 |
|
|
 |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
490 a.a.
Key reference
DOI no: 10.1074/jbc.M309411200 J Biol Chem 279:7287-7295 (2004) PubMed id: 14638688
Structural and enzymatic analysis of soybean beta-amylase mutants with increased pH optimum. A.Hirata, M.Adachi, A.Sekine, Y.N.Kang, S.Utsumi, B.Mikami. ABSTRACT
Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.
Selected figure(s)
Figure 3.
FIG. 3. Stereoviews of the structural conformation of SBA and the mutants complexed, respectively, with maltose. A, SBA (pH 5.4); B, M51T (pH 5.4); C, E178Y (pH 5.4); D, E178Y (pH 7.1); and E, N340T (pH 5.4). The electron density map (green, 2F[o] - F[c], contoured at 1.0) and the residues around the base catalyst and maltose are illustrated. The hydrogen bonds are indicated by broken lines (black). The mutated residues and the disordered conformations of maltose and the side chain of Glu380 in M51T are cyan. The disrupted hydrogen bonds in each mutants are indicated by broken lines (pink). This figure was generated using BOBSCRIPT (56) and Raster3D (55).
Figure 5.
FIG. 5. Stereoviews of the active site residues (green) and substrate (cyan) in SBA, GA, and BCX. The acid/base and nucleophile catalysts are shown in dark gray. CW refers to the catalytic water molecule. A, the structure of SBA complexed with maltose (Protein Data Bank code 1Q6C). B, the structure of GA complexed with acarbose (Protein Data Bank code 1AGM [PDB] ). C, the structure of BCX complexed with 2FXb [PDB] (Protein Data Bank code 1BVV [PDB] ). The hydrogen bonds are indicated by broken lines. This figure was generated using MOLSCRIPT (54) and Raster3D (55).The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 7287-7295) copyright 2004. Figures were selected by an automated process.
Literature references that cite this PDB file's key reference
PubMed id Reference
16636455 H.Takarada, Y.Kawano, K.Hashimoto, H.Nakayama, S.Ueda, M.Yohda, N.Kamiya, N.Dohmae, M.Maeda, and M.Odaka (2006).
Mutational study on alphaGln90 of Fe-type nitrile hydratase from Rhodococcus sp. N771.Biosci Biotechnol Biochem, 70, 881-889.
PDB code: 2zcf The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.
 |