Structural constraints on protein self-processing in L-aspartate-alpha-decarboxylase.
Aspartate decarboxylase, which is translated as a pro-protein, undergoes intramolecular self-cleavage at Gly24-Ser25. We have determined the crystal structures of an unprocessed native precursor, in addition to Ala24 insertion, Ala26 insertion and Gly24-->Ser, His11-->Ala, Ser25-->Ala, Ser25-->Cys and Ser25-->Thr mutants. Comparative analyses of the cleavage site reveal specific conformational constraints that govern self-processing and demonstrate that considerable rearrangement must occur. We suggest that Thr57 Ogamma and a water molecule form an 'oxyanion hole' that likely stabilizes the proposed oxyoxazolidine intermediate. Thr57 and this water molecule are probable catalytic residues able to support acid-base catalysis. The conformational freedom in the loop preceding the cleavage site appears to play a determining role in the reaction. The molecular mechanism of self-processing, presented here, emphasizes the importance of stabilization of the oxyoxazolidine intermediate. Comparison of the structural features shows significant similarity to those in other self-processing systems, and suggests that models of the cleavage site of such enzymes based on Ser-->Ala or Ser-->Thr mutants alone may lead to erroneous interpretations of the mechanism.
Schmitzberger
Florian
F
Department of Biochemistry, 80 Tennis Court Road, Cambridge CB2 1GA, UK. florian@cryst.bioc.cam.ac.uk
Kilkenny
Mairi L
ML
Lobley
Carina M C
CM
Webb
Michael E
ME
Vinkovic
Mladen
M
Matak-Vinkovic
Dijana
D
Witty
Michael
M
Chirgadze
Dimitri Y
DY
Smith
Alison G
AG
Abell
Chris
C
Blundell
Tom L
TL
D000409:Alanine; D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C019104:aspartate-alpha-decarboxylase; D005968:Glutamate Decarboxylase; D000409:Alanine