Biosynthesis of the Penicillins

The role of cysteine in penicillin biosynthesis was first demonstrated by Arnstein and Grant 210,211) who synthesised L- and D-cystine labelled in the P-position with or with or When these compounds were fed to cultures of Penicillium chrysogenum the L-isomer was found to be better incorporated into the resulting benzylpenicillin. Using a series of chemical degradations (Fig. 3), they were able to demonstrate the specific incorporation of cystine into benzylpenicillin. It was concluded that cystine is a direct precursor of penicillin, probably after reduction to cysteine. 

In a double-labelled experiment it was shown that when cystine was fed to P. chrysogenum the isotope ratio of the starting material was retained unchanged in the isolated benzylpenicillin. This demonstrated that cystine (rather than penicillamine) was the source of the thiazolidine sulphur atom. Further evidence on the origin of the penicillin sulphur atom was provided by Stevens etal, 212) who showed that the label from p S]-cysteine was better incorporated into benzylpenicillin than label from P S]-sulphate. In competition experiments it was shown that L-cysteine or L-cystine were utilised in preference to p S]-sulphate, whereas D-cysteine and DL-penicillamine had no effect on the incorporation of label from sulphate into benzylpenicillin. 

In a similar experiment Aberhart et al. (218) fed DL-cystines chirally labelled at carbon-3 with tritium to P. chrysogenum in combination with C-cystine and found that with the (3R) compound 76% of the tritium label was retained whereas with the (3iS) compound only 21% of the label was retained. 

In summary, L-cysteine is the specific precursor of the P-lactam moiety of benzylpenicillin. The amino acid is incorporated with retention of configuration at the P-carbon and a 2,3-dehydro intermediate is not involved. 

Warren etal. (225) achieved results similar to those above using a Cephalosporium sp. that produced penicillin N (260). They found that 

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As seen in the structure, moxalactam disodium has three asymmetric centers. Two centers in the ring system, C6 and C7, are stereospecifically defined during the biosynthesis of the penicillin used to produce the compound. A third asymmetric center exists adjacent to the amide carbon on the side chain of the antibiotic. The configuration of this center is free to equilibrate and thus a pair of diasterioisomers is possible for moxalactam disodium. The rate of interconversion between the isomers increases with increasing acidity, with the maximum rate occurring at a pH of about 2.5. For solutions with pH lower than 2.5, the rate of interconversion is decreased only slightly from the maximum rate. 

The relationship between the biosynthesis of the penicillin and cephalosporin nuclei [127] is shown in Fig. 8.25. The common intermediate in the biosynthesis of penicillins and cephalosporins is isopenicillin N (IPN), which in Penicil-lium is converted into penicillin G by replacement of the L-2-aminoadipyl side-chain with externally supplied phenylacetic acid, mediated by IPN acyl transferase (IPN AcT). In the cephalosporin-producing Ammonium chrysogenum, IPN is subjected to an enzymatic ring expansion. 

Fig. 8.25 Biosynthesis of the penicillin and cephalosporin nuclei. Compounds Aad, L-2-aminoadipate IPN, isopenicillin N. Enzymatic activity IPN AcT, IPN acyltransferase.

Although total syntheses of the penicillins have been elaborated, they are too expensive relative to the extraction from moulds or to semisynthetic procedures. This also applies to biomimetic syntheses, i.e. to total syntheses based on the biosynthesis of the compounds [108]. The biosynthesis of the penicillins starts from a peptide 2 which is derived from the amino acids cysteine and valine ... 

Baxter, R.L., G. A. Thomson, and A.I. Scott Synthesis and Biological Activity of 5-(L-a-Aminoadipoyl)-L-cysteinyl-N-hydroxy-D-valine a Proposed in the Biosynthesis of the Penicillins. J. Chem. Soc. Chem. Commun. (London) 1984, 32. 

D-Penicillamine, (CH3)2-CSH-CH -NH2-C02H, which was the first degradation product to be obtained from penicillin and is one of the starting materials for total chemical synthesis, plays no part in the biosynthesis of the penicillin molecule and does not appear to be formed in nature as an independent unit. 

The difficulty of determining the role of intermediates, free or enz5une-bound, in the biosynthesis of the penicillins and cephalosporin C has depended partly on the rapid turnover and very low concentrations of such substances in the mycelial cell and partly on the existence of permeability barriers which complicate the interpretations of results obtained when possible intermediates are added to the extracellular fluid. Some of these difficulties would be removed by a cell-free system in which all or part of the bios5mthetic pathways could be followed. Mycelium of the Cephalosporium sp. which has been disrupted by ultrasonic treatment in buffered saline is able to produce penicillin N and cephalosporin C at a rapid rate after a period of lag (Demain, 1963 Abraham et al, 1964). Centrifugation of these preparations yield a supernatant fraction and a sediment. The supernatant alone produces no antibiotics and the sediment alone relatively little. Thus it appears that the enzyme systems necessary for the production of penicillin N and cephalosporin C are present in the sediment and precursors in the supernatant. Further studies are necessary to determine how far, if at all, the permeability barriers of the normal cell are permanently broken down in the sediment fraction. 

Although a range of penicillins could be produced by directed biosynthesis using precursor feeding, this approach is limited by the toxicity of the precursors, the ability of the penicillin producing cells to take up the precursor and by the capability of the acyltransferase to transfer the acyl groups onto the 6-aminopenicillanic add moiety. 

An example for proteases are the (3-lactamases that hydrolyse a peptide bond in the essential (3-lactam ring of penicillins, cephalosporins, carbapenems and monobac-tams and, thereby, iireversibly inactivate the diug. 13-lactamases share this mechanism with the penicillin binding proteins (PBPs), which are essential enzymes catalyzing the biosynthesis of the bacterial cell wall. In contrast to the PBPs which irreversibly bind (3-lactams to the active site serine, the analogous complex of the diug with (3-lactamases is rapidly hydrolyzed regenerating the enzyme for inactivation of additional (3-lactam molecules. 

Studies on the mode of action of the penicillins in inhibiting bacterial cell-wall biosynthesis suggest that the members of this class of antibiotics (including the closely related cephalosporins) are conformationally restricted substrate analogs... 

Due to the importance of penicillins and cephalosporins, which certainly saved the lives of hundreds of millions of people, especially the mechanism of the penicillin biosynthesis was intensively investigated (24). Although total syntheses of penicillins are known now for more than 50 years, this reaction in which both rings of the penicillin structure are formed in one step is still impossible to reproduce for... 

The shikimate pathway was identified through the study of ultraviolet light-induced mutants of E. coli, Aerobacter aerogenes, and Neurospora. In 1950, using the penicillin enrichment technique (Chapter 26), Davis obtained a series of mutants of E. coli that would not grow without the addition of aromatic substances.4 5 A number of the mutants required five compounds tyrosine, phenylalanine, tryptophan, p-aminobenzoic acid, and a trace of p-hydroxybenzoic acid. It was a surprise to find that the requirements for all five compounds could be met by the addition of shikimic acid, an aliphatic compound that was then regarded as a rare plant acid. Thus, shikimate was implicated as an intermediate in the biosynthesis of the three aromatic amino acids and of other essential aromatic substances.6 7... 

The biosynthesis of the p-lactam antibiotic penicillin (Fig. 65), and also of cephalosporin, involves incorporation of L-valine and the question arises as to which of the two diastereotopic terminal methyl groups of the valine occupies which position in the penicillin. (In the case of cephalosporin, the question is as to which methyl group is incorporated into the six-membered ring and which becomes the methylene group of the carbinyl acetate.) The problem has been solved by two groups 65d,141) by synthesis of specifically 13C methyl labeled valine (cf. Fig. 42, and p. 35) which was then biosynthetically incorporated in the antibiotics. The position of the 13C in the resulting antibiotic molecules was determined by 13C NMR spectroscopy. 

Cell walls in bacteria are porous structures that provide structural integrity and physically protect the cell from swelling and bursting in a hypotonic environment. If the cell wall is removed, the bacteria assume a spherical shape. Cell walls contain specific antigens useful for the diagnosis of some infectious diseases. Inhibitions of the biosynthesis of the bacterial cell wall is the basis for the bactericidal action of penicillin and some other antibiotics. 

Inhibitions of Cell Wall Biosynthesis. All bactericidal modes of action involve the alteration or destruction of some component (s) of the cell whose physiological function is of vital importance and cannot be compensated for or repaired by other cell constituents. This has been described above for the bacterial chromosome (DNA) and for the total population of bacterial ribosomes. The most prominent bactericidal effect, however, is caused by interferences with the biosynthesis of the cell wall polymer. Such a mode of action was proposed for penicillin on the basis of morphological observations in 1946s°)when the underlying biochemistry was still unknown. 

Carbon atom 5 of the penicillins [as (198)] has been shown to derive from C-3 of cyst(e)ine [as (201)]. In feeding experiments with (2R, 3R)-[2,3- H2]cysteine, 2R, 3S)-[3- H]cysteine, and [3,3 - H2]cystine the fates of the hydrogen atoms originally present at C-3 of cysteine have been examined. As expected tritium label was to some extent lost from C-2 of cysteine. More importantly, retention of nearly one half of the tritium from [3,3 - H2]cystine indicated that hydrogen loss from C-3 was stereospecific. Further, high retention of tritium at C-5 of the penicillin G (199) derived from (2R, 3l )-[2,3- H2]cysteine and low retention in the experiment with (2R, 3S)-[3- H]cysteine demonstrates that it is the 3-pro-S hydrogen of cysteine which is lost in penicillin biosynthesis, and overall the transformation occurs with retention of configuration, as it does for valine. 

The molecular targets for the antibacterial activity of the penicillin and related j8-lactam antibiotics such as the cephalosporins are a group of bacterial enzymes known as penicillin-binding proteins (PBPs). The PBPs are essential to the final stages of bacterial cell wall biosynthesis. Penicillin and other j8-lactam antibiotics inhibit PBPs, thereby inhibiting bacterial cell wall biosynthesis, which eventually results in bacterial cell lysis. (Vancomycin and cycloserine are nonpenicillin antibiotics that also inhibit bacterial cell wall biosynthesis through other mechanisms.)... 

Synthesis of samples of L-cysteine 134 stereospecifically labeled with tritium was first achieved by ourselves in connection with studies on the biosynthesis of the -lactam antibiotics penicillin and cephalosporin (133, 134). The key steps in this synthesis were conversion of the imines 130 and 130, = H, to... 

In our work (133, 134) and that of Aberhart etal. (135) and Baldwin et al. (136), the samples of cysteine were used to examine the biosynthesis of the -lactam antibiotic penicillin 142. All three groups found that the ring closure step that gave rise to the j -lactam in the antibiotic occurred with retention of configuration (Scheme 44). We have also shown (137) that the... 

The biosynthesis of cephalosporins is analogous to that of the penicillins. It is derived from the peptide 5-(a-aminoadipoyl)cysteinylvaline (see p 160). First, the P-lactam ring is formed. This is followed by a C-H bond fission, not at the p-position but at the y-position of the valine building-block. However, enzymatic conversion of the penam system into the ceph-3-em system cannot be excluded. 

This is just in contrast to another approach of modifying the molecular structure of peptidic compounds on the biosynthetic level, namely the directed biosynthesis of the desired compound structures by feeding of appropriate biosynthetic precursors or stimulating agents during fermentation, as it is routinely performed for production of penicillin V und G by the addition of phenoxyacetic acid or phenyl-acetic acid, respectively, as precursors to facilitate penicillin extraction from the culture broth. 

Newbert R W, Barton B, Greaves P, Harper J, Turner G (1997). Analysis of a commercially improved Penicillium chrysogenum strain series involvement of recombi-nogenic regions in amplification and deletion of the penicillin biosynthesis gene cluster. J. Ind. Microbiol. Biotechnol. 19 18-27. 

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