Relevant Double Mutations in Bioengineered Streptomyces clavuligerus Deacetoxycephalosporin C Synthase Result in Higher Binding Specificities Which Improve Penicillin Bioconversion

Generation of mutant ScDAOCSs via site-directed mutagenesis.The cefE gene of S. clavuligerus NRRL 3585 was previously cloned into the pGK expression vector and expressed as a fusion protein with glutathione S-transferase (GST) (pscEXP-GST) (19). Site-directed mutagenesis was performed using a QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, with previously reported modifications (17). The recombinant expression vector pscEXP-GST was used as the mutagenesis template for creating single and C-terminal double mutants. The mutagenic oligonucleotide primers were synthesized by Sigma-Proligo (Singapore) (Table 1). Recombinant vectors carrying N304X (where X is alanine, leucine, methionine, lysine, or arginine), R306L, and R307L single mutations were obtained from previous studies (2, 3).

Double mutations consisting of either V275I or C281Y with the single C-terminal mutations were created by restriction digestion, followed by ligation. Recombinant vectors carrying V275I, C281Y, and the C-terminal single mutations (N304X, I305M, R306L, and R307L) were digested using the PfoI and BamHI restriction enzymes (Fermentas) according to manufacturer's instructions. The digested samples were separated by agarose gel electrophoresis. After this, the desired DNA fragments were excised and purified from the gel using a MinElute gel extraction kit (Qiagen). The purified fragments were then ligated using T4 DNA ligase according to manufacturer's recommendations (Invitrogen).

The site-directed mutagenized or restriction-ligated recombinant plasmids were subsequently transformed into Escherichia coli strain BL21(DE3). Clones harboring the desired mutations were screened by extracting the plasmid DNA using the Wizard Plus SV Minipreps DNA purification system (Promega), followed by automated DNA sequencing. DNA sequencing was performed using an ABI PRISM dye terminator cycle sequencing kit and an ABI PRISM 3100 genetic analyzer as previously described (3). The mutated genes were fully sequenced to ascertain that no random mutation had been incorporated during the site-directed mutagenesis or restriction-ligation process.

Expression and purification of wild-type and mutant ScDAOCS fusion proteins.Recombinant clones of wild-type and mutant ScDAOCSs were cultivated for high levels of protein expression and harvested as previously described (4, 19). Wild-type and mutant ScDAOCS fusion proteins were purified from soluble cell extracts by affinity chromatography using a MicroSpin GST purification module (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The cell lysates were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to ensure that the mutations did not have any adverse effects on the soluble expression of the fusion proteins. Gel images were captured using the Bio-Rad Molecular Imager Gel Doc XR system, which was controlled by the Quantity One software (Bio-Rad). This program was also used to estimate the relative expression levels of the wild-type and mutant ScDAOCSs and the purity of the purified fusion proteins. Protein concentrations were determined by the Bio-Rad protein assay, and bovine serum albumin (Sigma-Aldrich) was used as the reference protein standard.

Determination of penicillin conversion activities of wild-type and mutant ScDAOCSs.The standard reaction mixtures described by Sim and Sim were used because these conditions are optimal for ring expansion of a hydrophobic penicillin to obtain its cephalosporin moiety (19). All the chemical reagents were obtained from Sigma-Aldrich unless otherwise indicated. Briefly, the enzyme reaction mixture contained 50 mM Tris (pH 7.4), 1.8 mM iron(II) sulfate, 0.8 mM ATP, 1.28 mM α-ketoglutarate, 4 mg ml⁻¹ penicillin substrate and 0.8 mg ml⁻¹ purified GST-ScDAOCS. The penicillin substrates used were ampicillin, penicillin G, phenethicillin, and carbenicillin. The reaction mixtures were incubated at 30°C with shaking at 200 rpm for 15 min. The reaction was stopped by adding an equal volume of methanol. The amounts of product formed by wild-type and mutant ScDAOCSs were determined by a hole plate bioassay as previously described (19). E. coli strain Ess (a gift from A. L. Demain, Drew University) was used as the test organism and seeded at a final concentration of 1% (vol/vol) using a culture diluted to an optical density at 600 nm of 0.1. Difco Penase was added to a final concentration of 100,000 IU ml⁻¹ to the bioassay plates to remove the unconverted penicillin substrates. Cephalosporin C was used as the reference standard. One unit of enzyme activity was defined as the amount of enzyme that formed the equivalent of 1 μg of cephalosporin C per min under the prescribed conditions. The mean specific activity of each enzyme was determined using the data from at least three independent experiments with duplicates.

The high-performance liquid chromatography (HPLC) system used included an ÁKTA purifier (Amersham Biosciences) consisting of a UV-900 detector and an A900 autosampler and a SunFire C₁₈ column (5 μm; 4.6 by 150 mm; Waters, United States). The operation of the system and the acquisition of data in the chromatographic process were controlled by the UNICORN 5.0 program (Amersham Biosciences). The chromatographic conditions described by Chin and Sim were used (2). One unit of enzyme activity was defined as the amount of enzyme that formed 1 μg of cephalexin per min under the prescribed conditions. The specific activity of each enzyme was determined using the data from at least three independent experiments with duplicates.

Determination of kinetic parameters of wild-type and mutant ScDAOCSs.The kinetic assays were performed using a spectrophotometric method described by Dubus et al., assuming that ε is 6,100 M⁻¹ cm⁻¹ for the conversion of ampicillin to cephalexin (6). The final concentration of enzyme used was 2.5 μM. The final concentrations of the ampicillin substrates used ranged from 0.1 to 10 mM. The kinetic assay was performed using a 96-well format filter-based GENios spectrophotometer (Tecan, Austria) with a 260-nm absorbance filter and the temperature fixed at 30°C. The kinetic parameters were determined using the data from at least three independent measurements with triplicates and were calculated using a Hanes-Woolf plot.

Computational analysis.Comparative analyses of protein sequences were carried out using the CLUSTAL W multiple-sequence alignment program (version 1.7) (21). The amino acid sequences of annotated DAOCSs from different organisms were retrieved from the GenBank database, and the accession numbers were as follows: S. clavuligerus, P18548; A. chrysogenum, P11935; S. chartreusis, AY318743; S. ambofaciens, AY318742; Streptomyces jumonjinensis, AF317908; Nocardia lactamdurans, Q03047; and Lysobacter lactamgenus, CAA39984. Viewing and manipulation of three-dimensional structures were performed using the SwissPdb Viewer program, version 3.7 (10). The crystal structure of ScDAOCS was retrieved from the Protein Data Bank (accession number 1UNB) (22). Amino acid residues interacting with the penicillin substrate were determined using the LIGPLOT program (25). This program automatically generates a schematic diagram of protein-ligand interactions mediated by hydrogen bonds and hydrophobic contacts. Mutational models were generated using SwissPdb Viewer and were further assessed using LIGPLOT to validate the changes in the protein-ligand interactions.

Heterologous expression and purification of wild-type and mutant ScDAOCSs.After the mutation sites were confirmed via full DNA sequencing, recombinant E. coli BL21(DE3) clones harboring the wild-type and mutant ScDAOCS constructs were induced for protein expression. High-level soluble expression of wild-type and mutant ScDAOCS-GST fusion proteins was obtained (∼10% of the total soluble protein), as revealed by intense protein bands (∼60 kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses (data not shown). The cell lysates containing GST-ScDAOCS fusion proteins were then subjected to affinity chromatography purification using the MicroSpin GST purification module. The molecular masses of the purified fusion proteins were approximately 60 kDa, and the proteins were at least 90% pure as estimated by the Quantity One program (data not shown). These results showed that the double mutations did not significantly affect the soluble expression of the enzyme, which allowed subsequent determination of the properties of the mutant enzymes.

Determination of specific activities of wild-type and mutant ScDAOCSs by the hole plate bioassay.The conversion of hydrophobic penicillin substrates (ampicillin, penicillin G, phenethicillin, and carbenicillin) by wild-type and mutant ScDAOCSs was determined by a hole plate bioassay using E. coli strain Ess as the test microorganism. All experimental data were confirmed by at least three independent measurements. The specific activities of wild-type ScDAOCS for ampicillin, penicillin G, phenethicillin, and carbenicillin conversion were 0.64, 6.72, 3.57, and 0.76 U mg⁻¹, respectively. The specific activities of the single- and double-mutant enzymes were expressed as percentages relative to the activity of the wild-type enzyme (Table 2).

Specific activities and substrate specificities of V275I, V275L, C281Y, C281F, and I305M mutants.Wei et al. demonstrated that the V275I and C281Y mutations improved the conversion activity of ScDAOCS only with penicillin G (27). In this study, we explored the basis of this improvement using kinetic studies and showed that the substrate specificities of the enzymes included ampicillin, phenethicillin, and carbenicillin. The improvement observed for the C281Y and I305M single mutants for conversion of different penicillins was greater than that observed for the V275I mutant (Table 2). Compared to the relative activities of other single mutants, the relative activity of C281Y showed the greatest improvement for carbenicillin conversion (572%), followed by the I305M mutant (450%). For ampicillin conversion and phenethicillin conversion, the activities of the N304R mutant were dominant. However, this mutant was only as efficient as the C281F mutant for penicillin G conversion.

Structural analyses of these three residues using SwissPdb Viewer revealed that I305 is approximately 4.5 Å from the β-lactam ring of the penicillin substrate (Fig. 2). After mutation to methionine, the side chain, which is longer than that of isoleucine, is closer to the β-lactam ring of the penicillin substrate (Fig. 3A and 3B). Thus, the methionine residue could potentially stabilize or orient the penicillin substrate in a more receptive manner for ring expansion. However, the molecular mechanisms of V275I and C281Y single mutants that contribute to the increased catalytic activity of the enzyme are still unclear. Since the two sites are not located at the active site of ScDAOCS, it is reasonable to postulate that their effects are structure based.

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FIG. 2.

Spatial orientation of the V275, C281, N304, I305, R306, and R307 residues (orange) in ScDAOCS (PDB accession number 1UNB). Residues V275 and C281 are located in a small subdomain not at the active site. Residues N304, I305, R306, and R307 are located at the C-terminal end which forms part of the β-barrel structure that defines the active site. (Top inset) Close-up of residues V275 and C281. Residue V275 is located on the β13 strand and is surrounded by residues F273, V285, and L287. Residue C281 is located along the polypeptide chain extending between the α9 helix and the β14 strand. (Bottom inset) Modeled structures of (A) V275I, (B) V275L, (C) C281Y, and (D) C281F mutations obtained using SwissPdb Viewer. Brown, ampicillin; purple, α-ketoglutarate; dark green, H183 D185 H243 iron binding motif; light green, R258 S260 α-ketoglutarate binding motif; dotted green line, hydrogen bond.

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FIG. 3.

Mutational models of I305M, N304R, and N304K ScDAOCS mutants generated using SwissPdb Viewer. (A) Orientation of the side chains of residues N304 and I305 with respect to the ampicillin substrate and Fe(II) ion in wild-type ScDAOCS (PDB accession number 1UNB). (B to D) Predicted orientations of (B) the methionine side chain at position 305 and the (C) arginine and (D) lysine side chains at position 304 of ScDAOCS.

Residue V275 is located on the β13 strand and is surrounded by residues F273, V285, and L287 in the polypeptide chain extending from strand β13 to strand β14 (Fig. 2). The interaction of these residues is important in maintaining the integrity of this small subdomain, which extends to the C-terminal end of the enzyme. When V275 was replaced with leucine, which is structurally similar to isoleucine, comparable relative activity was observed. Since multiple-sequence alignment of all annotated DAOCSs showed that the V275 residue is strictly conserved (data not shown), this finding corroborates the hypothesis that an aliphatic residue at this position is important in the interaction with the surrounding residues to maintain the integrity of this subdomain. However, the relative activities of the V275I mutant for conversion of all penicillins are about twofold higher than those of V275L. This suggests that the branching variation in the side chains of isoleucine and leucine at this position is important for influencing the enzyme activity.

Residue C281 is also located within the small subdomain between the α9 helix and the β14 strand (Fig. 2). In silico analyses revealed that when this residue was changed to a tyrosine, a hydrogen bond formed between the hydroxyl group on the side chain of tyrosine and the backbone oxygen of V238. This hydrogen bond could possibly help stabilize the polypeptide chain leading to residue H243, which is critical for iron(II) coordination. This is consistent with the results reported by Wei et al. (26). To further validate the contribution of the hydrogen bond, C281 was changed to phenylalanine, which resembles tyrosine structurally except for the absence of the hydroxyl group on the aromatic ring. Interestingly, the C281F mutant exhibited specific activity similar to that of the C281Y mutant. This implied that the improved activity obtained was not due to the predicted hydrogen bond formation. Since phenylalanine is nonpolar, the interactions of either tyrosine or phenylalanine at position 281 with its surrounding residues are likely to be due to hydrophobic and van der Waals forces.

Comparative analyses of V275I N304X, C281Y N304X, and N304X R306L double mutants.The effects of selected single mutations on the activity of ScDAOCS have been demonstrated in various studies, and two observations were made. The C-terminal residues (i.e., N304, I305, R306, and R307) are close to the penicillin substrate. Thus, mutations at these sites would have a direct effect on enzyme catalysis through interactions with the substrate. In contrast, mutations at the V275 and C281 residues, which are not located at the catalytic site, would have indirect consequences. In the N304 complete library study, it was found that there were five different mutations that enhanced the catalytic activity of ScDAOCS (3). The specific activities of two of the five mutants, the N304R and N304K mutants, for ampicillin conversion were more than twofold greater than the specific activity of the wild-type enzyme, whereas the specific activities of the other mutants (N304A, N304L, and N304M) showed only modest increases (less than twofold). Since N304 of ScDAOCS has been thoroughly studied for beneficial amino acid substitutions, it is a good candidate for investigating the effects of combining such mutations on the catalytic activity of the enzyme and the molecular effects of the mutations on each other. Therefore, two types of mutation combinations were tested; N304X mutations (where X is alanine, leucine, methionine, lysine, or arginine) were combined with distal site mutations (V275I and C281Y), as well as a mutation at a site close to site 304 (R306L).

A comparative analysis of the V275I N304X and C281Y N304X double mutants with the corresponding single mutants revealed that the specific activities of the double mutants were higher than those of the corresponding single mutants for all the substrates tested. The trend observed was that the greatest increases in the conversion activities resulting from double mutations involved the single mutation that resulted in the greatest improvement (i.e., combination of C281Y with N304R or N304K). This suggests that combining relevant improved single mutants can have additive or synergistic effects on the resulting enzymatic capability.

In contrast, some of the N304X R306L mutants differed from the improved V275I N304X and C281Y N304X double mutants. For example, for ampicillin conversion, the relative activities of the N304K R306L (245%) and N304R R306L (256%) double mutants were lower than those of the corresponding single mutants, the N304K (270%) and N304R (346%) mutants. Only slight increases in the specific activity were observed for the N304A R306L (214%) and N304M R306L (194%) double mutants compared to the corresponding single mutants, the N304A (192%), N304M (120%), and R306L (186%) mutants. For phenethicillin conversion, the N304A R306L, N304K R306L, and N304M R306L mutants had higher relative activities than the corresponding single mutants, and for carbenicillin conversion, the N304A R306L, N304K R306L, and N304R R306L double mutants had higher relative activities than the corresponding single mutants. These results suggest that the N304L R306L double mutation was not an ideal combination as the activity of the N304L R306L double mutant for any of the three conversions was lower than that of the corresponding single mutants. Intriguingly, all N304X R306L double mutations were countereffective for penicillin G conversion, and the relative activities were similar to or lower than that of the wild-type enzyme. These results suggested the possibility that one mutation affects the environment of another mutation in close proximity, leading to undesirable effects.

Effects of V275I or C281Y with other C-terminal mutations.Since improvement was obtained by combining the V275I or C281Y mutation with N304X mutations, it can be extrapolated that when V275I and C281Y are combined with other C-terminal mutations (namely, I305M, R306L, and R307L), similar outcomes should be observed. As expected, V275I-C terminus double mutants exhibited further improvement compared with the corresponding single mutants. Although the trend for most C281Y-C terminus double mutants was similar to the trend for the V275I-C terminus double mutants, the C281Y R306L double mutant did not show further improvement in the relative penicillin G conversion activity (191%) compared to the C281Y single mutant (251%). Nevertheless, these results supported the conclusion that the V275I and C281Y mutations increased the ScDAOCS activity when they were combined with other beneficial C-terminal mutations.

Determination of specific activities of wild-type and mutant ScDAOCSs by HPLC.HPLC, a more sensitive and quantitative nonbiological method, was used to confirm the results obtained from bioassays for ampicillin conversion. The substrate (ampicillin) and product (cephalexin) could be discriminated by resolved retention times of 19 and 18 min, respectively (data not shown). The fact that the retention time of the converted product coincided with that of the cephalexin standard authenticated the identity of the product in the enzyme reactions. Furthermore, the chromatographic results demonstrated that only cephalexin was produced by the double-mutant ScDAOCSs (data not shown). These results were comparable to those obtained with bioassays (Table 3).

Kinetic parameters of wild-type and mutant ScDAOCSs for ampicillin conversion.The K_m values of all mutant enzymes were lower than that of the wild-type ScDAOCS (6.94 mM) for ampicillin conversion, indicating that the substrate binding affinities were higher (Table 4). In the single mutants, the N304R (0.26 mM), N304K (0.54 mM), and I305M (1.00 mM) mutations increased the substrate binding affinity of the enzyme most effectively. This property was further enhanced when these mutations were combined with the V275I, C281Y, or R306L mutation.

Interestingly, the I305M mutation doubled the turnover rate of the enzyme (0.104 s⁻¹) compared to that of the wild-type ScDAOCS (0.059 s⁻¹). This is the only mutation that significantly increased the turnover rate of ScDAOCS's ring expansion activity. Similar k_cat values were obtained regardless of the type of mutation with which I305M was combined; the k_cat for V275I I305M was 0.105 s⁻¹, and the k_cat for C281Y I305M was 0.122 s⁻¹. However, the other mutations affected the turnover rate of ScDAOCS to various degrees. It was observed that the double mutants with the R306L mutation had the lowest k_cat values (0.026 s⁻¹ for the N304M R306L mutant and 0.037 s⁻¹ for both the V275I R306L and C281Y R306L mutants). In contrast, the R306L single mutation (0.050 s⁻¹) did not affect the turnover rate of the ring expansion activity as much as the double mutations. In the crystal structure of ScDAOCS, the side chain of the R306 residue that is oriented away from the catalytic site is surrounded by five hydrophobic residues, which help preserve the interaction between the β7 (Y184 and L186), α10 (W297 and I298), and β15 (V303) strands (9, 22, 23). This hydrophobic cleft is postulated to be important for stabilizing the integrity of the iron(II) binding motif (H183 D185 H243). Hence, in the N304X R306L double mutants, the N304X mutations probably influence the environment of R306L and affect the integrity of this hydrophobic cleft. This, in turn, may destabilize the iron binding platform, leading to a lower turnover rate.

In general, the k_cat/K_m values of all double-mutant ScDAOCSs were higher than that of the wild-type enzyme for ampicillin conversion. Four double mutants, C281Y N304R, C281Y I305M, V275I N304R, and C281Y N304K, showed the greatest improvement in the enzyme's catalytic efficiency (101-, 68-, 42-, and 42-fold increases, respectively). These kinetic data supported the hypothesis that the ring expansion activity of these double mutants is superior.