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Expression, purification, crystallization and preliminary X‐ray diffraction analysis of acylpeptide hydrolase from Deinococcus radiodurans
Author(s) -
Are Venkata Narayana,
Ghosh Biplab,
Kumar Ashwani,
Yadav Pooja,
Bhatnagar Deepak,
Jamdar Sahayog N.,
Makde Ravindra D.
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14017944
Subject(s) - deinococcus radiodurans , hydrolase , enzyme , thermophile , biochemistry , active site , peptide , monomer , molecular replacement , biology , serine , chemistry , stereochemistry , gene , organic chemistry , polymer
Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of an N ‐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 group P 2 1 , with unit‐cell parameters a = 77.6, b = 189.6, c = 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å 3 Da −1 corresponds 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 in D. radiodurans .