Institute for Protein Research, Osaka University, Suita, Osaka 565-0871 and 2 Hayashibara Biochemical Laboratories, Inc., Amaseminami, Okayama 700-0834, Japan
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Abstract |
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Keywords: -amylase/maltotetraose-forming amylase/product-complexed mutant/reaction mechanism/X-ray structure
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Introduction |
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The product-complexed two crystal forms (type 1 and 2) of the E219Q mutant of G4-amylase, where the catalytic glutamic acid has been converted to glutamine, were obtained by co-crystallization with maltopentaose. Their structure analyses (Yoshioka et al., 1997) revealed the following three points: (i) the binding mode of each of the non-reducing end four glucose residues (the product maltotetraose) after substrate cleavage; (ii) the structural mode of the recognition of the non-reducing end of the substrate amylose, that determines the exo-acting specificity of this enzyme and (iii) a deformation of a sugar ring at the cleaved reducing end. In other substrate analogue or inhibitor-complexed amylase structures (Kadziola et al., 1994
; Machius et al., 1995
; Qian et al., 1995
; Fujimoto et al., 1998
), this kind of deformation has not yet been observed, except in the case of glucoamylase complexed with D-gluco-dihydro-acabose (Stoffer et al., 1995
).
Further, the fact that the catalytic residue mutated enzyme cleaved the maltopentaose and a bound maltotetraose was observed in the crystal (Yoshioka et al., 1997), implies that even such a mutant enzyme possesses non-zero activity. In G4-amylase, it has been shown that maltotetraose could also be a substrate for a transglycosylation reaction in the synthesis of a maltooligosaccharide derivative (Usui and Murata, 1988
). This fact suggests the possibility of observing the near transition state of this enzyme with bound maltotetraose by using mutant enzymes. Deformation of the reducing end glucose unit in the E219Q mutant of G4-amylase may provide experimental support for this view.
We have now crystallized the product complexes of four different single mutants of the three catalytic residues Glu219, Asp193 and Asp294E219G, D193N, D193G and D294Nand determined the structures of these complexes by the single crystal X-ray diffraction method to elucidate the detailed structural modes of sugar binding at the active site. These structures are compared and the possible roles of mutated residues in the catalytic and substrate binding mechanism are discussed. These findings may be generalized to other enzymes in the -amylase family.
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Materials and methods |
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The activity of each mutant G4-amylase was measured as previously described (Nakada et al., 1990), using 5 ml of 1% soluble starch in 20 mM phosphate buffer (pH 7.0) as substrate, and 0.2 ml of an enzyme solution. The reaction was carried out for 20 min at 40°C and the reducing power of maltotetraose produced was measured by the SomogyiNelson method. One unit of enzyme activity is defined as the amount of enzyme which liberates 1 µmol of maltotetraose per min.
Crystallization
All complexed mutants of G4-amylase were crystallized under the same conditions as those employed for the previously reported E219Q complex, using 14 mg/ml protein in 20 mM TrisHCl buffer pH 7.5, and 0.7 to 0.9 M ammonium sulfate in the presence of 10 mg/ml maltopentaose by the hanging drop vapor diffusion technique at 20°C. The crystals were grown to a size suitable for X-ray diffraction within 12 weeks. All crystals obtained belonged to the orthorhombic complex type 2 form (Yoshioka et al., 1997) and had essentially the same cell dimensions, except for small but significant differences in the D193G crystal, as shown in Table I
.
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All X-ray intensity data were collected with an imaging plate diffractometer, Rigaku RAXIS-IIC, up to 2 Å resolution by using graphite monochromatized Cu-K radiation from a rotating anode generator operated at 40 kV and 100 mA with a focus size of 0.3x0.3 mm2. A summary of data collection is given in Table I
. As shown in this table, we collected two kinds of data sets (D294N0 and D294N) for the Asp294Asn
mutant complex crystal for the reasons described in the Results and discussion.
Structure determination and refinements
Because the crystals of E219G, D193N and D294N mutants co-crystallized with maltopentaose were isomorphous with the previously reported E219Q type 2 complexed crystal (Yoshioka et al., 1997), the refinements carried out were straightforward, starting from the E219Q structure, excluding bound maltotetraose and water molecules and with a replaced mutated residue. Because the crystal of the D193G mutant was not isomorphous with the type 2 crystal, the molecular replacement method was applied by using the program AUTOMR (Matsuura, 1991
). The solution of the molecular replacement was unambiguous and the refinement was carried out using the coordinates derived from the MR solution as the initial model. The molecular model of bound maltotetraose was obtained by fitting the initial model into the (2Fo Fc) map calculated for each complexed crystal, by omitting maltotetraose using the program CHAIN (Sack, 1988
). The bound water molecules were checked against the (Fo Fc) map calculated after incorporating maltotetraose by using the program SEAWAT (Y.Matsuura, unpublished program) to obtain the starting coordinates list of water molecules. We used the restrained least-squares program PROFFT (Hendrickson and Konnert, 1980
; Finzel, 1987
) for refinement calculations of every complexed mutant structure. The manual model corrections for the polypeptide chain were made by using the program CHAIN with interactive computer graphics. After manual model corrections, water molecules were added or removed from the list at points of convergence of refinement based on peak height and hydrogen bond geometry, which were checked using the same system. Reflections in the lowest resolution range (d > 10 Å) were omitted from all calculations. The final refinement statistics are summarized in Table II
. The average positional errors were estimated from the Luzzati plots (Luzzati, 1952
) to be approximately 0.2 Å for every structure (plots not shown).
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The coordinates and the structure factors of E219G, D193N, D193G and D294N complexes have been deposited in the Protein Data Bank (Bernstein et al., 1977) (accession code: 1qi4, r1qi4sf; 1qi3, r1qi3sf; 1qpk, r1qpksf; 1qi5, r1qi5sf, respectively).
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Results and discussion |
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The results of the activity measurements of the mutant enzymes are shown in Table III. All the mutants tested showed a significant decrease in activity, especially for the mutants of the three catalytic residues D193N, E219Q and D294N, where the activities were non-detectable (shown as <0.1 U/mg). These catalytic residue mutants were further checked for residual activity by increasing the concentration of enzyme >1000-fold, which resulted in a significant amount of activity. This fact agrees with the findings in the present structure analyses where maltopentaose used for co-crystallization was hydrolysed into maltotetraose that bound in the active site cleft.
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The overall structural deviations of the polypeptide structure of each of the present mutants from that of the E219Q mutant were calculated by the least-squares method based on the main chain atoms, as shown in Table IV. The overall deviations are small, but significant positional differences (>2 Å) were observed for the side chain atoms, especially in the case of arginine residues, although these mutant proteins form nearly isomorphous crystals.
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For the present structure analyses, single mutations, E219G, D193N, D193G and D294N, have been introduced at the catalytic residues which surround the reducing end glucose unit (Glc4) of the bound product maltotetraose. Major structural changes have occurred in the enzyme active site region and in Glc4. The r.m.s. deviations of the atoms from the E219Q complex in Glc4 were significantly larger (except for E219G) than those in the three non-reducing end glucose units (Glc1Glc3), as shown in Table IV. A superimposed picture of the five complexed mutant enzymes at the active site is shown in Figure 1
, depicting only the reducing end glucose unit of the bound maltotetraose and the three catalytic amino acid residues. As shown in Figure 1
, the Glc4 ring is deformed, adopting a half-chair conformation in all mutant complexes except in D294N. The average position of Glc4 with respect to the enzyme active site is similar to that of the E219Q complex (Yoshioka et al., 1997
) in the present E219G and D294N complexes. However, in D193G the position moved downward to the bottom of the cleft, and in D193N it moved upward from the bottom. The torsion angles about the glucosidic linkages,
and
, of maltotetraose for each complex structure are shown in Table V
. These values are more dispersed among mutants at the Glc34 as compared with those at 12 and 23, but all lie in a reasonable region of conformational energy space (Tran et al., 1989
).
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Possible roles of the catalytic residues
The three catalytic residues Glu219, Asp193 and Asp294 in the present enzyme, and in all -amylase family enzymes (Henrissat, 1991
; Takata et al., 1992
; Svensson, 1994
) are fully conserved in amino acid sequences and in the three-dimensional structures so far known (Katsuya et al., 1998
). This suggests that these catalytic residues share their individual roles and possibly cooperate with each other in the catalytic process in the same way in all family enzymes. Possible mechanism and roles of the catalytic residues deduced from the discussion below are schematically shown in Figure 3
.
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It is implied that the intermediate formation involving an amino acid residue is not necessarily or absolutely indispensable, from the fact that even in the D193G mutant complex crystal, which has no side chain atoms to form a reaction intermediate, the maltopentaose present during the crystallization was hydrolysed to maltotetraose. A probable explanation for this mechanism is that some other component, like an ion or a water molecule, from the solvent replaces the group to form the intermediate.
As described in the previous section, in the D294N mutant complex structure, maltotetraose showed a low occupancy, as demonstrated by high B-values, in binding onto the cleft. This fact suggests that the mutation of Asp294Asn lowered the binding constant of maltotetraose, even though the side chain atoms of Asn294 seem to maintain hydrogen bonds between both O2 and O3 of Glc4. This suggestion is supported by the experiment in which the occupancy was raised (B-values lowered) by soaking the crystal in the substrate solution during data collection. As shown in Table V, in the D294N complex the torsion angle
(Glc34) adopts a small value compared with the others, detwisting the Glc4 ring. This means that Asp294 compels to twist this ring, with the glucosidic oxygen to be cleaved (between 1 and +1) orienting toward an optimum position for the proton donation by Glu219. Furthermore, the most remarkable fact in this mutant complex is that the deformation of Glc4 was not observed. These findings, taken together, make it highly probable that Asp294 is working predominantly to tightly bind the substrate in cooperation with other amino acid residues involved in binding the substrate (Yoshioka et al., 1997
). This tightening and twisting might induce the ring distortion of the reducing end glucose unit (Glc4) in order to lower the reaction potential of the hydrolysis. Thus it may be possible to define the role of Asp294 as fixing the substrate in the catalytic process to help accelerate the acidbase hydrolysis reaction (Figure 3
).
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Notes |
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3 To whom correspondence should be addressed
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References |
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Received May 1, 1999; revised June 8, 1999; accepted June 21, 1999.