Title

The purification, crystallization, and characterization of the enzyme penicillin G acylase from {\it Providencia rettgeri\/}

Date of Completion

January 1995

Keywords

Biology, Molecular|Chemistry, Biochemistry|Biophysics, General

Degree

Ph.D.

Abstract

In its mature state, the enzyme penicillin G acylase (EC 3.5.1.11) from Providencia rettgeri ATCC 31052 (strain Bro1) is composed of two dissimilar subunits, $\alpha$ and $\beta$, much like the heterodimeric penicillin G acylases from Escherichia coli and Kluyvera citrophila. Improved homogeneity compared to previous efforts to purify this penicillin G acylase was achieved with an additional step of anion exchange chromatography. Two isoforms of comparable enzymatic activity that differed slightly in the molecular weight and pI of their respective $\alpha$ subunit were separated. The origin of these differences was traced to the partial conversion of the N-terminal Gln of the $\alpha$ subunit to pyrrolidonecarboxylic acid (pyro-Glu). The boundaries of the mature enzyme within the translated DNA sequence of the wild-type propeptide (GenBank entry 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 $\alpha$ and $\beta$ subunits, based on these termini, were 23.7kDa and 62.2kDa respectively. Both isoforms were crystallized independently as hexagonal bipyramids up to 0.60mm in diameter in either space group P6$\sb1$22 or P6$\sb5$22 (a = b = 140.5A and c = 209.5A) from ammonium sulfate solutions buffered by 50mM potassium phosphate at pH 7.5. The presence of glycerol, while not required, facilitated crystal growth. Native and heavy atom data sets were collected to 3.0A resolution. Heavy atom positions were located and refined for the KAuCl$\sb4$ derivative. Single isomorphous replacement phases with anomalous scattering and solvent flattening were calculated. The interpretation of the electron density map is under way, as is the search for additional phase information from other derivatives. ^

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