Purification and Characterization of PBP4a, a New Low-Molecular-Weight Penicillin-Binding Protein from Bacillus subtilis

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Abstract
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 β-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.