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, Frere, 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.