title: Structural constraints on protein self-processing in L-aspartate-alpha-decarboxylase.
abstract: 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.
authors.affiliation: Department of Biochemistry, 80 Tennis Court Road, Cambridge CB2 1GA, UK. florian@cryst.bioc.cam.ac.uk
authors.forename: Florian
authors.initials: F
authors.lastname: Schmitzberger
authors.forename: Mairi L
authors.initials: ML
authors.lastname: Kilkenny
authors.forename: Carina M C
authors.initials: CM
authors.lastname: Lobley
authors.forename: Michael E
authors.initials: ME
authors.lastname: Webb
authors.forename: Mladen
authors.initials: M
authors.lastname: Vinkovic
authors.forename: Dijana
authors.initials: D
authors.lastname: Matak-Vinkovic
authors.forename: Michael
authors.initials: M
authors.lastname: Witty
authors.forename: Dimitri Y
authors.initials: DY
authors.lastname: Chirgadze
authors.forename: Alison G
authors.initials: AG
authors.lastname: Smith
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
authors.forename: Tom L
authors.initials: TL
authors.lastname: Blundell
mesh_terms: 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
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C019104:aspartate-alpha-decarboxylase; D005968:Glutamate Decarboxylase; D000409:Alanine