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

Abstract

Two isoforms of the heterodimeric enzyme penicillin G acylase (EC 3.5.1.1 I ) 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 a-subunit. The origin of this difference was traced to the partial conversion of the N-terminal Gln of the a-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 cy-and 0-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 P6,22 or P6522 ( a = b = 140.5 A and c = 209.5 A) 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 A resolution, and the calculation of isomorphous replacement phases is under way.

Penicillin acylases revisited: importance beyond their industrial utility

“…However, now it is known that many bacteria (Shewale & Sudhakaran, 1997) produce PVAs also. PGAs from E. coli , Alcaligenes faecalis (Verhaert et al, 1997;Varshney et al, 2012), Providencia rettgeri (Klei et al, 1995;McDonough et al, 1999) and Kluyvera citrophila (Kim et al, 2004a,b;Varshney et al, 2013) have been well-studied with respect to their structure and post-translational processing. While these enzymes are targeted at the periplasmic space, the PGAs from Grampositive bacteria Bacillus megaterium (Senthilvel & Pai, 1998;Yang et al, 2006) and Arthrobacter viscosus are extracellular (Konstantinovic et al, 1994;Ohashi et al, 1989).…”

Improved X-ray diffraction from Bacillus megaterium penicillin G acylase crystals through long cryosoaking dehydration

“…It is produced as either an intracellular or an extracellular enzyme by a variety of microorganisms. PGAs from Gram-negative bacteria such as Escherichia coli (Cole, 1969), Kluyvera cryocrescens (formerly K. citrophila; Barbero et al, 1986), Providencia rettgeri (Klei et al, 1995) and Alcaligenes faecalis (Verhaert et al, 1997) accumulate in the periplasmic space, whereas PGAs from Gram-positive bacteria such as Arthrobacter viscosus (Ohashi et al, 1988) and Bacillus megaterium (Chiang & Bennett, 1967) are generally secreted outside of the cells. Interestingly, primary-structure analysis indicates that the PGAs of Gram-positive and Gram-negative bacteria are distinct, although they belong to the same protein family.…”