1 European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany and 3 Department of Enzyme Design,Novo Nordisk A/S, Novo Alle 1, 2880 Bagsværd, Denmark
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: -amylase/electrostatics/pH/activity profile/pKacalculations/protein dynamics
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
-Amylases
The -amylases consist of three domains called A, B and C. Domain A is a TIM-barrel [(
/ß)8-barrel], which is interrupted by an irregular ß-domain (domain B) inserted between the third ß-strand and the third
-helix of the TIM-barrel. Domain C is a Greek key motif which is located approximately on the opposite side of the TIM-barrel with respect to domain B. The active site is situated in a cleft at the interface between domains A and B.
The active site consists of a large number of charged groups, among which are three acids essential to catalytic activity. Two of these, Asp231 and Glu261 (numbering according to the BLA sequence), are believed to be the two catalytic groups. Asp231 is the catalytic nucleophile, while evidence has been presented for Glu261 being the catalytic hydrogen donor (McCarter and Withers, 1996; Uitdehaag et al., 1999
). The third essential acid (Asp328) is believed to assist catalysis by hydrogen bonding to the substrate and by elevating the p Ka of Glu261 (Klein et al., 1992
; Knegtel et al., 1995
; Strokopytov et al., 1995
; Uitdehaag et al., 1999
).
The catalytic reaction is believed to consist of three steps (Sinnot, 1990; McCarter and Withers, 1994
; Davies and Henrissat, 1995
; McCarter and Withers, 1996
) (see Figure 1
). Step one is the protonation of the glycosidic oxygen by the proton donor (Glu261). This is followed by a nucleophilic attack on the C1 of the sugar residue in position 1 by Asp231 [nomenclature as described by Davies et al. (Davies et al., 1997
)]. After the aglycone part of the substrate has left, a water molecule is activated, presumably by the now deprotonated Glu261. This water molecule hydrolyses the covalent bond between the nucleophile oxygen (of Asp231) and the C1 of the sugar residue in position 1, thus completing the catalytic cycle.
|
Changing the pHactivity profile
If we accept the assumption that -amylase catalysis is limited at low pH by protonation of the nucleophile (Asp231) and at high pH by deprotonation of the hydrogen donor (Glu261), then the pHactivity profile is determined by the pKa values of the these two active site groups (Kyte, 1995
). We consider two types of pHactivity profiles: kcat profiles and the kcat/Km profiles. The kcat profile of an enzyme is determined by the pKa values of the active site groups in the substrate bound form of the enzyme (Kyte, 1995
).
Since the substrate is present in high concentrations in the majority of industrial processes where -amylases are used, it is obviously the kcat profile and not the kcat/Km profile that limits the activity of the enzyme. In order to change the kcat profile, we therefore have to identify the factors that determine the pKa values of the active site residues when the substrate is bound. The protein environment certainly is important and although not much is known about the effect of the substrate, it is undoubtedly also important, since shifts in
-amylase pHactivity profiles have been measured when changing the substrate (Keating et al., 1998
). Also, elegant experiments with Bacillus circulans xylanase have shown that the pKa value of the catalytic hydrogen donor `cycles' during the catalytic reaction in order to fulfil its dual role as hydrogen donor and hydrogen acceptor (McIntosh et al., 1996
).
Changing the substrate is unfortunately not an option when trying to engineer the pHperformance profile of an -amylase for a specific industrial process and we are therefore left with the option of changing the enzyme (in our case by site-directed mutagenesis) and in that way changing the pKa values of the active site groups and thereby the kcat profile.
Changing pKa values
The pKa value of a residue depends on the free energy difference between the neutral and the charged states of the residue in the protein. The free energy difference between these two states is influenced both by desolvation effects and by the charges and dipoles in the protein and the substrate. Desolvation effects and the interaction with dipoles are short-ranged and therefore result mainly from interactions with residues in the immediate environment. Mutations that aim at changing the pKa value of a titratable group by changing these energies should therefore be placed in the vicinity of the titratable group.
Mutations that introduce or remove unit charges can be placed further away from the titratable group, because the interaction energy between a titratable group and a unit charge (which may itself be a part of another titratable group) is less dependent on distance.
The direction of the expected pKa shift when perturbing the environment of an uncoupled titratable group is summarized in Figure 2. Using this figure, we can therefore easily predict the direction of an
-amylase pHactivity profile shift, if we assume that the active site residues of the
-amylases are relatively uncoupled or if the charged residue is placed a short distance from the active site. Thus, placing a negative charge near the hydrogen donor will elevate the pKa of the hydrogen donor and give a basic shift in the high-pH limb of the pHactivity profile. Placing a positive charge close to the hydrogen donor will shift the basic limb of the pHactivity profile to more acidic values.
|
Previously (Nielsen et al., 1999a), we constructed 15 mutants in and around the active site of Bacillus licheniformis
-amylase (BLA), in an attempt to change its pHactivity profile. The mutations in the active site were conservative in nature and were an attempt to change the pHactivity profile of the enzyme by changing the hydrogen-bond interactions and the solvent accessibility of the active site residues. The mutations outside the active site aimed at changing the pHactivity profile by introducing or removing a charge and in this way perturbing the pKa values of the active site acids.
The mutations in the active site were found to be highly deleterious to the activity of the enzyme, whereas most of the mutations further away from the active site did not change the activity of the enzyme significantly, but nevertheless they produced some changes in the pHactivity profile. Unfortunately, the changes in the pHactivity profiles did not correlate with the predictions from electrostatic potential calculations, prompting us to suggest that effects other than electrostatics were important for determining the pHactivity profile.
In this paper, we describe how these `other effects' are indeed important for determining the pHactivity profile. We designed mutations at four positions around the active site of a chimeric Bacillus -amylase (Ba2). This enzyme was chosen as a model system because high-resolution structures are available for both the apo and holo forms of this enzyme (Brzozowski et al., 2000
). We mutated the original neutral residues at the four positions to both neutral and charged residues and examined the effects on the pHactivity profile.
The results indicate that point mutations that are likely to change the dynamics of the active site can change the pHactivity profile. The effects on the pHactivity profile of a neutral neutral mutation are so large that effects caused by neutral
charged mutations in this study are also likely to be dominated by the associated differences in active site dynamics.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The `MegaPrimer' method for site-directed mutagenesis (Sarkar and Sommer, 1990) was used to construct a DNA fragment carrying the mutation. Mutagenic primers were designed so as to introduce or remove a unique site in the gene encoding the hybrid
-amylase (Ba2). The mutant DNA fragments were inserted into a Bacillus expression plasmid in the context of the Bacillus licheniformis
-amylase promoter, signal sequence and transcriptional terminator. The resulting mutant plasmids were transformed into Bacillus subtilis. Mutant colonies were identified by endonuclease digests of colony polymerase chain reaction (PCR) fragments and confirmed by DNA sequencing throughout the region covered by the mutagenic primer.
Fermentation
Fermentation was carried out at 37°C for 5 days in shake flasks containing a complex medium mainly consisting of potato flour, barley flour and sucrose soy meal (Bang et al., 1999; Beier et al., 2000
).
Protein purification
The supernatant of the fermentation mixture was isolated by flocculation and centrifugation. During ultrafiltration the buffer was changed to 20 mM sodium acetate, pH 5.5. The protein was subsequently applied to an SP-Sepharose Hi-Load column (Pharmacia). A dialysis step was used to change the buffer to 20 mM H3BO3 + 5 mM KCl, pH 9.6. The final step of the purification procedure consisted of anion-exchange chromatography on a Mono-Q Hi-Load column (Pharmacia). All protein preparations were at least 95% pure as shown by SDSPAGE.
Activity assays
The activity as a function of pH was measured over the range pH 4.010.5, using the Phadebas -amylase test kit (Pharmacia). Measurements were carried out in duplicate at 30°C in 50 mM BrittonRobinson buffer (50 mM H3PO4 + 50 mM CH3COOH + 50 mM H3BO3) containing 0.1 mM CaCl2. The Phadebas
-amylase test kit is based on the release of blue colour from the substrate (blue-coloured starch) upon cleavage. Hydrolysis is stopped by adding 1/6 volume of 1 M NaOH to the reaction mixture. After removal of the unhydrolysed blue starch by filtration, the amount of hydrolysed substrate is proportional to the absorbance at 620 nm.
Since the substrate is insoluble (and added in large quantities), the activity measurements obtained by this method can be regarded as kcat for insoluble starch.
Stability assays
Stability was measured as the residual activity after incubation at 30°C for 15 min. Measurements were carried out at pH 4.5, 7.0 and 9.0 and were performed in duplicate. None of the mutants showed any detectable differences from the wild-type stability.
pKa calculations
pKa calculations were performed with the WHAT IF pKa calculation routines (Nielsen and Vriend, 2001). The routines apply the hydrogen-bond optimization procedure of Hooft et al. (Hooft et al., 1996) in order to model each of the protein protonation states accurately. DelPhi II (Nicholls and Honig, 1991
) was used to calculate electrostatic energies. The parameters for DelPhi II were set as described previously (Nielsen and Vriend, 2001), namely protein dielectric constant, 16 (for residues that participate in crystal contacts, have an alternative accessible rotamer or an average B-factor of >20) or 8 (all other residues); ionic strength, 0.160 M; solvent probe radius, 1.4 Å; and solvent dielectric constant, 80. The calculations were performed without water molecules, because the inclusion of water molecules was found to decrease the accuracy of the pKa calculations for a test set of nine proteins (Nielsen and Vriend, 2001).
A model of Ba2 in complex with malto-nonaose was constructed from the Ba2 -amylasesacarbose X-ray structure (Brzozowski et al., 2000
). The few changes that were necessary in order to convert the inhibitor to a substrate were made using Quanta. The CHARMm 22 parameter set was used as the source of charges and radii for the substrate in the pKa calculations.
Preparation of mutant structures
Mutant structures for use in the pKa calculations were designed using the WHAT IF position-specific rotamer libraries (Chinea et al., 1995). Mutant structures were inspected visually.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
N190 is positioned in domain B in a turn that points into the edge of the substrate binding cleft (Figure 3). The residue does not hydrogen-bond to any other residue, except that its NH2 group points to Tyr193 and most likely interacts with the
-electrons of the aromatic ring. The rotamer distributions for Asp and Lys at this position reveal that the Asp and Lys side chains will most likely point in the same direction as the Asn side chain, thereby causing almost no changes in the local structure. The unfavourable interaction of the Asp side chain with the
-electrons of Tyr193 is, however, likely to cause some rearrangements.
The pHactivity profiles of N190D, N190K and N190G show no significant changes compared with the wild-type pHactivity profile (Figure 4a).
|
F290 is situated in the -helix that lies between ß-strand 6 and
-helix 6 of the TIM-barrel. This `in-between'
-helix is not a part of the classical TIM-barrel. Phe290 sits next to His289, which hydrogen bonds to Ser337. Ser337 is positioned in the loop that covers the active site and its mutation to glycine results in a protein with 2% of wild-type activity at pH 7.0, thus hinting at an important role played by the hydrogen bond between Ser337 and His289.
The pHactivity profiles for F290K, F290E and F290A are shown in Figure 4b. Both F290E and F290A are more active than the wild-type and both enzymes are much more active at basic pH than they would have been if they had had a pHactivity profile shaped as the wild-type protein. F290K is slightly less active than the wild-type, but its pHactivity profile has also been shifted slightly to more basic pH values.
N326L/A/D
Asn326 sits in the loop that covers the active site and is fairly close to Asp328 (Table I). Based on the position-specific rotamer distributions and a bump analysis, it was judged that neither a lysine nor an arginine could be accommodated at this position. Furthermore, the rotamer distribution for leucine gave only two hits at this position, although visual inspection suggested that only minor adjustments in the local structure are needed to accommodate this mutation.
The pHactivity profile of N326A is almost identical with that of the wild-type (Figure 4c), although the mutant has only ~60% of the wild-type activity at pH 7.0. N326D has relatively higher activity than the wild-type at basic pH, which is in agreement with the results reported previously by Takase (1993).
The pHactivity profile of N326L has lost the characteristic peak around pH 5.5 and has become almost completely flat. Furthermore, the basic limb of the pHactivity profile is shifted slightly towards more basic pH values.
Q360K/A/E
The mutants Q360A and Q360K (Figure 4d) have ~50% of the wild-type activity and their pHactivity profiles are very similar to that of the wild-type. Q360E, on the other hand, is more than twice as active as the wild-type at pH 6.0 and has a pHactivity profile which is much more bell-shaped than the wild-type profile.
pKa calculations
pKa calculations were carried out as described in the Materials and methods section. The titration curves for the active site residues were mostly irregular and did not follow the classical HendersonHasselbalch shape (Figure 5). It was therefore not possible to determine a pKa value for any of the active site residues. Instead, we have given a qualitative description of the differences in the titration curves. The detailed titration curves for the active site residues in all mutant structures can be found at http://www.cmbi.kun.nl/gv/nielsen/amylase/pKa/.
|
pKa calculations for the mutants
The pKa calculations for the mutants were carried out with the holo structure of the enzyme to give the pKa shifts for the active site acids when the substrate is bound. The perturbations in the titration curves for the active site residues were fairly small for all mutations of Asn190 and Phe290. This is in perfect agreement with the experimental results for the mutations at position 190, but does not correlate with the results for the mutations at position 290. Changes were observed for the mutations of Asn326 and Gln360. These changes are described in more detail below.
N326D/A/L
The calculations for N326A show an upward shift in the pKa value of His327 and a downward shift in the pKa value of Asp328. The calculations for N326L show a slight upward shift in the pKa value of Asp328 and an even smaller upward shift in the pKa value of Glu261. The N326D pKa calculations predict that the pKa values of Asp231, His327, Asp328 and Glu261 increase.
Q306A/E/K
The pKa calculations for the mutants in position 360 predict that the pKa value of Asp231 becomes elevated upon all three mutations. In the case of Q360A and Q360K, the shift is very large, whereas a smaller shift is seen for Q360E. The pKa values of His327 and Asp328 are predicted to become slightly lower for Q360A. The calculations for Q360K predict that the pKa values for His327 and Asp328 increase.
Correlation of the calculated pKa shifts with the experimentally observed pHactivity profile shifts
The magnitude and direction of the pHactivity profile shifts for the Ba2 mutants are not reproduced by the pKa calculations. This is most clearly seen when comparing the calculations with the experimental results for F290A and N326A. F290A gives the largest shift in the pHactivity profile, but the pKa calculations produce insignificant changes in the active site pKa values for this mutation. In the case of N326A, the pKa calculations show a significant perturbation of the pKa values of Asp328 and His327, but the pHactivity profile for this mutant is almost identical with that of the wild-type.
The calculations for the mutations that introduce charges also do not correlate well with the experimental pHactivity profile shifts and this strongly suggests that long-range electrostatics are less important for the pHactivity profile of the -amylases than previously thought.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The His289Ser337 hydrogen bond
His289 hydrogen bonds to Ser337 and the change in the pKa, and possibly in the dynamics of His289, is likely to affect the strength of this hydrogen-bond. Ser337 is situated in a loop that covers the active site and it is therefore likely that a change in the Ser337His289 hydrogen bond could affect the dynamics of the active site. The hydrogen bond between His289 and Ser337 is known to be important, since the mutation of Ser337 to glycine produces an enzyme with a 50-fold reduction activity (J.E.Nielsen, G.Vriend and T.V.Borchert, unpublished results).
The reason for the discrepancy between the pKa calculations and the experiments for the mutants F290K and F290E now becomes clear, since these mutations are also likely to change the solvent accessibility and the dynamics of His289. The pHactivity profile shifts resulting from these two mutations are therefore the combined effects of the charge and the change in the mobility of His289 that are induced by the mutations. The pKa calculations do not attempt to model such effects and are therefore unable to reproduce the pHactivity profile shifts.
Asn326
Takase constructed the N326D mutation (Takase, 1993) in the Bacillus stearothermophilus
-amylase and reported the same downward shift in the pHactivity profile as we found. This shift is not readily explainable and is possibly due to changes in the mobility. The neutral
neutral mutations in this position (N326A and N326L) are both likely to influence the active site dynamics, but only N326L shows a significant change in the pHactivity profile.
Gln360
The mutations at position 360 are remarkable in that Q360E has a large impact on the pHactivity profile, whereas the mutations Q360K and Q360A, which are expected to change the dynamics of the enzyme more than Q360E, have wild-type pHactivity profiles. This suggests that the effect of Q360E is purely electrostatic, although it is difficult to understand why Q360K does not have an equally large effect on the pHactivity profile.
Effect of introducing a point mutation on a tightly coupled system of titratable groups
In constructing the point mutations in BLA and BA2 we have silently assumed that the effect of inserting a titratable group near the active site could be predicted using the well-established rules that are summarized in Figure 2. The pKa calculations for Q360K show that this is not always the case, as the pKa of Asp328 is calculated to increase when Gln360 is mutated to a lysine. This is clearly not what would be expected and shows that counterintuitive effects can indeed be achieved when a system of tightly coupled titratable groups is perturbed. The phenomenon is illustrated in Figure 6
, where the rules of Figure 2
are shown to break down for an AspGlu system much like the two catalytic acids in the
-amylases. It is therefore essential in many cases to use pKa calculations to predict the effect of charged point mutations on the pKa values in the active site.
|
We have shown that significant changes in the activity of pHactivity profiles of a Bacillus -amylase can indeed be achieved using site-directed mutagenesis. The shifts in the pHactivity profiles for the mutants did not agree with the calculated changes in the active site electrostatics.
We speculate that changes in the dynamics of the active site residues are at least as important for the pHactivity profile as the changes in the active site electrostatics caused by the introduction of a charged residue. This is corroborated by the mutations F290A and N326L, which change the pHactivity profile without changing the net charge on the molecule. This implies that the pKa values of the active residues can be changed significantly by altering the dynamics of the active site and thus suggests an alternative approach to the engineering of pHactivity profiles.
A detailed explanation of the effects of the neutral neutral mutations is difficult, as we do not know the motions that are important for the catalytic activity of the
-amylases. It is likely that very accurate and long molecular dynamics (MD) simulations could provide insights into this, but in view of the present day MD force fields and computer speeds, this is not a feasible solution. The pKa calculation method that we used does not model heavy atom mobility and the pKa shifts induced by mobility changes can therefore not be reproduced in the calculations. Furthermore, we may well have underestimated the value of the dielectric constant in the active site in our calculations. Employing a higher dielectric constant in the pKa calculations would reduce the magnitude of the calculated shifts in the titration curves, but it is unlikely that it would give a better qualitative agreement with the experimental data.
The pKa calculations also show that the -amylase active site is a strongly connected system of titratable groups. The example with a strongly coupled AspGlu system given in Figure 6
shows that the effect of inserting a titratable group cannot always be predicted by using the simple scheme of Figure 2
. If the circumstances are right, one can observe the exact opposite of what was predicted. We speculate that the shifts in the `wrong' direction seen for several of the mutants in this and in the previous study (Nielsen et al., 1999a
) could be partly due to such effects. It seems more likely, though, that the change in the pHactivity profiles result primarily from the change in the active site dynamics.
The findings of this study stress the point that dynamics are an essential part of every enzyme and that rational engineering of enzyme activity is more likely to succeed if a detailed description of the enzyme mobility is available when designing the point mutations.
![]() |
Notes |
---|
Email: jnielsen{at}mccammon.ucsd.edu
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beier,L., Svendsen,A., Andersen,C., Frandsen,T.P., Borchert,T.V. and Cherry,J.R. (2000) Conversion of the maltogenic alpha-amylase Novamyl into a CGTase. Protein Eng., 13, 509513.
Brzozowski,A.M., Lawson,D.M., Turkenburg,J.P., Bisgaard-Frantzen,H., Svendsen,A., Borchert,T.V., Dauter,Z., Wilson,K.S. and Davies,G.J. (2000) Structural analysis of a chimeric bacterial -amylase. High resolution analysis of native and ligand complexes. Biochemistry, 39, 90999107.[ISI][Medline]
Chinea,G., Padron,G., Hooft,R.W.W., Sander,C. and Vriend,G. (1995) The use of position-specific rotamers in model building by homology. Proteins, 23, 415421.[ISI][Medline]
Davies,G.J. and Henrissat,H. (1995) Structures and mechanisms of glycosyl hydrolases. Structure, 3, 853859.[ISI][Medline]
Davies,G.J., Wilson,K.S. and Henrissat,B. (1997) Nomenclature for sugar-binding subsites in glycosyl hydrolases. Biochem. J., 321, 557559.[ISI][Medline]
Fang,T.Y. and Ford,C. (1998) Protein engineering of Aspergillus awamori glucoamylase to increase its pH optimum. Protein Eng., 11, 383388.[Abstract]
Guzman-Maldano,H. and Paredes-Lopez,O. (1995) Amylolytic enzymes and products derived from starch; a review. Crit. Rev. Food. Nutr., 36, 373403.
Hooft,R.W., Sander,C. and Vriend,G. (1996). Positioning hydrogen atoms by optimising hydrogen-bond networks in protein structures. Proteins, 26, 363376.[ISI][Medline]
Keating,L., Kelly,C. and Fogarty,W. (1998). Mechanism of action and the substrate-dependent pH maximum shift of the -amylase of Bacillus coagulans. Carbohydr. Res., 309, 311318.[ISI][Medline]
Klein,C., Hollender,J., Bender,H. and Schulz,G.E. (1992) Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis. Biochemistry, 31, 87408746.[ISI][Medline]
Knegtel,R.M, Strokopytov,B., Penninga,D., Faber,O.G., Rozeboom,H.J., Kalk,K.H., Dijkhuizen,L. and Dijkstra,B.W. (1995) Crystallographic studies of the interaction of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 with natural substrates and products. J. Biol. Chem., 270, 2925629264.
Kyte,J. (1995) Mechanism in Protein Chemistry. Garland Publishing, New York, pp. 258283.
McCarter,J.D. and Withers,S.G. (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr. Opin. Struct. Biol., 4, 885892.[ISI][Medline]
McCarter,J.D. and Withers,S.G. (1996) Unequivocal identification of Asp-214 as the catalytic nucleophile of Saccharomyces cerevisiae -glucosidase using 5-fluoro glycosyl fluorides. J. Biol. Chem., 271, 68896894.
McIntosh,L.P., Hand,G., Johnson,P.E., Joshi,M.D., Korner,M., Plesniak,L.A., Ziser,L., Wakarchuk,W.W. and Withers,S.G. (1996) The pKa of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of Bacillus circulans xylanase. Biochemistry, 35, 99589966.[ISI][Medline]
Nicholls,A. and Honig,B. (1991) A rapid finite difference algorithm, utilizing successive over-relaxation to solve the PoissonBoltzmann equation. J. Comput. Chem., 12, 435445.[ISI]
Nielsen,J.E. and Borchert,T.V. (2000) Protein engineering of bacterial -amylases. Biochim. Biophys. Acta, 1543, 253274.[ISI][Medline]
Nielsen,J.E. and Vriend,G. (2001) Optimising the hydrogen-bond network in PoissonBoltzmann equation based pKa calculations. Proteins, 43, 403412.[ISI][Medline]
Nielsen,J.E., Beier,L., Otzen,D., Borchert,T.V., Frantzen,H.B., Andersen,K.V. and Svendsen,A. (1999a) Electrostatics in the active site of an -amylase. Eur. J. Biochem., 264, 816824.
Qian,M., Haser,R., Buisson,G., Duee,E. and Payan,F. (1994) The active centre of a mammalian -amylase. Structure of the complex of a pancreatic
-amylase with a carbohydrate inhibitor refined to 2.2-Å resolution. Biochemistry, 33, 62846294.[ISI][Medline]
Sarkar,G. and Sommer,S.S. (1990). The `megaprimer' method of site-directed mutagenesis. Biotechniques, 8, 404407.[ISI][Medline]
Shaw,A., Bott,R. and Day,A.G. (1999) Protein engineering of -amylase for low pH performance. Curr. Opin. Biotechnol., 10, 349352.[ISI][Medline]
Sinnot,M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev., 90, 11711202.[ISI]
Strokopytov,B., Penninga,D., Rozeboom,H.J., Kalk,K.H., Dijkhuizen,L. and Dijkstra,B.W. (1995) X-ray structure of cyclodextrin glycosyltransferase complexed with acarbose. Implications for the catalytic mechanism of glycosidases. Biochemistry, 34, 22342240.[ISI][Medline]
Takase,K. (1993) Effect of mutation of an amino acid residue near the catalytic site on the activity of Bacillus stearothermophilus alpha-amylase. Eur. J. Biochem., 211, 899902.[Abstract]
Uitdehaag,J.C., Mosi,R., Kalk,K.H., van der Veen,B.A., Dijkhuizen,L., Withers,S.G. and Dijkstra,B.W. (1999) X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the -amylase family. Nature Struct. Biol., 6, 432436.[ISI][Medline]
Vriend,G. (1990) WHAT IF: a molecular modelling and drug design program. J. Mol. Graphics, 8, 5256.[ISI][Medline]
Wind,R.D., Uitdehaag,J.C., Buitelaar,R.M., Dijkstra,B.W. and Dijkhuizen,L. (1998) Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1. J. Biol. Chem., 273, 57715779.
Received September 25, 2000; revised April 24, 2001; accepted May 14, 2001.