Purification and preliminary crystallographic studies of penicillin G acylase from Providencia rettgeri

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Abstract
Two isoforms of the heterodimeric enzyme penicillin G acylase (EC 3.5.1.11) from Providencia rettgeri ATCC 31052 (strain Brol) were purified to near homogeneity. The isoforms exhibited comparable enzymatic activities but differed slightly in the molecular weight and pI of their respective α‐subunit. The origin of this difference was traced to the partial conversion of the N‐terminal Gln of the α‐subunit to pyrrolidonecarboxylic acid (pyro‐Glu). The boundaries of the mature enzyme within the translated DNA sequence of the wild‐type propeptide (GenBank M86533) were determined. The results conclusively identified the length of the signal peptide and the position of the spacer cleaved from the propeptide to form the active heterodimer. The molecular weights of the α‐ and β‐subunits, based on these termini, were 23.7 and 62.2 kDa, respectively. Both isoforms were crystallized independently as hexagonal bipyramids up to 0.60 mm in diameter in either space group P6122 or P6522 (a = b = 140.5 Å and c = 209.5 Å) from ammonium sulfate solutions buffered by 50 mM potassium phosphate at pH 7.5. The presence of glycerol, although not required, facilitated crystal growth. Native and heavy atom derivative data were collected to 3.0 Å resolution, and the calculation of isomorphous replacement phases is under way.