Two-step Dimerization for Autoproteolysis to Activate Glycosylasparaginase

Abstract

Glycosylasparaginase (GA) is an amidase and belongs to a novel family of N-terminal nucleophile hydrolases that use a similar autoproteolytic processing mechanism to generate a mature/active enzyme from a single chain protein precursor. From bacteria to eukaryotes, GAs are conserved in primary sequences, tertiary structures, and activation of amidase activity by intramolecular autoproteolysis. An evolutionarily conserved HisAsp-Thr sequence is cleaved to generate a newly exposed N-terminal threonine, which plays a central role in both autoproteolysis and in its amidase activity. We have recently determined the crystal structure of the bacterial GA precursor at 1.9-Å resolution, which reveals a highly distorted and energetically unfavorable conformation at the scissile peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not understood how the polypeptide chain distortion was generated and preserved during the folding of GA to trigger autoproteolysis. An obstacle to our understanding of GA autoproteolysis is the uncertainty concerning its quaternary structure in solution. Here we have revisited this question and show that GA forms dimers in solution. Mutants with alterations at the dimer interface cannot form dimers and are impaired in the autoproteolytic activation. This suggests that dimerization of GA plays an essential role in autoproteolysis to activate the amidase activity. Comparison of the melting temperatures of GA dimers before and after autoproteolysis suggests two states of dimerization in the process of enzyme maturation. A two-step dimerization mechanism to trigger autoproteolysis is proposed to accommodate the data presented here as well as those in the literature.

Crystallographic Snapshot of Glycosylasparaginase Precursor Poised for Autoprocessing

“…15 Another important aspect is that the structure of the T152C mature enzyme had been shown to be identical with that of the wild-type enzyme. 35,36 Thus, the T152C precursor appears to be an excellent candidate for a crystallographic snapshot of a GA pre-autoproteolysis structure.…”

Identification of Small Molecule Compounds for Pharmacological Chaperone Therapy of Aspartylglucosaminuria

“…In the case of the mutant AGA precursors, this rearrangement is severely reduced, preventing the cleavage into subunits. It has been shown that dimerization of two precursor molecules is a prerequisite for the cleavage, and some AGU mutations in the dimer interface prevent dimerization and thus activation22930. However, this appears not to be the case with T122K and AGU-Fin, which both become processed into subunits when coexpressed with the wildtype AGA.…”

Release of an HtrA-Like Protease from the Cell Surface of Thermophilic Brevibacillus sp. WF146 via Substrate-Induced Autoprocessing of the N-terminal Membrane Anchor

“…Cross-linking of proteins was carried out according to the method of Wang and Guo (2003) with slight modification. Briefly, cross-linking was performed at approximately 0.1 mg/ml protein and 0.001% glutaraldehyde in 50 mM potassium-phosphate buffer (pH 7.5), and incubated for 4 h at room temperature.…”