Expression, purification, crystallization and preliminary X-ray diffraction analysis of acylpeptide hydrolase fromDeinococcus radiodurans

Abstract

Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of anN-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the β-amyloid peptides implicated in Alzheimer's disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium,Deinococcus radiodurans(APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space groupP21, with unit-cell parametersa= 77.6,b= 189.6,c= 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å3 Da−1corresponds to four monomers, each with a molecular mass of ∼73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme inD. radiodurans.