Change in the crystal packing of soybean beta-amylase mutants substituted at a few surface amino acid residues.
In spite of the high similarity of amino acid sequence and three-dimensional structure between soybean beta-amylase (SBA) and sweet potato beta-amylase (SPB), their quaternary structure is quite different, being a monomer in SBA and a tetramer in SPB. Because most of the differences in amino acid sequences are found in the surface region, we tested the tetramerization of SBA by examining mutations of residues located at the surface. We designed the SBA tetramer using the SPB tetramer structure as a model and calculating the change of accessible surface area (DeltaASA) for each residue in order to select sites for the mutation. Two different mutant genes encoding SB3 (D374Y/L481R/P487D) and SB4 (K462S added to SB3), were constructed for expression in Escherichia coli and the recombinant proteins were purified. They existed as a monomer in solution, but gave completely different crystals from the native SBA. The asymmetric unit of the mutants contains four molecules, while that of native SBA contains one. The interactions of the created interfaces revealed that there were more intermolecular interactions in the SB3 than in the SB4 tetramer. The substituted charged residues on the surface are involved in interactions with adjacent molecules in a different way, forming a new crystal packing pattern.
Kang
You-Na
YN
Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Adachi
Motoyasu
M
Mikami
Bunzo
B
Utsumi
Shigeru
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D013025:Glycine max / Q000201:enzymology*; D001614:beta-Amylase / Q000737:chemistry* / Q000235:genetics*
D000596:Amino Acids; D001614:beta-Amylase