models of the pro segments could be built, it is clearly shown that the number of polar and van der Waals contacts is greatly diminished in the intermediate mutants (see below). It is likely that conformational constraints in precursor may be confined within the pro segment during protein folding with expenditure of the noncovalent interaction energies, which are released upon primary autocleavage.

Primary Autoproteolytic Site.

The active sites of mutants formed upon primary autocleavage display structural features similar to the mature enzyme. First, an intact catalytic pseudotriad of Ser-His-Glu becomes registered in all of the mutants, involving the free α-amino group of (Ser-1), not the usual hydroxyl group, as in the wild-type structures (11, 24). The side chain of (His-23) in the wild-type contributes to a reduction in the pK_a of the α-amino group of (Ser-1), which then acts as a general base to enhance the nucleophilicity of its own hydroxyl group (data not shown). Second, Ser-170(Ser-1), which is buried in the precursor (11, 18), becomes partially exposed to solvent in the mutants; it is completely open to solvent in mature enzyme (11). The differential burial of Ser-170 may correspond to a structural activation signal for enzyme catalysis. Third, despite the partial enclosure of the active site by the pro segment, a solvent molecule emerges near the hydroxyl group of (Ser-1) at the active site in Y202L and R226K. The solvent molecule at this position plays a crucial role in the enzyme activity of mature CA (10, 11, 24), as recent studies revealed the role of the bound water near the catalytic residue in Ntn hydrolases (18, 21, 26).

Secondary Autoproteolysis: Is It Intermolecular or Intramolecular?

In contrast to the primary autocleavage mechanisms for the Ntn hydrolases (11, 19, 20, 22), the secondary cleavage mechanism of CA is not clearly understood. An intermolecular cleavage mechanism for CA activation was proposed based on reconstitution experiments (23). CA takes glutaryl 7-aminocephalosporanic acid (7-ACA) as a primary substrate and catalyzes its hydrolysis into glutaric acid and 7-ACA. It was reported that the mutations in the substrate binding site that inhibited deacylation activity also prevented secondary autoproteolysis (25). CA secondary autoproteolysis might be correlated with catalytic activity. Nevertheless, the question remains: how is secondary cleavage initiated in the absence of the active mature enzyme? Importantly, the release rate of the pro segment of Y202L was unchanged, and the autocleavage of S170C was not restored when incubated with the mature enzyme (Fig. 1C, Y202L+WT and S170C+WT), providing experimental evidence against intermolecular secondary cleavage. More convincingly, Y202L crystals were incubated at 37°C for 24 h, where intermolecular autocleavage is not allowed because of crystal packing, and SDS/PAGE analysis of the incubated Y202L crystals demonstrated the generation of the cleaved α subunit (Fig. 1C, Y202L Inc/Rec, lane 3).

In the precursor structure, the carboxyl group of Glu-159 is within hydrogen-bonding distance of Gln-75 (Fig. 3, S170A). The side chain of Asp-161 is hydrogen-bonded to those of Ser-152 and Arg-155. Moreover, Leu-165 is involved in hydrophobic contacts with Leu-148, Tyr-199, and Phe-200. There are extensive contacts between the pro segment and protein residues in the deep cleft (see Table 2). In the intermediate mutants, however, there are fewer contacts and no hydrophobic interaction is found between Leu-165 and protein residues. Notably, Gln-75 points away toward the solvent and its hydrogen-bonding to Glu-159 is absent. In Y202L, Glu-159 is linked to Thr-76 by a network of hydrogen bonds involving a series of water molecules (Fig. 3, Y202L). A positive density at the F_o − F_c difference Fourier map could be modeled as solvent molecule WAT1, which is hydrogen-bonded to the carbonyl oxygen of Asp-161 and another solvent molecule. Interestingly, WAT1 is also close to the side chain of Glu-159 (4.0 Å) and is positioned on top of the carbonyl carbon of Gly-160 (4.0 Å) at the scissile peptide bond of secondary autocleavage. R226K has a very similar backbone configuration to that of Y202L at the secondary cleavage site (Fig. 3). Notably, a solvent molecule corresponding to WAT1 of Y202L could be placed in hydrogen-bonding distance to Glu-159 (3.5 Å) and close to the carbonyl carbon of Gly-160 (4.0 Å). Both mutant structures thus reveal that WAT1 could be modeled only 3.5–4.0 Å from Glu-159 adjacent to the conserved secondary site, suggesting that it is well within the range to facilitate proton abstraction.

Fig. 3.

Secondary autocleavage site of CA proteins. The interactions between pro-segment and protein residues in Y202L, precursor S170A, R226K, S170C, and E159Q. Those involved in hydrophobic interactions in S170A are superimposed in a transparent space-filling representation. Hydrogen bonds are indicated by dotted lines, and water molecules are represented as red spheres. The distances between the side chain of Glu-159 and the backbone carbon atom of Gly-160 via WAT1 in Y202L and R226K are highlighted by thick dotted lines.

In the S170C mutant structure, however, strong hydrogen bonds could be made between a water molecule and the backbone oxygen and nitrogen atoms of Glu-159 and Gly-160, respectively, instead of the side chain of Glu-159 (Fig. 3). S170C displays different backbone conformation of the secondary cleavage site, in contrast to other mutants (see Fig. 7B). In E159Q, where the local backbone conformation is similar to that of Y202L or R226K, no significant electron densities for solvent molecules could be observed at the secondary site.

Carboxyl Proteolytic Autocleavage.

In an effort to derive a detailed molecular mechanism of secondary autocleavage, we further carried out a number of biochemical and mutational experiments. At first, it appeared that the sequential arrangement of two carboxylic acids, Glu-159 and Asp-161, together with WAT1 has some resemblance to aspartyl protease. A carboxyl protease inhibitor, a carbodiimide-mediated modification of Y202L, blocked the secondary cleavage (Fig. 1C, Y202L+EDC). Monitoring the pH dependence of the secondary autocleavage also indicated that deprotonation of the carboxyl group was required for autocatalysis (Fig. 1C, Y202L pH 5.0–7.0). However, the mutants D161L or D161N gave a fully processed enzyme (Fig. 1B). Mutations in other candidate residues that are close to the scissile peptide bond, S152A, R155K, and R155M, also produced a successfully processed enzyme. Only two mutants, E159M and E159Q, lost their secondary autocleavage activities. To test the possibility of general base catalysis by glutamate, the α′ subunits from S170C and E159Q mutants were purified separately in the denatured condition (α′_S170C and α′_E159Q, respectively) and reconstituted with the denatured β subunit (β_mature) of the mature enzyme by refolding (Fig. 1C, Y202L Inc/Rec, lanes 4–6). The secondary cleavage occurred only when the enzyme was reconstituted with the combination of α′_S170C and β_mature subunits, but did not when it was reconstituted with the α′_E159Q and β_mature subunits. Our results convincingly demonstrated that Glu-159 is pivotal for this secondary processing.

Cation–π Interaction.

The oxyanion binding of Arg-155 could be stabilized by the carbonyl oxygens of Tyr-149 and Val-150 (Fig. 3, Y202L). Importantly, the side chains of Tyr-149 and (Phe-177) form a near edge-face aromatic interaction and (Phe-177) is in a favorable arrangement for a cation–π interaction (27) with (Arg-57) in the substrate binding site (Fig. 4). Similar interactions are recognized as crucial for the structural stabilization of caspase BIR domain (28) and neutrophil collagenase (29). Moreover, (Phe-177) is the only residue of 682 modeled amino acid residues in a disallowed region of the Ramachandran plot in all mutant crystal structures as well as in precursor and mature CA (see Fig. 8, which is published as supporting information on the PNAS web site). F177P mutant remains a nonprocessed precursor with no catalytic activity (25). Notably, Y202L, R226K, and S170C mutants have the defects localized in this region, where Tyr-202(Tyr-33), Arg-226(Arg-57), and (S1) are replaced by leucine, lysine, and cysteine, respectively. In Y202L, solvent water molecules are incorporated in hydrogen-bonding networks, similar to precursor or mature CA. The cation–π interaction in R226K may become weak because of the mutation, resulting in a much slower processing mutant than Y202L (data not shown). S170C and E159Q, on the other hand, lack the hydrogen-bonding networks, probably interrupted by an unexplained strong positive density found midway between (Arg-57) and (Cys-1) or (Ser-1). Therefore, the perturbation in the integrity of this region caused by mutation may result in a serious defect in the secondary autocatalytic activity, suggesting that the cation–π and hydrogen-bonding interactions may play an important structural role in supporting proper autocatalysis.

Fig. 4.

Close-up view of the active site in Y202L, R226K, S170C, and E159Q structures. Cation–π and aromatic interactions are shown in a ball-and-stick representation. The electron densities from the 2 F_o − F_c maps were contoured at 1.0 σ. Hydrogen bonds are indicated by dotted lines, and water molecules are represented as red spheres.

Discussion

A glutamate residue acting as a general base was first discovered in histone acetyltransferase, where the conserved glutamic acid is an essential catalytic residue by deprotonating the histone substrate (30). N-myristoyl transferase also has a conserved glutamic acid residue that is essential in catalysis (31). Interestingly, chloramphenicol acetyltransferase uses a histidine residue as a general base to abstract a proton from the primary hydroxyl group of chloramphenicol. A glutamic acid replacement of this histidine can substitute as the general base, albeit with lower catalytic activity (32). Mutational studies in other homologous CA proteins support our notion that Glu-159 plays a critical role in secondary autoproteolysis: The Glu-159 mutants, E159L, E159M, and E159Q, of CA from P. diminuta also underwent primary autocleavage but lost the secondary autocleavage activity (18). In the crystal structure of E159Q, the backbone conformation from Gly-158 to Asp-161 is fairly similar to that of the Y202L and R226K forms (Fig. 3).

Based on the intermediate structures, we therefore propose that CA may have evolved separate autoproteolytic mechanisms, the first a serine proteolysis and the second a carboxyl proteolysis. Upon protein folding, precursor CA undergoes the primary autoproteolysis that uses a N → O acyl shift (Fig. 5A). Initially, a high-energy state of the pro segment caused by conformational constraints may trigger a peptide flip. WAT1, held by hydrogen bonds in pseudotetrahedral geometry, acts as a general base to deprotonate the hydroxyl group of Ser-170 to enhance its nucleophilicity. The hydroxyl group carries out the nucleophilic attack on the peptide bond between Gly-169 and Ser-170. This primary cleavage results in generating a helix-to-loop transition of the pro segment, which consequently triggers an activation signal switch for initiation of the secondary carboxyl autoproteolysis. Glu-159 acting as a general base may accept a proton from WAT1 to enhance its nucleophilicity (Fig. 5B). The nucleophilic attack on the peptide bond between Gly-160 and Asp-161 is carried out by WAT1, producing a tetrahedral transition state. Formation of the ester intermediate (a N → O acyl shift) requires stabilization of the oxyanionic transition state, which may be undertaken by the backbone nitrogen of Arg-155. The secondary autoproteolysis results in cleavage and subsequent dissociation of the pro segment, producing the α subunit. Autoproteolytic cleavage in Ntn hydrolases is mediated by intramolecular autocleavage reactions. CA then truly belongs to the Ntn hydrolase family of intramolecular self-cleavage.

Fig. 5.

Proposed autoproteolytic activation mechanisms for CA. (A) Primary serine autoproteolytic cleavage. A peptide flip (open arrow) under the influence of the strained conformation creates a hydrogen bond between WAT1 and the carbonyl group of Gln-168. WAT1, held by hydrogen bonds in pseudotetrahedral geometry, acts as a general base and deprotonates the hydroxyl group of Ser-170 for initiation of primary cleavage (curved arrow). Dotted lines indicate hydrogen-bond interactions. WAT and WAT1 represent a water molecule. (B) Secondary carboxyl autoproteolytic cleavage. It is initiated by a general-base-catalyzed attack of WAT1 on the Gly-160–Asp-161 peptide bond, where Glu-159 acts as a general base (curved arrows). The main-chain NH of Arg-155 stabilizes the oxyanion of Gly-160.

Two mutants, Y202L and R226K, showed significant decreases in the rates of autocleavage reactions, thus being capable of kinetically trapping the uncleaved and the cleaved forms of the pro segment. We postulate that Y202L and R226K may represent intermediate structures in transition from an immature to a mature arrangement during activation, whereas S170C and E159Q may be regarded as trapped intermediates. However, we cannot rule out the possibility that the pro segment containing the Glu-159–WAT1 pair in Y202L or R226K may not represent a legitimate intermediate during autoproteolysis of CA or may adopt a slightly altered conformation from that of a native intermediate. Nevertheless, our results are strongly suggestive that the proposed mechanisms represent a common molecular mode of action for CA autoproteolysis.

In conclusion, our finding of dual catalysis at intervals of 9 aa, both serine and a previously undiscovered carboxyl autoproteolysis, may have novel implications for understanding the mechanisms of autoproteolysis that are important in such diverse biological processes.

Materials and Methods

Plasmids, Strains, and Site-Directed Mutagenesis.

The pET23d plasmids harboring the cloned mature CA gene from Pseudomonas sp. strain GK16 and the S170A mutant gene have been described (11). The mutants S152A, R155K, R155M, E159M, E159Q, D161L, D161N, S170C, Y202L, and R226K were constructed by site-directed mutagenesis, using a PCR. Each PCR product was cloned into vector pET22b or pET23d (Novagen), which carries a sequence coded with a C-terminal His tag downstream from the T7 RNA polymerase promoter site. The construct was then transformed into an Escherichia coli DH5α. The bacteria were grown at 37°C, and the plasmids were purified by using a plasmid mini kit (Qiagen, Valencia, CA). All mutations were verified by automated DNA sequencing (data not shown). E. coli strains were grown in LB medium.

Protein Expression and Purification.

The clones were inserted into E. coli BL21/DE3 (Novagen) for protein overexpression, and bacteria were grown at 37°C until the culture reached an absorbance of 0.6 at 600 nm. Protein expression was induced by addition of isopropyl 1-thio-β-d-galactopyranoside (Sigma) to a final concentration of 0.4 mM, which was followed by an additional 4 h of incubation. The purification of all of the mutant CA proteins was performed according to published procedures (11). All of the mutant proteins yielded proteins of sufficient purity for crystallization.

Analysis of Autocleavage.

The extent of primary and secondary autocleavage for the purified mutants was analyzed by SDS/PAGE. Y202L showed an approximately equimolar mixture of cleaved and uncleaved α subunits subsequent to purification (Y202L early: Y202L). Both the α subunit linked with the pro segment (α′ subunit) and the α subunit itself were subjected to MALDI-TOF mass spectrometry (Korea Basic Research Institute, Seoul). The time-course monitoring of in vitro secondary processing of S170C or Y202L was carried out in 20 mM Tris (pH 8.0) at 37°C for 24 h. S170C remained in the uncleaved form because of the lack of secondary cleavage. For Y202L, it took ≈24 h at 37°C, to make a complete transition from the uncleaved to the cleaved form (Y202L late: Y202L, L). From the solution processed at 37°C, 5-μl aliquots were sampled at 3-h intervals. The addition of the mature enzyme to each mutant solution or 0.1 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to Y202L in 1.0 M glycine ethyl ester at pH 5.0 at 25°C was used to detect changes in secondary processing. For pH analysis of the secondary autocleavage of Y202L, time-course monitoring was carried out at different pH values, ranging from 5.0 to 8.0. For reconstitution experiments, α, α′, or β subunit was separately purified from mature CA, S170C, and E159Q in the denatured condition (α_mature and β_mature, α′_S170C, and α′_E159Q, respectively). They were reconstituted with the combination of α_mature and β_mature, α′_S170C and β_mature, or α′_E159Q and β_mature by refolding. The results of reconstitution and time-course monitoring experiments were analyzed by SDS/PAGE.

Crystallization and Data Collection.

Unlike autocleavage analysis, only four mutants were crystallized for structural analysis at the cleavage sites. The crystals of the two Y202L forms (Y202L and Y202L, L), R226K, S170C, and E159Q were grown at 22°C, using the hanging-drop vapor diffusion method. One to two microliters of protein solution (30 mg/ml) was mixed with an equal volume of well solution and equilibrated against 1 ml of the well solution of 0.1 M Na-cacodylate (pH 6.5), 0.2 M MgCl₂, and PEG 6000. For Y202L, PEG 3000 was used. Crystals were produced with a well developed bipyramidal morphology within 3–4 days. Diffraction data were collected with the crystals flash cooled at 100 K by using either a laboratory x-ray source or a synchrotron radiation source, beamline 4A at Pohang Light Source (Pohang, Korea). All intensity data were processed and scaled by using the programs denzo and scalepack (33). The unit cell dimensions of the mutant protein crystals of P4₁2₁2 in this study were almost identical (see Table 1).

Structure Solution and Refinement.

The mutant crystal structures of Y202L, Y202L, L, R226K, S170C, and E159Q were solved by using cns (34) for molecular replacement, using the mature structure as a search model (11). The initial solution was optimized by rigid body refinement, which produced a clearly interpretable electron density for the overall α and β subunit structures and the mutation site. Manual adjustment of the backbone and side chain and the manual building of the pro-segment residues was conducted in o (35). Crystallographic refinement was carried out by using the program refmac5 (36). The sigma A weighted 2 F_o − F_c maps were used to perform a visual inspection during the rebuilding of the pro segment with o (35). After a few rounds of model rebuilding, a continuous electron density for the residues of the pro segment could be obtained at the 0.5- to 0.8-σ level depending on mutants. At 1.0 σ, there was no electron density visible for the region. In the case of Y202L, the pro-segment atoms were modeled with half-occupancy, based on the equimolar ratio of the cleaved and uncleaved α subunits on SDS/PAGE (Fig. 1C, Y202L Inc/Rec, lanes 1 and 2). Nevertheless, the electron density for the pro-segment backbone was quite clear in the mutant structures. The residues up to Ala-166, Leu-165, Asp-164, and Asp-164 of the pro segments could be modeled in the Y202L, R226K, S170C, and E159Q structures, respectively. More importantly, the electron density map for the secondary cleavage site including residues from Glu-159 to Gly-160 was readily interpretable. Water molecules were added by using the F_o − F_c map peaks >3.0 σ, if the putative water molecule made possible hydrogen-bonding contacts and had no adverse van der Waals contacts. Some water molecules contacting the pro segment were added by using the F_o − F_c map peaks <3.0 σ, if the B factors were <60 Ų after refinement. The free R value was used as an indicator to validate the water picking and refinement procedure and to guard against possible overfitting of the data (37). The stereochemical analysis of all of the refined structures by using procheck (38) showed four prolines [Pro-131 of the α subunit and (Pro-253), (Pro-379), and (Pro-466) of the β subunit] in a cis-peptide conformation and one residue (Phe-177) of the β subunit in a disallowed region of the Ramachandran plot (see Fig. 8). Fig. 3 was prepared with the programs molscript (39) and raster3d (40). Figs. 2, 4, 5, 6B, and 7 were prepared with the program pymol (www.pymol.org; ref. 41).

Abbreviations

CA

cephalosporin acylase

Ntn

N-terminal nucleophile