152 Davey Laboratory, Department of Chemistry, The Pennsylvania State University, University Park, PA 16802-6300, USA
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Abstract |
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Keywords: DNA shuffling/GAR transformylase/hybrid enzyme/protein engineering
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Introduction |
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Glycinamide ribonucleotide (GAR) transformylase is one such enzyme which may have arisen as a result of recombination of protein domains. In Escherichia coli the purN encoded GAR transformylase, a 23 kDa enzyme, catalyzes the transfer of the formyl group from 10-formyltetrahydrofolate to the free amino of GAR to give formyl-GAR and tetrahydrofolate as products (Figure 1). Because of the importance of GAR transformylase in purine biosynthesis and the potential therapeutic benefit from its inhibition, the enzyme has been thoroughly characterized. A high-resolution crystal structure of the enzyme bound to a multisubstrate adduct inhibitor (Klein et al., 1995
) and protein labeling studies (Inglese et al., 1990
) have allowed the identification of the regions involved in substrate and cofactor binding. From mutagenesis (Warren et al., 1996
) and kinetic studies (Shim and Benkovic, 1998
) a catalytic mechanism for the enzyme has been proposed.
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To investigate the possibility of utilizing evolutionary strategies such as domain recruitment in the design of biocatalysts, we have previously described the creation of a hybrid enzyme with GAR transformylase activity (Nixon et al., 1997). Using a modular approach in which the protein domains were treated as discrete elements, a hybrid enzyme was created from the GAR binding domain of the purN derived GAR transformylase of E.coli and the formyltetrahydrofolate binding domain from purU. Although this enzyme was capable of formyl transfer, there was a strong preference for hydrolysis of the formyltetrahydrofolate cofactor to give formate and tetrahydrofolate. The most likely explanation of this observation is that this hybrid enzyme exists in two states, a closed conformation that is capable of formyl transfer and an open form that is capable of cofactor hydrolysis but not formyl transfer. We reasoned that in the open form hydrophobic surfaces of the protein would be exposed to solvent, which would lead to the solubility problems we encountered. We anticipated that a shift in equilibrium towards the closed conformation should improve the ratio of formyl transfer to hydrolysis. Here we describe efforts to increase the efficiency of formyl transfer of the hybrid GAR transformylase.
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Materials and methods |
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Restriction enzymes were obtained from Promega and New England Biolabs. T4 DNA ligase and Taq polymerase were obtained from Promega. Ultrapure dNTPs were obtained from Boehringer Mannheim. Agarose for analytical gel electrophoresis was obtained from Kodak. For preparative gel electrophoresis, Nusieve GTG from FMC bioproducts was used.
Bacterial strains
DH-5 (GIBCO/BRL-Life Technologies), BL21(DE3) pLys S (Novagen) and MW12 [ara
(gpt-pro-lac) thi rbs-221 ilvB2102 ilvHI2202 purN'-lacZ+Y+::KanR purT (DE3)] were used.
Plasmid construction
The stop codon was removed from the purN/PurU fusion using the megaprimer mutagenesis method (Sarkar and Sommer, 1990; Pont-Kingdom, 1994
). A first PCR reaction was performed using purU reverse (ATC GAT AAG CTT TAC GTT GAG AAA AAT GAA C) and a mutagenic oligo in which the stop codon was mutated to a XhoI restriction site (CGA TTA TTC TCG AGT CGC CAG CAA AAA TAA CTG G). The product of this PCR reaction was first purified using the Wizard PCR prep system (Promega) and then used as the reverse oligo in a second PCR reaction using a purN forward primer (5' GAT ATA CAT ATG AAT ATT GTG GTG CTT ATT TCC) and the purN/purU fusion as template. The product of this reaction was again purified using the Wizard PCR prep system and directly ligated into a commercial T-vector (pGEM-T, Promega) to yield vector pGEM14. A purN/purU hybrid enzyme-lac Z
-peptide fusion construct was prepared by subcloning the SacII/XhoI fragment from the vector pGEM14 into SacII/XhoI digested pBC KS (Stratagene).
DNA shuffling
DNA shuffling was carried out essentially as described previously (Stemmer, 1994). Briefly, 927 µg of pGEM14 were digested with SacII and XhoI and the 1 kb DNA fragment containing the hybrid enzyme gene purified using the Wizard PCR prep system. A 1 µg amount of this fragment was digested with 0.15 units of DNase I for up to 15 min in a final reaction volume of 100 µl. The digest was run out on a 2% low melting point agarose gel and fragments in the <50 bp range were isolated from the gel slice by spinning in a Millipore ultrafree filter unit. The isolated fragments were then reassembled using a program consisting of 30 cycles of 1 min 95°C, 2 min 52°C, 2 min 72°C. The primers purN forward (5' GAT ATA CAT ATG AAT ATT GTG GTG CTT ATT TCC) and XhoI mutant reverse (5' CCA GTT ATT TTT GCT GGC GAC TCG AGA ATA ATC G) were then used to amplify the assembled hybrid enzyme gene in a further 30 cycles using the same PCR program as described above.
ß-Galactosidase assays
Blue/white screening was performed essentially as described (Sambrook et al., 1989). Briefly, DH5-
transformed with the fusion construct were plated on LB-agar plates containing chloramphenicol (35 µg/ml), IPTG (1 mM) and X-Gal (50 µg/ml). The plates were then incubated at 37°C for 16 h, generally sufficient time for the blue color to develop. The chemiluminescent ß-galactosidase substrate Galacton-Star (Tropix, MA, USA) was used in accordance with the manufacturer's protocol.
Protein preparation
Expression of the shuffled hybrid enzymelacZ fusion was in the GAR transformylase auxotroph MW12. Cells containing the hybrid enzymelacZ fusion expression system were grown in minimal media containing 35 mg/ml chloramphenicol at 37°C until an OD600 of 0.40.6 was reached. Expression was then induced by the addition of IPTG to a final concentration of 1 mM and the cells allowed to grow on for a further 4 h at 37°C. Cells were harvested by centrifugation and the pellet frozen. Expression and purification of the His-tagged hybrid enzyme was as previously described (Nixon et al., 1997).
Enzyme assays
Enzyme activity was determined by monitoring the deformylation of 10-formyl-5,8-dideazafolate (fDDF) ( = 18.9 mM1 cm1 at 295 nm). Assays were performed in 50 mM Tris, 1 mM EDTA at pH 7.5 and 20°C using a Gilford 252 spectrophotometer. Steady-state parameters were determined using a hybrid enzyme concentration of 2 µM. The GAR concentration was varied between 4 and 160 µM and the fDDF concentration between 2 and 195 µM. For specific activity determination, an excess of substrates was used.
Formyl transfer
Hybrid enzyme (1 µM) was incubated with 0.7 mM [14C]fDDF (388 µCi/µmol) alone or in addtion to 0.8 mM GAR for 12 h at 37°C. The reaction products were separated by TLC using PEI-Cellulose plates (EM Separations, Gibbstown, NJ) eluting with 50 mM K2HPO4 (pH 7.0) and quantitated using a Molecular Dynamics PhosphorImager.
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Results |
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Using a substrate which contained a radiolabeled formyl group, it was possible to follow partitioning between the possible products, either by hydrolysis to free formate or transfer to GAR to yield formyl-GAR (fGAR). We determined that the hybrid enzyme favors hydrolysis of the cofactor over transfer to GAR, probably because the GAR binding domain is not aligned to facilitate capture of the formyl group from 10-formyltetrahydrofolate. This misalignment and the observed solubility problems most likely have the same root cause, faulty interactions along the domain interface. Since it is difficult to identify the key residues involved in such interdomain contacts, we chose to use random mutagenesis, specifically DNA shuffling (Stemmer, 1994), to introduce mutations that may improve solubility and in turn improve formyl transfer efficiency.
DNA shuffling has previously been shown to have utility in improving the solubility of the green fluorescent protein (Crameri et al., 1996). In this case increased fluorescence of the bacterial colonies expressing the mutated protein was used to screen for improved solubility. We chose to use a similar strategy to improve the solubility of the GAR transformylase hybrid enzyme in this study. We developed a system to allow screening of improvements in protein solubility based on
-complementation.
-Complementation refers to the ability of the
-peptide, which consists of residues 1146 of ß-galactosidase, to restore fully the activity of an enzymatically inactive ß-galactosidase created by the deletion of residues 1141 of ß-galactosidase. Our system creates a fusion between the
-peptide and the hybrid GAR transformylase. Cells which contain an insoluble fusion protein will lack ß-galactosidase activity. This can be visually assessed using a blue/white selection system. When the ß-galactosidase substrate X-Gal is metabolized it produces an insoluble blue precipitate, which results in the colony developing an intense blue color. One limitation of this approach is that the assay has a fairly limited dynamic range, a little ß-galactosidase is sufficient to yield a blue colour and so the assay typically generates binary data: blue or white.
The long hybrid enzyme was fused N-terminal to the -peptide of ß-galactosidase and was expressed in the E.coli strain DH5-
which has an inactive ß-galactosidase and as such is competent for
-complementation. DH5-
containing the hybrid enzyme
-peptide fusion construct were plated on LB-agar plates containing IPTG, to induce protein expression and X-Gal. This initial construct resulted in white colonies only, indicative of no
-complementation.
The hybrid enzyme gene was then subjected to random mutagenesis through DNA shuffling. The shuffled gene was then cloned to produce a fusion to the -peptide, yielding a library of ~105 cfu. From this library seven colonies were identified that demonstrated ß-galactosidase activity using the blue/white screen. To ensure that the identified mutants were still capable of GAR transformylase activity, the vectors encoding the seven mutants were transformed into the auxotrophic E.coli, expressed and the specific activity determined. All mutants were found to have a similar level of GAR transformylase activity.
The DNA from the colonies was prepared and sequenced (Table I). Of the eight base substitutions identified, five resulted in amino acid replacement. Three of the mutations, two of which were neutral, fall in the region of the purU domain that has no homologous sequence in purN and that is believed to loop out into solution (Figure 2
). All the mutations identified were a result of point mutations; no frameshifts were identified. The mutations were mapped on to the structure of glycinamide ribonucleotide transformylase using a combination of sequence alignments and also a model of the purU formyltetrahydrofolate binding domain. The model was prepared using the automated, knowledge-based tool Swiss Model (Peitsch, 1995
). Four of the five mutations fall around the interface of the two domains, although none appear to make direct interfacial contact.
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Discussion |
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We previously described the creation of a GAR transformylase hybrid enzyme that was capable of formyl transfer although the partitioning of the formyl group between free formate and fGAR was ~40:1 in favor of free formate. The most likely explanation of this observation is that the two domains of the hydrid enzyme are not orientated to facilitate efficient transfer of the formyl group to the ribonucleotide. Difficulties in ensuring correct interactions across protein domain interfaces has previously been noted for oligomeric protein assembly (Murata and Schachman, 1996). It seems reasonable to expect that the problems which exist in the formation of heterologous oligomeric proteins would also exist in the formation of hybrid enzymes from domains of two different proteins. Based on the observations for oligomeric protein assembly, it was expected that modification of purNpurU domaindomain interface should produce a more soluble enzyme. In addition to improved solubility, improved domain assembly should result in a hybrid GAR transformylase that is capable of efficient formyl transfer.
Given the general complexity of interactions across protein domain interfaces, rather than target discrete residues to be mutated, point mutations were introduced in a random fashion over the whole of the hybrid enzyme gene using DNA shuffling. Mutants were then screened using a ß-galactosidase-based screening system that would identify those mutants that were more soluble. It was proposed that the more soluble mutants would also be more efficient in transfer of formyl to GAR. Clone 6 (Table I) was found to have the highest level of ß-galactosidase activity. With the exception of one addition neutral point mutation found in clone 5, clone 6 has the same pattern of mutations as clone 5. It is likely, therefore, that the difference in ß-galactosidase activity stems from differences in expression levels of the two mutant proteins. Four of the mutations in clone 6 fall around the interface of the two domains, although from an analysis of the purN crystal structure none of these residues make any direct inter-domain contacts. In the absence of a crystal structure of the hybrid enzyme or of purU it is difficult to fully explain how these mutants could act to stabilize the interaction of the two domains.
The rate of fDDF deformylation of both the parent hybrid enzyme and the shuffled hybrid enzyme is three orders of magnitude lower than that for wild-type purN. It is worth noting that although this value is close to that measured for purU, the source of the catalytic module, it is not expected that this would be an upper limit on the rate of formyl transfer. Rather, we would expect that the hybrid enzyme should be capable of activity similar to that of wild-type purN since the amino group of the ribonucleotide should be a more potent nucleophile than water.
At the simplest level domain swapping to create novel biocatalysts offers the potential of vast numbers of novel biocatalysts, many of which would have industrial and perhaps therapeutic value. The modular approach that has been described here and elsewhere has demonstrated the power of the domain swapping technique. A more refined domain swapping technology has recently been described (Hopfner et al., 1998). This work describes the creation of a hybrid enzyme generated by combining the N-terminal sub-domain of the coagulation factor X with the C-terminal subdomain from trypsin. Although successful, this approach relies on detailed information of the proteins involved. A combinatorial approach to the fusion of protein domains has recently been described (Ostermeier et al., 1999a
,b
). This approach has the potential to move beyond the limitations inherent in a rational domain swapping venture.
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Notes |
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2 To whom correspondence should be addressed. Email: sjb1{at}psu.edu
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References |
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Received March 24, 1999; revised December 10, 1999; accepted February 20, 2000.