Activity of wheat alpha-amylase inhibitors towards bruchid alpha-amylases and structural explanation of observed specificities.
Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five alpha-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect alpha-amylases and porcine pancreatic alpha-amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect alpha-amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human alpha-amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.
Franco
O L
OL
Depto. de Biologia Celular, Instituto de Química, UnB, Brasilia, Brazil.
Rigden
D J
DJ
Melo
F R
FR
Bloch
C
C
Silva
C P
CP
Grossi de Sá
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D007313:Insecta / Q000187:drug effects / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010179:Pancreas / Q000201:enzymology; D010571:Pest Control; D011485:Protein Binding; D011487:Protein Conformation; D012463:Saliva / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D014908:Triticum / Q000737:chemistry*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000737:chemistry
D004791:Enzyme Inhibitors; D000516:alpha-Amylases