Abstract
Soluble, truncated mutant and wild-type forms of penicillin-binding protein 5 (sPBP 5) from Escherichia coli were produced in large amounts by placing the dacA gene that encodes PBP 5 under the control of the trp-lac fusion promoter. The 3' end of the dacA gene used in this study contains a stop codon that results in the deletion of 15 amino acids from the carboxyl terminus and the production of a soluble protein. Using oligonucleotide-directed mutagenesis, the role of cysteine 115 in the mechanism of sPBP 5 was investigated. Alkylation of cysteine 115 with sulfhydryl reagents has previously been shown to inhibit severely the D-alanine carboxypeptidase activity of PBP 5. Alkylation also inhibits the hydrolysis of bound penicillin G, with only a slight effect on its binding. Cysteine 115 in sPBP 5 was changed to either a serine (sPBP 5C-S) or an alanine (sPBP 5C-A) residue. The wild-type and mutant sPBPs were purified in milligram amounts from induced cultures by ampicillin affinity chromatography. The mutant PBPs showed only a 2-fold increase in the half-life of the penicilloyl-PBP complex, and had a binding affinity for penicillin G identical to wild-type PBP 5. The Km for the release of D-alanine from the peptide L-Ala-D-gamma-Glu-L-Lys-D-Ala-D-Ala was 5.0, 3.5, and 7.8 mM for PBP 5, PBP 5C-S, and PBP 5C-A, respectively, while the values for Vmax were 2.5, 3.3, and 5.1 mumol/min/mg. From these data it was concluded that the cysteine residue does not directly participate in the enzymatic mechanism.
Highlights
Soluble, truncated mutant and wild-type forms of D-Ala-D-AlaCOOH terminus of the pentapeptide [2]
Forms the covalent bond with both substrate and penicillin has been identified as serine 44 [17, 18].A recent study on the interaction of PBP 5 with the cytoplasmic membrane has shown that the removal of as little as 10 amino acids from the carboxyl terminus is sufficient to result in the release of Nascent peptidoglycan in Escherichia coli consists of alternating @ 1,4-linked N-acetylglucosamine and N-acetylmuramic acid residues, in which the N-acetylmuramic acid residues are substitutedwith the pentapeptide L-Ala-D-y-Glu-mdiaminopimelic acid-D-Ala-D-Ala [1].Cellwall synthesizing enzymes,which are located in the cytoplasmic membrane, react with the pentapeptide to form an acyl-enzyme intermediate, with the concomitant release of the carboxyl-terminal D-alanine residue
Osmotic shock of E . coli HBlOl harboringpRN20.4 resulted in therelease of a significant amount of sPBP 5', when assayed by SDSPAGE and fluorography (Fig. 2). sPBP 5' was isolated in yields approaching 1 mg of protein/liter of bacterial culture and was identical to detergent-soluble PBP 5' in its binding affinity for penicillin G
Summary
Cloning and Purification of Soluble PBP 5'"Pratt e t al. [19] have shown that thedeletion of as few as 10 amino acids from the carboxyl terminus of PBP 5 results itnhe production of a soluble form of the protein. When the lowcopy number plasmid pBS42-4, which encodes soluble, truncated wild-type PBP 5, is transformed into E. coli, sPBP 5 is produced at only 3-5 times the level of endogenous PBP 5 [19]. This amount was deemedunsatisfactory for large scale purification of wild-type and mutantproteins, and the dacA gene was put under the transcriptional control of an inducible promoter. The EcoRI-BstEII fragmentfrom pATG4 was used to replace the corresponding fragment of pKAN20.4 This new construction was designated pATG20.4 (Fig. 1).The level of sPBP 5’ in the osmotic shock fluid from XA90/pATG20.4 was compared to that from XA90/pKAN20.4 and HB101/pRN20.4. SPBP 5 showed a muchlower stoichiometry of penicillin binding (-0.65 mol/mol protein) than did sPBP 5‘“ or sPBP
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