1 Departments of Chemistry and 2 Biochemistry, University of Minnesota, Minneapolis, MN 55455, USA
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Abstract |
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Keywords: aminotransferase/enzyme design/fatty acid binding protein/pyridoxamine/semisynthetic enzyme
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Introduction |
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Versatile cofactors found in nature are pyridoxal phosphate (PLP) and pyridoxamine phosphate (PAP), which catalyze reactions such as transamination, decarboxylation, racemization and aldol condensation. To achieve highly efficient catalysis, interactions with neighboring functional groups are critical. In PLP-dependent enzymes, two highly conserved amino acid residues are found. First, a lysine, which forms, via its -amino group, a Schiff base linkage to the aldehyde group of PLP and interacts in several catalytic steps with the reactive intermediates. Second, a carboxylate side chain from aspartate or glutamate is positioned within hydrogen bonding distance to the pyridine-nitrogen of PLP (Goldberg and Kirsch, 1996
; Hayashi et al., 1998
). Thus, the positive charge in the aromatic ring is stabilized, forming an electron sink. Substitution of one of these residues via mutagenesis results in enzyme mutants with virtually no catalytic activity (Yano et al., 1992
; Malashkevich et al., 1996).
A prerequisite to position functional groups around a catalytic center in an artificial enzyme is a sphere-shaped scaffold that is easily manipulated. Fatty acid binding proteins are a well-characterized family of intracellular transport proteins involved in lipid metabolism (Banaszak et al., 1994; Veerkamp and Maatman, 1995
). These proteins bind single fatty acid molecules in a cavity consisting of a ß-barrel and a flexible
-helical lid. Intestinal fatty acid binding protein from rats (IFABP) is a cysteine-free, 15 kDa protein with a cavity volume of 600 Å3. Its established structure (Scapin et al., 1992
; Hodson et al., 1996
) and the convenient overexpression of mutants in Escherichia coli make IFABP a conceptually simple framework for the design and assembly of an active site.
Recently, we have demonstrated that a pyridoxal group (Px) can be tethered inside an IFABP cavity with a lysine mutation specifically designed to form a Schiff base with the cofactor (IFABP-PxK38 and IFABP-PxK51; Figure 1) (Häring and Distefano, 2001
).
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Materials and methods |
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Pyridoxamine dihydrochloride was converted into 1-methyl-5-(2-pyridyldithio)pyridoxamine as described previously (Kuang et al., 2000). The mutagenic primers were obtained from the Microchemical Facility of the Institute of Human Genetics (University of Minnesota). The site-directed mutagenesis kit (Quick-Change) was obtained from Stratagene. The expression vector pMON-IFABP was used previously for the preparation of IFABP-V60C (Jiang and Frieden, 1993
; Frieden et al. 1995
; Kuang and Distefano, 1998
). Protein purification was carried out at 4°C unless noted otherwise.
Modeling
Molecular modeling experiments were performed with InsightII 97.0/Discover 3.00 (Molecular Simulations). The potential energy was calculated on basis of a consistent valence forcefield and minimized sequentially by steepest descents and conjugate gradients. During the optimization process, the protein backbone and residues were fixed, except the mutated residues V60C, L38K, E51K or F93E.
Mutagenesis and expression of IFABP mutants
Site-directed mutations were introduced in the expression vector of pMON-IFABP recombinant plasmid with the QuickChange mutagenesis kit. In general, the parental plasmid was subjected to temperature cycling using the non-strand-displacing action of Pfu DNA polymerase to extend and incorporate the primers resulting in nicked circular strands. The non-mutated, dam-methylated DNA template was digested with DpnI endonuclease. Finally, the circular, nicked plasmid was transformed in supercompetent cells. The sequence for the mutagenic oligomers was 5'-CATGACAACTTGAAAAAGACGATCACACAGGAAG-3' for L38K, 5'-AAATAAATTCACAGTCAAAAAATCAAGCAACTTC-3' for E51K and 5'-GAAATAAACTTGTTGGAAAAGAGAAACGTGTAGACAATG-3' for F93E. The mutations were confirmed by sequencing of the complete cDNA encoding the IFABP mutant. The proteins were overexpressed in E.coli strains JM105, which were grown under shaking at 37°C in Luria-Bertani (LB) media (6x1 l) with 100 mg/l ampicillin. At an OD of 0.4 the recA promoter was induced with nalidixic acid (50 mg/l), the cells grown for an additional 3.5 h and harvested by centrifugation (15 min at 3600 g). The cell paste was resuspended in 50 ml of 25 mM imidazoleHCl, pH 7.0, 50 mM NaCl, 5.0 mM EDTA, 10 mM 2-mercaptoethanol and 0.5 mM PMSF; cell lysis was accomplished by sonication (10x30 s, 50% duty cycle, at 0°C).
Purification of IFABP-V60C/E51K
This IFABP mutant protein was found in the supernatant after centrifugation (30 min at 20 400 g) of the cell lysate. Solid (NH4)2SO4 was slowly added to a final saturation of 60% at 0°C and the solution was equilibrated for 1 h. The suspension was centrifuged (15 min at 10 000 g) and the pellet discarded. The supernatant which contained the IFABP was extensively dialyzed in buffer A (20 mM potassium phosphate, pH 7.3, 1.0 mM EDTA and 1.0 mM DTT) to remove excess ammonium sulfate. The resulting protein solution was concentrated to 10 ml by ultrafiltration (Amicon YM3 filter) and applied to a QAE Sephadex A-50 ion exchange column pre-equilibrated in buffer A. The IFABP mutant eluted with this buffer immediately after the dead volume. After ultrafiltration to a volume of 5.0 ml, the sample was further purified on a Sephacryl S-100 gel filtration column using 20 mM HEPES, pH 7.5, 250 mM NaCl, 1.0 mM EDTA and 1.0 mM DTT (buffer B). The fractions containing the pure IFABP (as determined by SDSPAGE) were pooled and concentrated by ultrafiltration to 5.0 ml. Based on a dye-binding assay (Bio-Rad) with BSA as a standard, 80 mg of IFABP-V60C/E51K were obtained.
Purification of IFABP-V60C/L38K
This IFABP mutant protein was obtained as inclusion bodies, which were separated after cell lysis by centrifugation at 3000 g for 20 min. They were pelleted twice (3000 g for 20 min) and resuspended in buffer C (0.5 M urea, 50 mM Tris, pH 7.5, 10 mM EDTA, 5.0 mM DTT and 1.0% Triton X-100) and a further twice in buffer C without urea and Triton X-100. The white pellet was solubilized in 20 ml of 3 M guanidine in buffer D (20 mM HEPES, pH 7.5, 10 mM EDTA and 10 mM DTT). Undissolved particles were removed by centrifugation (15 000 g for 20 min). Refolding was achieved by dropping (2.0 ml/h) the protein solution in a 25-fold volume of buffer D containing 250 mM NaCl. Small amounts of precipitated protein were removed by centrifugation and the solution was concentrated by ultrafiltration. The IFABP mutant was finally isolated after gel filtration chromatography with a Sephacryl S-100 column using buffer B for elution. The fractions containing the pure IFABP were pooled and concentrated by ultrafiltration to 7 ml. Based on a dye-binding assay (Bio-Rad) with BSA as standard, 70 mg of IFABP-V60C/E51K were obtained.
Purification of IFABP-V60C/E51K/F93E and IFABP-V60C/L38K/F93E
Both of these IFABP mutants were expressed as inclusion bodies and washed with buffer C and solubilized in buffer D as described above. However, various attempts to refold the denatured proteins yielded large amounts of precipitate. In each case, <3 mg of pure protein were isolated from a 6 l culture.
Conjugation with MPx
Prior to conjugation, DTT was removed from the IFABP mutants by gel filtration (Bio-Gel P6-DG, 20 mM HEPES, pH 7.5) and the concentration of free thiol was determined by titration with 5,5'-dithiobis(2-nitrobenzoic acid) as described previously (Kuang et al., 1996). The conjugation reagent 1-methyl-5-(2-pyridyldithio)pyridoxamine (Kuang et al., 2000
) was added in 10-fold excess and the reaction was allowed to proceed for 20 h at 20°C in 20 mM HEPES (pH 7.5). The excess of conjugation reagent was removed by gel filtration (Bio-Gel P6-DG, 20 mM HEPES, pH 7.5) and the concentration of the conjugated pyridoxamine determined by its UV absorbance (
338 = 6210 M1 cm1 in 5.25 M guanidine, 20 mM HEPES, pH 7.5).
Catalytic transamination reactions
All reactions were performed in a total volume of 100 µl at 37°C. The amount and enantiomeric distribution of glutamate were determined by RP-HPLC with a fluorescence detector after derivatization with N-acetyl-L-cysteine and o-phthaldialdehyde (Buck and Krummen, 1987; Kuang et al., 1996
). For the MichaelisMenten analysis, the rate data were directly fitted to the standard expression using a non-linear least-squares algorithm.
pH profiles
The reactions were performed with 50 µM catalyst, 5 mM phenylalanine and 50 mM -ketoglutarate acid in MHP buffer (25 mM HEPES, 25 mM MES, 50 mM 4-hydroxy-N-methylpiperidine, 0.1 M KCl) with a constant ionic strength of I = 0.2 in the pH range studied (Gloss and Kirsch, 1995
). The formation of the glutamate was monitored by HPLC as described above and the data points of the initial rate were fitted to a bell-shaped curve.
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Results |
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To mimic the cationic pyridoxamine electron sink feature present in most transaminases, we first explored the possibility of placing an Asp or Glu residue in the active site of our artificial transaminase IFABP-Px (Route A in Figure 2). As guidance to identify a suitable position in the IFABP cavity, the active site geometry of native aminotransferases (AT) was studied. Several crystal structures of D- and L-aspartate AT as well as aromatic amino acid AT in their pyridoxal, Schiff base and glutamate ketimine form are known. Several geometric parameters, which define the position of the Asp or Glu in the active site of these enzymes, were selected, measured and are presented in Table I
.
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Based on the molecular modeling studies, the IFABP mutants V60C/L38K, V60C/E51K, V60C/L38K/F93E and V60C/E51K/F93E were prepared by site-directed mutagenesis. The proteins were overexpressed in E.coli on a 6 l scale. After cell lysis, only V60C/E51K was found in the supernatant, whereas the other mutant proteins were expressed as inclusion bodies. To purify IFABP-V60C/E51K a literature protocol (Sacchettini et al., 1990) was optimized using ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography. The overall yield was
80 mg of protein.
The brown inclusion bodies of the other mutants were separated from most other cellular debris by fractional centrifugation. Most impurities could be removed by repeated suspension and repelletting in a washing buffer. The resulting white pellet was finally dissolved in 3 M guanidine and refolded by slowly dropping the concentrated protein solution into a large volume of buffer. However, IFABP-V60C/L38K/F93E and -V60C/E51K/F93E precipitated heavily within a few hours. Attempts with 0.5 M guanidine, 0.5 M guanidine10% sucrose or 0.5 M NaCl-containing refolding buffers and also a variety of dialysis procedures resulted in large amounts of precipitated IFABP protein. Only IFABP-V60C/L38K was refolded to a stable protein and further purified by gel filtration chromatography. It was possible to attach the pyridoxamine cofactor efficiently to the IFABP-V60C/L38K/F93E and -V60C/E51K/F93E proteins under denaturing conditions. However, the resulting conjugates were not stable upon removal of the denaturant. Efforts were then made to study reactions catalyzed by these constructs in the presence of 0.5 M guanidine. Unfortunately, preliminary experiments with the parent system, IFABP-Px in the presence of guanidine, manifested significant reductions in the reaction rate and enantioselectivity. For these reasons, the IFABP-V60C/L38K/F93E and -V60C/E51K/F93E proteins were not studied further.
In earlier work, we demonstrated that a pyridoxamine cofactor can be tethered via a disulfide bond to C60 in the IFABP mutants. In order to introduce an N-methylated pyridoxamine derivative (Equation 1):
| (1) |
a synthesis of a suitable conjugation reagent has recently been developed (Kuang et al., 2000). Starting from commercially available pyridoxamine, 1-methyl-5-(2-pyridyldithio)pyridoxamine was synthesized in seven steps. After removing DTT from the purified IFABP-V60C/L38K and -V60C/E51K by gel filtration, a 10-fold excess of the conjugation reagent was added and the reaction allowed to proceed for 20 h at room temperature. The artificial transaminases IFABP-MPxK38 and IFABP-MPxK51 were finally isolated by gel filtration chromatography. The UV spectrum of the resulting conjugates showed the absorption bands of both the protein (280 nm) and the bound MPx (338 nm). The elution volumes obtained by gel filtration of the unconjugated and conjugated proteins were identical, suggesting that the overall folding of these chemically modified proteins resembles the native state.
Catalytic activity
Pyridoxamine-derived cofactors catalyze the transamination reaction between -amino and
-keto acids (Equation 2
):
| (2) |
Accordingly, the conjugates IFABP-MPxK38 and IFABP-MPxK51 were able to catalyze the transamination reaction between -ketoglutarate and various amino acids (Table II
). The unconjugated IFABP mutants showed no catalytic activity. Within 24 h of reaction time up to 17 catalytic turnovers were achieved, yielding L-glutamate with up to 96% ee as determined by HPLC. The artificial transaminases are also selective for the amino acid substrate, as was demonstrated by higher turnovers using L- instead of D-tyrosine.
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Discussion |
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First, we used the classical means of protein engineering and developed computational models of suitable IFABP mutants. In order to create an active site with an architecture similar to native aminotransferases, important geometric parameters were obtained from published AT crystal structures (Table I). With these data as guidelines, the neighborhood of pyridoxamine was searched for C
positions suitable for the introduction of an aspartate or glutamate. Only the mutation F93E resulted in a close positioning of a carboxylic group close to N-1 of the cofactor. The residue F93 is variable in the fatty acid binding protein family, e.g. Asn, Ser, Glu or Val are found in this position (Banaszak et al., 1994
; Veerkamp and Maatman, 1995
).
The IFABP mutants V60C, V60C/L38K and V60C/E51K have been overexpressed and prepared earlier and proven to be stable. However, when the F93E mutation was introduced into the double mutant background, both IFABP-V60C/L38K/F93E and -V60C/E51K/F93E were unstable. They were overexpressed in E.coli as inclusion bodies in quantities similar to IFABP-V60C/L38K. Attempts to refold them under various conditions resulted in large amounts of precipitated protein. Even highly diluted protein solutions showed precipitation within a few hours. An explanation might be linked to the high hydrophobicity of the IFABP cavity, which consists of only 2125% hydrophilic surface area residues (Likic and Prendergast, 1999); there are no anionic residues within the IFABP cavity. The change of a hydrophobic Phe to the negative Glu was apparently very destabilizing. While it was possible to prepare Px conjugates of these proteins under denaturing conditions, they could not be refolded to yield soluble products.
The only solution that genetic protein engineering offers to these problems is searching for other suitable mutations. Molecular modeling revealed no other promising mutation with a natural amino acid. At this point, the flexibility of our enzyme design concept offered an interesting alternative. In order to stabilize the positive charge in the pyridoxamine ring system, the cofactor itself can be tailored. The chemical N-methylation results in a permanent positive charge at N-1. Recently, a synthesis for a conjugation reagent has been developed to allow for the linkage of an N-methylated pyridoxamine (MPx) to a cysteine residue (Kuang et al., 2000). In model reactions with 1-methyl-3-hydroxy-4-formylpyridinium iodide, the quaternization of the nitrogen has enhanced the rate of transamination 20-fold compared with 3-hydroxy-4-formylpyridine (Maley and Bruice, 1970
). However, when native aspartate aminotransferase was reconstituted with N-methylated pyridoxal phosphate, less than 0.2% of the original activity was found (Furbish et al., 1969
; Fonda, 1971
).
Molecular modeling studies showed that the N-methylated pyridoxamine has a very similar orientation to the non-methylated cofactor. However, in all optimized models the N-methyl group was oriented out of the aromatic plane. As shown in Figure 4, this methyl group points toward the plane of the phenyl ring of F93. Such an unfavorable conformation indicates a compression of the pyridoxamine moiety due to limited space. When F93 was substituted by alanine and the conformation optimized again, the N-methyl group assumed a position coplanar with the pyridine ring, as illustrated in Figure 7
.
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When acidic or basic groups are introduced in a catalytic site, their influence can be seen in the pH dependence of the reaction rate. Previously, the positioning of a lysine residue close to the pyridoxamine in IFABP-PxK38 or -PxK51 resulted in a bell-shaped pH profile of the initial rates. The pKa1 and pKa2 of these curves were similar to those of native transaminases. The conjugate without such a lysine mutation (IFABP-Px) displayed a linear dependence of the rate versus pH (Table IV). The IFABP conjugates with the methylated pyridoxamine described here also showed a bell-shaped pH profile for the initial rate, demonstrating the influence of the lysine mutation in position 38 or 51. As a consequence of chemical modifications of the cofactor, a shift towards higher pH values was observed with these new constructs. Interestingly, there are subtle differences in the rate versus pH and enantiomeric excess versus pH profiles (Figure 5
) for the two different constructs. This probably reflects the differential positioning of K38 and K51 relative to the pyridoxamine cofactor. Molecular modeling of IFABP-PxK38 indicates that the
-amino group from K38 can interact with the
-ketoglutaratepyridoxamine complex at several positions whereas the
-amino group from K51 is closer to C-1 and C-2 of the bound
-ketoglutarate. These observations also suggest a reason for why the IFABP-PxK38 conjugate has a higher kcat' value: the
-amino group from K38 is better positioned to assist in several key steps in the catalytic reaction owing to its closer proximity to the pyridoxamine moiety.
In summary, two artificial transaminases were assembled by linking an N-methylated pyridoxamine derivative within an engineered fatty acid binding protein. The goal of mimicking a native transamination site by stabilizing a cationic pyridoxamine ring system was approached using two completely different routes. First, the IFABP scaffold was tailored to position a carboxylate group close to the pyridine nitrogen. When these mutants proved to be unstable, the cofactor was chemically modified. By N-methylation of the pyridoxamine, a cationic cofactor was created. Although only modest increases were obtained, the kcat values and the turnover numbers that are reported for these conjugates are the highest achieved to date in our system. This outcome was only made possible by the unique flexibility of the underlying concept. Our results suggest that a combination of both genetic and chemical methods used in concert provide the greatest chance for success in enzyme design.
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Notes |
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Acknowledgments |
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References |
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Received September 2, 2001; revised February 25, 2002; accepted April 1, 2002.