Exploring routes to stabilize a cationic pyridoxamine in an artificial transaminase: site-directed mutagenesis versus synthetic cofactors

Dietmar Häring1, Mason R. Lees2, Leonard J. Banaszak2 and Mark D. Distefano1,3*

1 Departments of Chemistry and 2 Biochemistry, University of Minnesota, Minneapolis, MN 55455, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two artificial transaminases were assembled by linking a 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 different strategies. First, the scaffold of intestinal fatty acid binding protein (IFABP) was tailored by molecular modeling and site-directed mutagenesis to position a carboxylate group close to the pyridine nitrogen of the cofactor. When these IFABP mutants (IFABP-V60C/L38K/E93E and -V60C/E51K/E93E) proved to be unstable, a second approach was explored. By N-methylation of the pyridoxamine, a cationic cofactor was created and tethered to Cys60 of IFABP-V60C/L38K and -V60C/E51K; this latter strategy had the effect of permanently installing a positive charge on the cofactor. These chemogenetic assemblies catalyze the transamination between {alpha}-ketoglutarate and various amino acids with enantioselectivities of up to 96% ee. The pH profile of the initial rates is bell shaped and similar to native aminotransferases. The kcat values and the turnover numbers for these new constructs are the highest achieved to date in our system. This success was only made possible by the unique flexibility of the underlying enzyme design concept employed, which permits full control of both the protein scaffold and the catalytically active group.

Keywords: aminotransferase/enzyme design/fatty acid binding protein/pyridoxamine/semisynthetic enzyme


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The construction of designed biocatalysts is the ultimate challenge to our understanding of enzymes. Various strategies have been developed to create enzyme mimics; some examples include fully synthetic active site mimics, cyclodextrin-based catalysts, imprinted polymers and catalytic antibodies. We are using a combination of chemical and genetic protein engineering to assemble novel biocatalysts (Distefano et al., 1998Go). In this strategy, a desired catalytically active group is chemically tethered within a protein cavity, which has been tailored by genetic engineering. This approach is based on a combination of several well established techniques: molecular modeling provides the theoretical design, organic synthesis creates a desired cofactor and site-directed mutagenesis tailors the protein cavity.

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 {varepsilon}-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, 1996Go; Hayashi et al., 1998Go). 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., 1992Go; 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., 1994Go; Veerkamp and Maatman, 1995Go). These proteins bind single fatty acid molecules in a cavity consisting of a ß-barrel and a flexible {alpha}-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., 1992Go; Hodson et al., 1996Go) 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 1Go) (Häring and Distefano, 2001Go).



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 1. Computational model of IFABP-PxK51 developed from the crystal structure of IFABP. The pyridoxamine cofactor is tethered by a disulfide bond to C60, which has been introduced by site-directed mutagenesis. K51 forms a Schiff base with the aldehyde group of the cofactor.

 
Here we explore two routes to stabilize a positive charge in the pyridoxamine ring, using either genetic or chemical engineering. The goal of these studies was to incorporate a second feature of native transaminases—the cationic pyridoxamine acting as electron sink—into this artificial transaminase. The unique flexibility of our design concept opens two routes to achieve this goal. The ‘classical’ protein engineering path can be taken by introducing a carboxylate group via mutagenesis into the IFABP cavity in an appropriate position for hydrogen bonding to the pyridine nitrogen (Figure 2AGo). However, our strategy allows not only for full control of the scaffold, but also for the cofactor. Using a synthetic N-methylated pyridoxamine derivative with a permanent positively charged ring would result in a similar electron sink effect (Figure 2BGo). Here we describe the results of experiments designed to explore both of these approaches.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 2. Two routes to a stabilized positive charge in the pyridoxamine ring. (A) Site directed mutagenesis; (B) synthetic cofactor.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

Pyridoxamine dihydrochloride was converted into 1-methyl-5-(2-pyridyldithio)pyridoxamine as described previously (Kuang et al., 2000Go). 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, 1993Go; Frieden et al. 1995Go; Kuang and Distefano, 1998Go). 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 imidazole–HCl, 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 SDS–PAGE) 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., 1996Go). The conjugation reagent 1-methyl-5-(2-pyridyldithio)pyridoxamine (Kuang et al., 2000Go) 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 ({varepsilon}338 = 6210 M–1 cm–1 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, 1987Go; Kuang et al., 1996Go). For the Michaelis–Menten 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 {alpha}-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, 1995Go). 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Molecular modeling

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 2Go). 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 IGo.


View this table:
[in this window]
[in a new window]
 
Table I. Critical geometric parameters for the placement of a glutamic acid residue near pyridoxamine in a transamination catalysta
 
The molecular modeling studies for the artificial enzymes were based on the crystal structure of IFABP (Scapin et al., 1992Go). Previously, the structures of a IFABP-V60C/L38K and -V60C/E51K with a pyridoxamine group bound to C60 have been modeled and the conjugates synthesized (Häring and Distefano, 2001Go). The neighborhood of the pyridine-nitrogen in IFABP-PxK38 and IFABP-PxK51 was searched for C{alpha}-atoms, which have a relative orientation similar to the C{alpha}-atoms of Asp or Glu in the active site of native aminotransferases. This search revealed only two suitable positions in the backbone of IFABP, namely C{alpha} of Y70 and F93. We introduced in both positions Asp as well as Glu and optimized their conformation. The Y70D and Y70E mutations put the carboxylic acid clearly above the plane of the pyridine ring and made hydrogen bonding with the pyridine nitrogen unfavorable. Both F93D and F93E revealed an acceptable orientation of the carboxylate, with the glutamate being closer to the nitrogen than the aspartate. The final geometric parameters of the IFABP-PxK38 and -PxK51 with the F93E mutation are similar to those of the native archetypes (see Table IGo). Figure 3Go provides a stereoview of the active site of aspartate aminotransferase in comparison with the model for IFABP-PxK51/E93.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Comparison of the active site of native aspartate aminotransferase (top, crystal structure showing D222 and K258) (Okamoto et al., 1994Go) and IFABP-Px-E51K/F93E (bottom, model; both in the Schiff base form). The carboxylic groups of Asp or Glu can form hydrogen bonds to the aromatic nitrogen of the pyridoxamine moiety as indicated by the lines (aspartate AT D222–PLP 3.20 Å; IFABP-Px-E51K/F93E Glu51–Px 3.35 Å).

 
A second approach to stabilize a positive charge in the pyridoxamine ring is via N-methylation (Route B in Figure 2Go). The synthesis of a cationic pyridoxamine derivative (MPx) suitable for conjugation to C60 in IFABP has been developed recently (Kuang et al., 2000Go). The cofactor in the computer models of IFABP-PxK38 and -PxK51 was N-methylated and the conformation optimized. The overall orientation of the cationic ring is very similar to that of the pyridoxamine without the N-methyl group as shown in Figure 4Go. However, the pyridine ring is shifted ~1 Å away from F93 and the N-methyl group points out of the plane of the heterocyclic ring owing to unfavorable steric interactions between the N-methyl group and the phenyl ring of F93.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4. Comparison of IFABP-MPxK38 (black) and IFABP-PxK38 (grey) (both in the pyridoxamine form). The N-methylated cofactor is shifted 0.7 Å towards K38 (top) owing to the proximity of F93 (bottom). In IFABP-MPxK38 the distance K38–MPx is 2.78 Å and F93–MPx is 3.31 Å, as indidicated by the lines.

 
Preparation and conjugation of IFABP mutants

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., 1990Go) 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 guanidine–10% 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 1Go):


(1)

a synthesis of a suitable conjugation reagent has recently been developed (Kuang et al., 2000Go). 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 {alpha}-amino and {alpha}-keto acids (Equation 2Go):


(2)

Accordingly, the conjugates IFABP-MPxK38 and IFABP-MPxK51 were able to catalyze the transamination reaction between {alpha}-ketoglutarate and various amino acids (Table IIGo). 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.


View this table:
[in this window]
[in a new window]
 
Table II. Catalytic transamination of {alpha}-ketoglutarate and various amino acids catalyzed by IFABP-MPx conjugates (5.0 mM amino acid, 50 mM {alpha}-ketoglutarate, 50 µM catalyst in 0.2 M HEPES, pH 7.5, at 37°C); the reactions were stopped after 24 h and the amount and enantiomeric distribution of glutamate determined by HPLC
 
The kinetic parameters for the reaction of {alpha}-ketoglutarate and phenylalanine were determined and the values are summarized in Table IIIGo. The concentration of the keto acid was varied while Phe was held at a constant, saturating concentration. The initial rates were determined by quantifying the amount of produced glutamate by HPLC. These rates followed saturation kinetics and were fitted to a standard Michaelis–Menten equation. Inspection of the kinetic parameters for these new constructs and comparison with earlier data reveal several interesting trends. First, the new constructs IFABP-MPxK38 and -MPxK51 have increased values for kcat (1.12 and 0.52 h–1) that are 1.2–2.5-fold higher compared with the conjugates containing the non-methylated cofactor and lysine mutations. In contrast, in the conjugates that lack K38 or K51, N-methylation has little effect on kcat (IFABP-Px, kcat = 0.29 h–1; IFABP-MPx, kcat = 0.23 h–1) Thus, introduction of the N-methylated cofactor increases the overall reaction rate only when the Schiff base forming lysine residues are present. Accompanying the increases in kcat for IFABP-MPxK38 and -MPxK51 were significant increases (17–36-fold) in Km compared with conjugates lacking the N-methylated cofactor. Such an increase was also observed with the conjugates lacking the active site lysine residues (IFABP-Px and-MPx).


View this table:
[in this window]
[in a new window]
 
Table III. Kinetic parameters for the transamination catalyzed by N-methylated pyridoxamine moieties in different environmentsa
 
As noted in our earlier work (Häring and Distefano, 2000), the rate of transamination and the enantioselectivities in pyridoxamine-mediated reactions are pH-dependent processes. The pH profiles were determined for the reaction of {alpha}-ketoglutarate and phenylalanine in a buffer with constant ionic strength over the studied pH range. The initial rates were plotted against the pH and the data were fitted to a bell-shaped curve with pKa1 and pKa2 as illustrated in Figure 5Go; the values for pKa1 and pKa2 are summarized in Table IVGo. The plot of the enantiomeric ratio of the produced glutamate versus pH could not be fitted to such a curve and the table lists only the experimentally determined maxima. The values for pKa1 and pKa2 of IFABP-MPx38 and -MPx51 are similar to those of native aspartate aminotransferase. Interestingly, they are both shifted to higher pH in comparison with the non-methylated IFABP-Px conjugates.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5. pH profiles of the initial rates and the enantiomeric ratios (e.r.) of IFABP-MPx (top), IFABP-MPxK38 (middle) and IFABP-MPxK51 (bottom).

 

View this table:
[in this window]
[in a new window]
 
Table IV. The initial rate and enantiomeric ratio of IFABP-conjugate catalyzed transaminations depend on the pH value
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mechanism of a variety of pyridoxamine-dependent transaminases was studied in detail and is summarized in Figure 6Go. The lysine and the aspartate or glutamate residues interacting with the cofactor are highly conserved throughout native aminotransferases (Banaszak et al., 1994Go; Veerkamp and Maatman, 1995Go). The {varepsilon}-amino group of lysine can form a Schiff base with the aldehyde intermediate during the catalytic cycle and interacts in several steps with the substrate and the cofactor. The carboxylic acid group of an Asp or Glu is positioned within salt-bridge formation or hydrogen-bonding distance to the aromatic nitrogen of the cofactor. Its negative charge stabilizes the positive charge at N-1 and hence enhances the ‘electron sink’ effect of the pyridoxamine. This is critical for the removal of the {alpha}-proton of the amino acid substrate (Yano et al., 1992Go).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 6. Simplified half-reaction of the catalytic transamination cycle. For more detailed information, see the literature (Goldberg and Kirsch, 1996Go; Hayashi et al., 1998Go).

 
In previous work, an artificial aminotransferase has been assembled by tethering a pyridoxamine derivative inside the large cavity of a mutated fatty acid binding protein. Using site-directed mutagenesis, a cysteine residue (C60) was introduced as a point of attachment (Kuang and Distefano, 1998Go) and a lysine mutation was made capable of forming a Schiff base linkage to the pyridoxal group (Häring and Distefano, 2000). Here we explored two routes to stabilize a positive charge in the pyridoxamine ring using either genetic or chemical engineering.

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 IGo). With these data as guidelines, the neighborhood of pyridoxamine was searched for C{alpha} 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., 1994Go; Veerkamp and Maatman, 1995Go).

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 21–25% hydrophilic surface area residues (Likic and Prendergast, 1999Go); 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., 2000Go). 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, 1970Go). 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., 1969Go; Fonda, 1971Go).

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 4Go, 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 7Go.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7. The N-methyl group in IFABP-MPxK51 (black) is not in the plane of the aromatic ring. When F93 (left) is substituted with alanine, the N-methylated pyridoxamine stretches into a planar, relaxed conformation (grey). The models show the aldimine form of pyridoxal with {alpha}-ketoglutarate and K51 on the right side.

 
The limited space within the cavity for the cofactor and especially its substrate ketimine form might be the reason for the high Km values of IFABP-MPxK38 and -MPxK51. These values were up to 37-fold higher than those for the non-methylated IFABP-PxK38 and -PxK51 (Table IIIGo). Introduction of the N-methylated cofactor into the mutant IFABP-V60C/L38K produced a conjugate with a kcat of 1.1 h–1. This value is 36-fold greater than the value for the free cofactor (MPx) in the absence of the protein scaffold and is the highest turnover number we have obtained to date in these designed systems. Owing to the high Km values, the overall efficiency (kcat/Km) of this generation of catalysts has not improved. However, the turnover numbers after 24 h of reaction with various amino acid substrates confirm the high kcat values, because the enzymes are saturated with substrates under the given conditions. This is particularly exciting because our earlier efforts to improve catalytic efficiency generally resulted in accomplishing this via decreases in Km. Increases in kcat have been more difficult to obtain. It is also interesting that the augmentation in kcat that we report here appears to require the presence of the catalytic lysine (K38 or K51). Earlier work in which the N-methylated cofactor was introduced into IFABP-V60C, a protein lacking engineered lysine residues, resulted in no increase in kcat (Table IIIGo) (Kuang et al., 2000Go). Hence there appears to be some functional synergy between N-methylation and lysine introduction. Although the increases reported here are modest, the fact that they were obtained only via the chemical modification approach makes them particularly interesting. In addition, it is likely that additional rounds of design (e.g. by incorporating an F93A mutation) will further increase the catalytic power of these semisynthetic constructs.

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 IVGo). 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 5Go) 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 {varepsilon}-amino group from K38 can interact with the {alpha}-ketoglutarate–pyridoxamine complex at several positions whereas the {varepsilon}-amino group from K51 is closer to C-1 and C-2 of the bound {alpha}-ketoglutarate. These observations also suggest a reason for why the IFABP-PxK38 conjugate has a higher kcat' value: the {varepsilon}-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.


    Notes
 
3 To whom correspondence should be addressed. E-mail: distefan{at}chem.umn.edu Back


    Acknowledgments
 
We thank D.P.Cistola for providing pMON-IFABP. The Minnesota Supercomputing Institute provided computer time for the modeling. This work was supported by the National Science Foundation (CHE9807495) and the National Institutes of Health (GM13925.) D.Häring gratefully acknowledges a fellowship from the Deutsche Akademische Austauschdienst (NATO-Programm).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Banaszak,L.J., Winter,N., Xu,Z., Bernlohr,D.A., Cowan,S. and Joner,T.A. (1994) Adv. Protein Chem., 45, 89–151.[ISI][Medline]

Buck,R.H. and Krummen,K. (1987) J. Chromatogr., 387, 255–266.[CrossRef][Medline]

Distefano,M.D., Kuang,H., Qi,D. and Mazhary,A. (1998) Curr. Opin. Struct. Biol., 8, 459–465.[CrossRef][ISI][Medline]

Fonda,M.L. (1971) J. Biol. Chem., 246, 2230–2240.[Abstract/Free Full Text]

Frieden, C., Jiang, N. and Cistola, D.P. (1995) Biochemistry, 34, 2724–2730.[ISI][Medline]

Furbish,F.S., Fonda,M.L. and Metzler,D.E. (1969) Biochemistry, 8, 5169–5180.[ISI][Medline]

Gloss,L.M. and Kirsch,J.F. (1995) Biochemistry, 34, 3990–3998.[ISI][Medline]

Goldberg,J.M. and Kirsch,J.F. (1996) Biochemistry, 35, 5280–5291.[CrossRef][ISI][Medline]

Häring,D. and Distefano,M.D. (2001) Bioconjug. Chem., 12, 385–390.[CrossRef][ISI][Medline]

Hayashi,H., Mizuguchi,H. and Kagamiyama,H. (1998) Biochemistry, 37, 15076–15085.[CrossRef][ISI][Medline]

Hodson,M.E., Ponder,J.W. and Cistola,D.P. (1996) J. Mol. Biol., 264, 585–602.[CrossRef][ISI][Medline]

Jiang, N. and Frieden, C. (1993) Biochemistry, 32, 11015–11021.[ISI][Medline]

Kuang,H. and Distefano,M.D. (1998) J. Am. Chem. Soc., 120, 1072–1073.[CrossRef][ISI]

Kuang,H., Brown,M.L., Davies,R.R., Young,E.C. and Distefano,M.D. (1996) J. Am. Chem. Soc., 118, 10702–10706.[CrossRef][ISI]

Kuang,H., Häring,D., Mazhary,A. and Distefano, M.D. (2000) Bioorg. Med. Chem. Lett., 10, 2091–2095.

Likic,V.A and Prendergast,F.G. (1999) Protein Sci., 8, 1649–1657.[Abstract]

Malashkevich,V.N., Strokopytov,B.V., Borisov,V.V., Dauter,Z., Wilson,K.S. and Torchinsky,Y.M. (1995a) J. Mol. Biol., 247, 111–124.[CrossRef][ISI][Medline]

Malashkevich,V.N., Jäger,J., Sauder,U., Gehring,H., Christen,P. and Jansonius,J.N. (1995b) Biochemistry, 34, 405–414.[ISI][Medline]

Maley,J.R. and Bruice,T.C. (1970) Arch. Biochem. Biophys., 136, 187–192.[ISI][Medline]

Miyahara,I., Hirotsu,K., Hayashi,H. and Kagamiyama,H. (1994) J. Biochem., 116, 1001–1012.[Abstract]

Okamoto,A., Higuchi,T., Hirotsu,K., Kuramitsu,S. and Kagamiyama,H. (1994) J. Biochem., 116, 95–107.[Abstract]

Okamoto,A., Nakai,Y., Hayashi,H., Hirotsu,K. and Kagamiyama,H. (1998) J. Mol. Biol., 280, 443–461.[CrossRef][ISI][Medline]

Peisach,D., Chipman,D.M., Van Ophem,P.W., Manning,J.M. and Ringe,D. (1998) Biochemistry, 37, 4958–4967.[CrossRef][ISI][Medline]

Sacchettini,J.C., Banaszak,L.J. and Gordon,J.I. (1990) Mol. Cell. Biochem., 98, 81–93.[ISI][Medline]

Scapin,G., Gordon,J.I. and Sacchettini,J.C. (1992) J. Biol. Chem., 267, 4253–4269.[Abstract/Free Full Text]

Suigo,S., Petsko,G.A., Manning,J.M., Soda,K. and Ringe,D. (1995) Biochemistry, 34, 9661–9669.[ISI][Medline]

Veerkamp,J.H. and Maatman,R.G.H.J. (1995) Prog. Lipid Res., 34, 17–52.[CrossRef][ISI][Medline]

Yano,T., Kuramitsu,S., Tanase,S., Morino,Y. and Kagamiyama,H. (1992) Biochemistry, 31, 5878–5887.[ISI][Medline]

Received September 2, 2001; revised February 25, 2002; accepted April 1, 2002.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Request Permissions
Google Scholar
Articles by Häring, D.
Articles by Distefano, M. D.
PubMed
PubMed Citation
Articles by Häring, D.
Articles by Distefano, M. D.