Purification and characterization of PBP4a, a new low-molecular-weight penicillin-binding protein from Bacillus subtilis.
Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits DD-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781-788, 1992), which is rapidly inactivated by many beta-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k(2)/K) for the acylation of the essential serine by benzylpenicillin is 300,000 M(-1) s(-1) for the Actinomadura sp. strain R39 peptidase, 1,400 M(-1) s(-1) for B. subtilis PBP4a, and 7,000 M(-1) s(-1) for Escherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k(2)/K = 46,000 M(-1) s(-1)). PBP4a is also much more thermostable than the R39 enzyme.
Duez
C
C
Centre d'Ingénierie des Protéines and Laboratoire d'Enzymologie, Institut de Chimie, Université de Liège, B-4000 Liège, Belgium.
Vanhove
M
M
Gallet
X
X
Bouillenne
F
F
Docquier
J
J
Brans
A
A
Frère
J
J
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D006602:Hexosyltransferases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007769:Lactams / Q000378:metabolism; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D010957:Plasmids; D013869:Thiolester Hydrolases / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007769:Lactams; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D013869:Thiolester Hydrolases; D002267:Muramoylpentapeptide Carboxypeptidase