1 Department of Food Science and Human Nutrition and 2 Department of Biochemistry, Michigan State University, East Lansing, MI 48824 and 3 Michigan Biotechnology Institute, Lansing, MI 48909, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: site-directed mutagenesis/thermal stability/thermozyme/xylose isomerase
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two XI groups have been identified; type I enzymes are shorter than type II enzymes by about 50 amino acids at their N-terminus (Vangrysperre et al., 1990). Xylose isomerases are significantly stabilized and activated in the presence of divalent cations, especially by Mg2+, Co2+ and Mn2+. Two type II XIs have been studied extensively in our laboratory: one from a thermophile, Thermoanaerobacterium thermosulfurigenes (TTXI) and the other from a hyperthermophile, Thermotoga neapolitana (TNXI). The genes encoding these enzymes (xylA genes) were cloned, sequenced and expressed in Escherichia coli (Lee et al., 1990a
,b
; Meng et al., 1991
; Vieille et al., 1995
). TNXI has a higher turnover number, a lower Km for glucose and is more thermostable than any other known type II xylose isomerases (Vieille et al., 1995
). Although TNXI and TTXI are highly similar (70.4% identity), TNXI is significantly more thermostable than TTXI with an optimum temperature of 95°C (85°C for TTXI) and melting temperature in the presence of 0.5 mM Co2+ of 126°C (82°C for TTXI) (Vieille et al., 1995
). No obvious differences in the enzyme structures can explain the differences in their stabilities (Gallay et al., manuscript in preparation) except for a few additional prolines and fewer Asn and Gln residues in TNXI (as seen from its alignment with TTXI).
Proteins can be stabilized by decreasing their entropy of unfolding (Matthews et al., 1987). Prolines, with their pyrrolidine ring, can only adopt a few configurations. They restrict the configurations allowed for the preceding residue, and they can decrease the entropy of a protein's unfolded state. Substituting Pro for another carefully chosen residue can thus increase protein stability, provided that the newly introduced proline does not create volume interferences, and does not destroy stabilizing non-covalent interactions. The effect of prolines on protein stabilization has been studied by site-directed mutagenesis. The two T4 lysozyme mutants Ala82Pro (Matthews et al., 1987
) and Ile3Pro (Dixon et al., 1992
) illustrate the importance of carefully selecting the mutation location. With mutation Ala82Pro, the two conditions listed above were addressed, and the mutation stabilized the protein mainly by decreasing its entropy of unfolding. In mutant Ile3Pro, the substitution eliminated a hydrogen bond and the hydrophobic interactions created by Ile. The degree of enthalpic destabilization was greater than the entropic stabilization and the mutation was destabilizing. In a study by Allen et al. (1998) of Aspergillus awamori glucoamylase, three proline mutantsSer30Pro, Asp345Pro and Glu408Prowere constructed. The Ser30Pro mutation stabilized the protein because the mutation site allowed a residue conformation compatible with a proline, and because residue 29 (a valine) could adopt one of the conformations allowed for the residue preceding a proline (Matthews et al., 1987
). The Glu408Pro mutation was destabilizing because the mutation site did not allow any conformation required for proline, whereas the Asp345Pro mutation did not affect stability due to the fact that the substitution destroyed the
-helix dipole in that region. Prolines 58 and 62 in TNXI are present in a large loop in which 14 hydrophilic residues surround Phe59. The corresponding TTXI residues are Gln58 and Ala62. The Phe59 loop participates in building the neighboring subunit's active site (Farber et al., 1989
; Whitlow et al., 1991
). In the present report we test the hypothesis that substituting Gln58 and Ala62 with prolines will stabilize TTXI, and that the reverse mutations will destabilize TNXI.
In a previous study, genetic engineering was used to improve TTXI activity on glucose (not the natural substrate): the enzyme's substrate-binding pocket was enlarged and the water-accessible surface area in the active site was altered (Meng et al., 1991, 1993
). Substituting Trp138 with Phe, a smaller residue, decreased TTXI's Km for glucose and increased its catalytic efficiency (kcat/Km) on glucose 2.6-fold. This mutant enzyme was less active than the wild-type TTXI on xylose. Interestingly, this mutation doubled TTXI's half-life at 85°C (Meng et al., 1993
). Substituting Val185 with Thr, a polar residue, improved TTXI's catalytic efficiency on glucose. This improvement was thought to result from the creation of an additional hydrogen bond to glucose's C6-OH group. The double mutation Trp138Phe/Val185Thr vastly improved TTXI's catalytic efficiency on glucose (5.7-fold). In this study, we will introduce these mutations in TNXI to determine if TNXI activity and stability can be further enhanced by genetic engineering. All mutations will be characterized by their kinetic parameters, optimum temperatures, half-lives at 85 or 95°C, and temperatures of 50% precipitation in order to compare their thermal stability and activity.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Escherichia coli strain BL21 (DE3) (Novagen, Madison, WI) was used to overexpress the recombinant T.thermosulfurigenes and T.neapolitana xylA genes cloned in the pET23a and pET22b+ vectors, respectively (Novagen). Media and growth conditions were the same as described by Vieille et al. (1995). Medium components and all other chemicals were reagent grade.
Site-directed mutagenesis and other DNA techniques
All DNA manipulations were performed using established protocols (Sambrook et al., 1989; Ausubel et al., 1993
). Point mutations were introduced into the T.neapolitana and T.thermosulfurigenes xylA genes using the QuickChangeTM Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and M13 single-stranded site-directed mutagenesis (Ausubel et al., 1993
). Mutagenic oligonucleotides (Table I
) were synthesized by the Macromolecular Structure Facility (Department of Biochemistry, Michigan State University). Mutations were verified by DNA sequencing using the Thermosequenase Sequencing kit (US Biochemical, Cleveland, OH).
|
Recombinant enzymes were purified using the procedure of Vieille et al. (1995) followed by two additional steps. Partially purified enzymes were applied to a Q-Sepharose column (2.5x15 cm) equilibrated with MOPS 50 mM (pH 7.0) containing 5 mM MgSO4 and 0.5 mM CoCl2 (buffer A) and enzymes were eluted using a 500 ml 0250 mM NaCl gradient in buffer A. The pooled fractions from the Q-Sepharose column were concentrated in a stirred ultrafiltration cell (MW cut-off 30 kDa) (Amicon, Beverly, MA), dialyzed twice against buffer A, applied to a Polybuffer column (Pharmacia, Uppsala, Sweden) equilibrated with 25 mM histidineHCl (pH 6.2) and eluted using a pH (6.04.0) gradient according to the manufacturer's instructions. The active fractions were pooled, concentrated in a stirred ultrafiltration cell (Amicon), and dialyzed twice against buffer A. Concentrated, homogenous enzymes were dispensed and stored frozen at 70°C.
Xylose isomerase assays
Xylose isomerase activity was routinely assayed with glucose as the substrate. The enzyme (0.06 mg/ml) was incubated in 50 mM MOPS (pH 7.0 at room temperature) containing 1 mM CoCl2 and 1 M glucose at 60°C for 20 min. The reaction was stopped by cooling the tubes in ice. The amount of fructose produced was determined by the cysteinecarbazolesulfuric acid method (Dische and Borenfreund, 1951). To determine the effect of temperature on XI activity, the assay mixtures were incubated at the temperatures of interest in a Perkin-Elmer Cetus GeneAmp PCR system 9600 (Perkin Elmer, Norwalk, CT) for 20 min. To determine the kinetic parameters, assays were performed in the presence of either 801400 mM glucose or 20900 mM xylose. The amounts of fructose and xylulose produced were determined as above. Absorbances were measured at 537 and 560 nm for xylulose and fructose, respectively. One unit of isomerase activity is defined as the amount of enzyme that produced 1 µmol product per min under the assay conditions.
Thermostability assays
The time course of irreversible thermoinactivation was measured by incubating the enzyme (0.55 mg/ml) in 10 mM MOPS buffer (pH 7.0) containing 50 µM CoCl2 (buffer B) at 85°C (TTXI derivatives) or 95°C (TNXI derivatives) in a Perkin-Elmer Cetus GeneAmp PCR system 9600 for various amounts of time, and by determining the residual glucose isomerase activity at 65 (TTXI derivatives) or 80°C (TNXI derivatives). The first order rate constant, k, of irreversible thermoinactivation was obtained by linear regression in semi-log coordinates. Enzyme half-life was calculated from the equation: t1/2 = ln 2/k.
Heat-induced enzyme precipitation
Heat-induced enzyme precipitation was monitored from 25 to 100°C by light scattering ( = 580 nm) using protein solutions (0.2 mg/ml) in buffer B. Absorbance measurements were conducted in 0.3 ml quartz cuvettes (path length = 1.0 cm), using a Gilford Response spectrophotometer (Corning, Oberlin, OH) equipped with a Peltier cuvette heating system. The increasing thermal gradient was 1.0°C min1. The temperature of 50% precipitation was the temperature at which the OD580 equals half of the difference between the baseline and the maximum ODs.
Analysis of TTXI and TNXI three-dimensional structures
Enzymes were visualized on an IRIS-4D25 computer (Silicon Graphics Computer System, Mountain View, CA) using the INSIGHT II graphic program (Biosym Technologies, San Diego, CA). Proteins Data Bank (PDB) files (#1A0C for TTXI and #1A0E for TNXI) were obtained from the Protein Data Bank website (www.rcsb.org/pdb).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TNXI Pro58 and Pro62 are present in a large loop in which 14 hydrophilic residues surround Phe59 (Figure 1). According to crystallographic data, Phe59 (Phe26 in Actinoplanes XI) participates in the architecture of the neighboring subunit's active site (Farber et al., 1989
; Whitlow et al., 1991
). The corresponding residues in TTXI, Gln58 and Ala62, have backbone dihedral angles [(66.57, 14.49) and (57.91, 138.87), respectively] (Gallay et al., manuscript in preparation) allowed for prolines (Nicholson et al., 1988
). With backbone dihedral angles of (139.73, 175.10) and (93.32, 176.20), respectively, TTXI residues Asp57 and Lys61 are in extended conformations, the most common conformation for residues preceding prolines (Nicholson et al., 1988
). This structural information suggests that mutations Gln58Pro and Ala62Pro would not create unfavorable backbone conformations and that they could stabilize TTXI.
|
|
|
|
The kinetic features of the wild-type and mutant xylose isomerases with glucose and xylose as substrates were determined at 65°C for the TTXI series and at 80°C for the TNXI series (Table III). With the exceptions of mutations Ala62Pro and Gln58Pro/Ala62Pro in TTXI, the mutations in TTXI and TNXI Phe59 loops did not significantly alter the enzymes' catalytic properties. The Ala62Pro mutation increased both TTXI's Vmax and Km on glucose, leaving its catalytic efficiency on glucose almost unchanged. This mutation had a much stronger effect on TTXI activity on xylose. It increased its affinity for xylose and its catalytic efficiency approximately 2.5-fold. Mutation Gln58Pro/Ala62Pro had a more pronounced effect on TTXI activity on glucose. A twofold increase in its Km for glucose decreased its catalytic efficiency almost threefold. These results indicate that mutations Ala62Pro and Gln58Pro/Ala62Pro altered TTXI's catalytic features and at the same time destabilized the enzyme.
|
The Val185Thr mutant and the double mutant Trp138Phe/Val185Thr TNXI derivatives had the same optimum temperature (97°C) as wild-type TNXI (Figure 4A). Interestingly, mutation Val185Thr almost doubled TNXI specific activity on glucose at 97°C (from 26.0 to 45.6 U/mg). Although the Trp138Phe mutation decreased TNXI optimum temperature to 87°C, the mutant enzyme still retained almost 80% of its activity at 97°C. The stability of these mutant enzymes was studied at 95°C (Figure 4B
). With a half-life of 69.3 min, the Val185Thr mutation did not affect TNXI stability at 95°C. The Trp138Phe mutant (half-life of 87 min) and the Trp138Phe/Val185Thr double mutant (half-life of 99 min) enzymes were 25 and 43% more stable, respectively, than wild-type TNXI.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two additional prolines are found in a large `Phe59 loop' region in TNXI (Figure 1). These prolines are substituted with Gln and Ala in TTXI. A protein can be stabilized by engineering proline into selected sites thereby decreasing the protein's conformational entropy of unfolding (Allen et al., 1998
). In order for a proline substitution to stabilize a protein, at least four criteria have to be considered: (i) the mutation site should allow one of the conformations allowed for proline; (ii) the preceding residue should be able to adopt one of the conformations allowed for the residue preceding a proline; (iii) proline should not create volume interferences and (iv) proline should not destroy stabilizing non-covalent interactions (Nicholson et al., 1988
). TTXI Gln58 and Ala62 have backbone dihedral angles allowed for prolines, and Asp57 and Lys61 have dihedral angles allowed for residues preceding prolines. Despite these first two conformational requirements being satisfied, only mutation Gln58Pro stabilized TTXI. As seen in Figure 5A
, the conformation of Gln58's side chain is very close to that of the proline pyrrolidine ring. No volume interference is created by the Gln58Pro mutation. In the wild-type TTXI structure, Gln58 is not involved in any potentially stabilizing, non-covalent interactions (not shown). No enthalpic destabilization is expected with mutation Gln58Pro, and the stabilization it provides to TTXI probably entirely results from a reduction of the entropy of unfolding. On the other hand, the Ala62Pro mutation had a destabilizing effect on TTXI. In TTXI, Ala62's side chain points toward a large cavity, and it is not in close vicinity of any other residues. So the Ala62Pro mutation does not eliminate any stabilizing non-covalent interactions. Detailed analysis of the Ala62Pro mutation modeled into the TTXI structure (Figure 6
) suggests that Pro62's pyrrolidine ring (C
atom) is in close contact (within 2.92 Å) with Lys61's side chain (Cß atom). Carbon atoms have van der Waal's radii of 1.701.78 Å in protein. Optimal van der Waal's interactions between two carbon atoms would take place at approximately 3.43.5 Å. The unfavorable van der Waals contact between Pro62-C
and Lys61-Cß probably leads to local conformational changes. Not only are these changes destabilizing, they also affect the active site structure and the enzyme's interaction with the substrate. The overall destabilizing nature of mutation Ala62Pro indicates that the conformational destabilization of the native enzyme more than cancels the benefits of a potential decrease in unfolding entropy.
|
|
|
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antrim,R.L., Colilla,W. and Schnyder,B.J. (1979) In Wingard,L.B. (ed.), Applied Biochemistry and Bioengineering. Academic Press, New York, pp. 97155.
Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (1993) In Struhl,K. (ed.), Current Protocols in Molecular Biology. Greene Publishing and Wiley-InterScience, New York.
Bucke,C. (1980) In Birch,G.G., Blackebrough,N. and Parker,J.K. (eds), Enzymes and Food Processing. Applied Science Publishers, London, pp. 5172.
Chen,W. (1980) Process Biochem., 15, 3035.
Creighton,T.E. (1993) Protein: Structures and Molecular Properties, 2nd Edn. W.H. Freeman, New York.
Dische,Z. and Borenfreund,E. (1951) J. Biol. Chem., 192, 583587.
Dixon,M.M., Nicholson,H., Shewchuk,L., Baase,W.A. and Matthews,B.W. (1992) J. Mol. Biol., 227, 917933.[ISI][Medline]
Farber,G.K., Glasfeld,A., Tiraby,G., Ringe,D. and Petsko,G.A. (1989) Biochemistry, 28, 72897297.[ISI][Medline]
Lee,C., Bagdasarian,M., Meng,M. and Zeikus,J.G. (1990a) J. Biol. Chem., 265, 1908219090.
Lee,C., Bhatnagar,L., Saha,B.C., Lee,Y.E., Takagi,M., Imanaka,T., Bagdasarian,M. and Zeikus,J.G. (1990b) Appl. Environ. Microbiol., 56, 26382643.[ISI][Medline]
Lee,C. and Zeikus,J.G. (1991) Biochem. J., 273, 565571.[ISI][Medline]
Matthews,B.W., Nicholson,H. and Becktel,W. (1987) Proc. Natl Acad. Sci. USA, 84, 66636667.[Abstract]
Meng,M., Lee,C., Bagdasarian,M. and Zeikus,J.G. (1991) Proc. Natl Acad. Sci. USA, 88, 40154019.[Abstract]
Meng,M., Bagdasarian,M. and Zeikus,J.G. (1993) Proc. Natl Acad. Sci. USA, 90, 84598463.
Nicholson,H., Becktel,W.J. and Matthews,B.W. (1988) Nature, 336, 651656.[ISI][Medline]
Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Takasaki,Y., Kosugi,Y. and Kanbayashi,A. (1969) Agric. Biol. Chem., 33, 15271534.[ISI]
Vangrysperre,W., van Damme,J., Vandekerckhove,J., De Bruyne,C.K., Cornelis,R. and Kersters-Hilderson,H. (1990) Biochem. J., 265, 699705.[ISI][Medline]
Vieille,C., Hess,M., Kelly,R.M. and Zeikus,J.G. (1995) Appl. Environ. Microbiol., 61, 18671875.[Abstract]
Vieille,C. and Zeikus,J.G. (1996) Trends Biotech., 183190.
Whitlow,M., Howard,A.J., Finzel,B.C., Poulos,T.L., Winborne,E. and Gilliland,G.L. (1991) Proteins, 9, 153173.[ISI][Medline]
Received August 19, 1999; revised January 27, 2000; accepted January 27, 2000.