Purification and preliminary crystallographic studies of penicillin G acylase from Providencia rettgeri.
Two isoforms of the heterodimeric enzyme penicillin G acylase (EC 3.5.1.11) from Providencia rettgeri ATCC 31052 (strain Bro1) were purified to near homogeneity. The isoforms exhibited comparable enzymatic activities but differed slightly in the molecular weight and pI of their respective alpha-subunit. The origin of this difference 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 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 23.7 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(1)22 or P6(5)22 (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.
Klei
H E
HE
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269-3125, USA.
Daumy
G O
GO
Kelly
J A
JA
D000595:Amino Acid Sequence; D002511:Cephalosporins / Q000378:metabolism; D018360:Crystallography, X-Ray; D015180:Electrophoresis, Gel, Two-Dimensional; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010399:Penicillin Amidase / Q000737:chemistry* / Q000302:isolation & purification; D010400:Penicillin G / Q000378:metabolism; D011532:Providencia / Q000201:enzymology*; D017421:Sequence Analysis
D002511:Cephalosporins; D007527:Isoenzymes; C025163:cephalosporin C; D010399:Penicillin Amidase; D010400:Penicillin G