INTRODUCTION ........................................................ 291 General ............... ......................................... 291 Cell Waill Structure and Synthesis ....................... ........................ 292 Identification of the Target of Penicillin Action .......... ........................ 293 PENICILLIN-BINDING COMPONENTS .............. .......................... 296 What Is Actually Bound? ....................................................... 298 How Much Penicillin Do Cells Bind? ................. ........................... 298 Is the Penicillin Bound Covalently? ................... .......................... 299 To What Group Does PenicillinBind?..... 299 Under What Conditions Can Penicillin Be Released from the PBCs?........ ........ 300 What Is the Physical Nature ofthe PBCs? ............. .......................... 300 Are PBCs Located in Peripheral Membranes, Mesosomes, or Both? ...... ........ 301 Do Organisms Have One or Multiple Proteins Which Bind Penicillin? ..... ...... 301 Isolation ofPBCs by Covalent Affinity Chromatography ........ ................. 303 What Is the Relationship of the PBCs to the Killing Site? ....... ................. 304 Biochemical Evidence for the Involvement ofPBCs in Peptidoglycan Synthesis . . 305 PENICILLIN-SENSITIVE ENZYMES ................ ........................... 306 Transpeptidase......................................................... 306 Transpeptidase assayed with natural acceptor in a coupled system ............ 306 Transpeptidase assayed with unnatural acceptor in a coupled system ..... ..... 307 Transpeptidase assayed in a simple, uncoupled system ....... ................. 307 D-Alanine Carboxypeptidase .................................................... 308 E. coli carboxypeptidase ...................................................... 309 Carboxypeptidases from Bacillus ................... .......................... 309 Streptomyces carboxypeptidases .................... .......................... 309 Endopeptidase......................................................... 313 Other Activities ........................................................ 314 Number of Enzymes Corresponding to the Different Penicillin-Sensitive Enzymatic Activities ........................................................ 314 Relationship ofthe Penicillin-Sensitive Enzymes to the PBCs ....... ............. 316 Physiological Functions ofthe Penicillin-Sensitive Enzymes ....... .............. 316 Physiological Evidence for the Existence of Multiple Transpeptidases ..... ...... 318 Kinetics of Penicillin Inhibition ................................................. 319 Models for the Interaction ofPenicillin with Its Target ........ .................. 321 MECHANISM OF KILLING BY PENICILLINS .......... ....................... 324 Mutant Analysis........................................................ 324 Evidence That Penicillin Kills by Inhibiting Cross-Linking ...................... 324 Killing by Amidino Penicillin ................................................... 325 Transfer of Penicillin Side Chain ................................................. 325 CONTROL OF CELL WALL SYNTHESIS ............. .......................... 326 SUMMARY ................ ....................................... 327 LITERATURE CITED ....................................................... 327

Interaction of penicillin with the bacterial cell: penicillin-binding proteins and penicillin-sensitive enzymes.

The most commonly used β-lactam antibiotics for the therapy of infectious diseases are penicillin and cephalosporin. Penicillin is produced as an end product by some fungi, most notably by Aspergillus (Emericella) nidulans and Penicillium chrysogenum. Cephalosporins are synthesized by both bacteria and fungi, e.g., by the fungus Acremonium chrysogenum (Cephalosporium acremonium). The biosynthetic pathways leading to both secondary metabolites start from the same three amino acid precursors and have the first two enzymatic reactions in common. Penicillin biosynthesis is catalyzed by three enzymes encoded by acvA (pcbAB), ipnA (pcbC), and aatA (penDE). The genes are organized into a cluster. In A. chrysogenum, in addition to acvA and ipnA, a second cluster contains the genes encoding enzymes that catalyze the reactions of the later steps of the cephalosporin pathway (cefEF and cefG). Within the last few years, several studies have indicated that the fungal β-lactam biosynthesis genes are controlled by a complex regulatory network, e.g., by the ambient pH, carbon source, and amino acids. A comparison with the regulatory mechanisms (regulatory proteins and DNA elements) involved in the regulation of genes of primary metabolism in lower eukaryotes is thus of great interest. This has already led to the elucidation of new regulatory mechanisms. Furthermore, such investigations have contributed to the elucidation of signals leading to the production of β-lactams and their physiological meaning for the producing fungi, and they can be expected to have a major impact on rational strain improvement programs. The knowledge of biosynthesis genes has already been used to produce new compounds.

Molecular Regulation of β-Lactam Biosynthesis in Filamentous Fungi

Publisher Summary This chapter describes the process of microbial transformation of antibiotics into useful compounds, which is mostly concentrated upon the production of 6-aminopenicillanic acid (6-APA) by enzymatic hydrolysis of biosynthetic penicillins. The chapter describes biotransformation processes of penicillins into 6-APA that use, as a source of acylase, intact bacterial cells, fungal mycelium, fungal spores, crude cell extracts, purified enzyme preparations, stabilized enzymes, and supernatant liquid of extracellular acylase-producing microorganisms. The chapter examines methods to isolate and purify penicillin acylases and to stabilize these enzyme preparations. It reviews the physicochemical properties and the degradation reactions in relation to the structure of penicillins and describes the reaction of cysteine and related compounds with penicillins, resulting in a loss of antibacterial activity. 6-APA was originally produced by fermentation synthesized in a purely chemical way. Enzymatic transformation of biosynthetic penicillins into 6-APA is an industrial process of economic importance as the main source of 6-APA used in the preparation of semisynthetic penicillins.

Microbial Penicillin Acylases

Control of antibiotic synthesis by phosphate

The acyl coenzyme A (CoA):6-aminopenicillanic acid (6-APA) acyltransferase of Penicillium chrysogenum AS-P-78 was purified to homogeneity, as concluded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The enzyme is a monomer with a molecular weight of 30,000 +/- 1,000 and a pI of about 5.5. The optimal pH and temperature were 8.0 and 25 degrees C, respectively. This enzyme converts 6-APA into penicillin by using phenylacetyl CoA or phenoxyacetyl CoA as acyl donors. The pure enzyme showed a high specificity and affinity for 6-APA and did not accept benzylpenicillin, 7-aminocephalosporanic acid, cephalosporin C, or isocephalosporin C as substrates. The enzyme converted isopenicillin N into penicillin G, although with a lower efficiency than when 6-APA was used as the substrate. It did not show penicillin G acylase activity. The acyl CoA:6-APA acyltransferase required dithiothreitol or other thiol-containing compounds, and it was protected by thiol-containing reagents against thermal inactivation. The acyltransferase was inhibited by several divalent and trivalent cations and by p-chloromercuribenzoate and N-ethylmaleimide. The activity was absent in four different mutants that were blocked in penicillin biosynthesis.

Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum.

Isotopic exchange of 35S between penicillins and 6-amino-penicillanic acid (6-APA) was observed in cell-free extracts of Penicillium chrysogenum. Sulfhydryl-containing compounds were required for activity. Dithiothreitol, dithioerythritol, mercaptoethanol, and glutathione served as activators. The acyltransferase was purified threefold by adsorption on calcium phosphate gel at pH 6 and elution at pH 8. The partially purified enzyme showed maximal activity at pH 8. The enzyme was stable at 25 C for at least 30 min at pH 8. Dissociable inhibitors or activators, other than the sulfhydryl-containing compounds, could not be demonstrated in the enzyme preparation. An apparent Michaelis constant of 1.5 ± 0.5 mm was determined for penicillin G at a 6-APA concentration of 5 mm. The enzyme did not appear to possess penicillin amidase activity. The exchange mechanism probably involves an acyl-enzyme intermediate. Penicillins V, G, K, X, and dihydro F showed isotopic exchange with 35S-6-APA. Penicillin N, methylpenicillin, and phenyl-penicillin did not show exchange. The level of acyltransferase in P. chrysogenum 51-20F3 was measured at times during the fermentation. The level of activity increased threefold between 40 and 55 hr, remaining high until about 90 hr.

Penicillin Acyltransferase in Penicillium chrysogenum

Pruess, David L. (University of Wisconsin, Madison), and Marvin J. Johnson. Enzymatic deacylation of S(35)-benzylpenicillin. J. Bacteriol. 90:380-383. 1965.-S(35)-benzylpenicillin, penicilloic acid, and penilloic acid were deacylated by cell suspensions of Escherichia coli and Micrococcus roseus. Both cultures deacylated penicillin most rapidly and penilloic acid least rapidly. The deacylase activity of M. roseus against penicilloic acid was cell-bound, probably requiring a metal ion for activity.

David L. Pruess

Papers

Penicillin Acyltransferase in Penicillium chrysogenum

Enzymatic deacylation of s35-benzylpenicillin.