6-APA Acyltransferase Research Articles

Phenoxymethylpenicillin amidohydrolases from Penicillium chrysogenum

A phenoxymethylpenicillin amidohydrolase which hydrolyses phenoxymethylpenicillin to 6-aminopenicillanic acid (6-APA) has been isolated from two species of Penicillium chrysogenum. The amidohydrolase had a molecular mass of approx. 42 kDa. Its activity with benzylpenicillin as substrate was only 1.5% of that with phenoxymethylpenicillin and it was inhibited by its products. No penicillin formation from 6-APA and phenoxyacetyl or phenylacetyl coenzyme A was observed. The enzyme is thus distinct from the phenylacetyl coenzyme A: 6-APA acyltransferase, which also has amidohydrolase activity and is involved in the final stages of the biosynthesis of penicillins.

Continuous cultivation of Penicillium chrysogenum. Growth on glucose and penicillin production

A series of constant-mass, continuous cultivations of the penicillin producing mold Penicillium chrysogenum was carried out using a chemically defined medium with glucose as the growth-limiting component. The stoichiometry for growth of P. chrysogenum on glucose was characterized in terms of mass-yield and maintenance coefficients. Saturation kinetics with respect to glucose was used to describe the glucose consumption rate at steady-state conditions. Transient data indicate that the maximum rate of glucose consumption at a particular set of operating conditions is correlated to the metabolic ‘capacity’ of the mold as reflected by its intracellular RNA content. A progressive loss in the penicillin productivity in glucose limited chemostat cultures was correlated to the formation of two mutants. The two mutants were characterized by their sporulation when grown as surface cultures and by Southern dot-tests for δ-( l-α-aminoadipyl)- l-cysteinyl- d-valine synthetase (ACVS), isopenicillin-N synthase (IPNS) and acyl-CoA:6-APA acyltransferase (AT). The loss of penicillin productivity was caused by an increasing fraction of mutants which had lost the genes encoding for all three enzymes needed in the penicillin synthesizing pathway.

Inhibition of penicillin biosynthetic enzymes by halogen derivatives of phenylacetic acid

The effect of phenylacetic acid (PAA) and several analogs on the activity of isopenicillin N synthase (IPNS) and acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum Wis 54-1255 has been tested. Whereas the substitution on the ring of a hydrogen atom by hydroxy-, methyl- or methoxy- groups did not cause any effect, the presence of halogens (Cl or Br) at positions 3 and/or 4 of PAA strongly inhibited these two enzymes. The replacement of hydrogen atoms by fluorine in certain positions also caused inhibition, but to a lesser extent.

Expression of genes and processing of enzymes for the biosynthesis of penicillins and cephalosporins.

The genes pcbAB, pcbC and penDE encoding the enzymes (alpha-aminoadipyl-cysteinyl-valine synthetase, isopenicillin N synthase and isopenicillin N acyltransferase, respectively) involved in the biosynthesis of penicillin have been cloned from Penicillin chrysogenum and Aspergillus nidulans. They are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of Penicillium notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. Each gene is expressed as a single transcript from separate promoters. Enzyme regulation studies and gene expression analysis have provided useful information to understand the control of genes involved in penicillin biosynthesis. The enzyme isopenicillin N acyltransferase encoded by the penDE gene is synthesized as a 40 kDa protein that is (self)processed into two subunits of 29 and 11 kDa. Both subunits appear to be required for acyl-CoA 6-APA acyltransferase activity. The isopenicillin N acyltransferase was shown to be located in microbodies, whereas the isopenicillin N synthase has been reported to be present in vesicles of the Golgi body and in the cell wall. A mutant in the carboxyl-terminal region of the isopenicillin N acyltransferase lacking the three final amino acids of the enzymes was not properly located in the microbodies and failed to synthesize penicillin in vivo. In C. acremonium the genes involved in cephalosporin biosynthesis are separated in at least two clusters. Cluster I (pcbAB-pcbC) encodes the first two enzymes (alpha-aminoadipyl-cysteinyl) valine synthetase and isopenicillin N synthase) of the cephalosporin pathway which are very similar to those involved in penicillin biosynthesis. Cluster II (cefEF-cefG), encodes the last three enzymatic activities (deacetoxycephalosporin C synthetase/hydroxylase and deacetylcephalosporin C acetyltransferase) of the cephalosporin pathway. It is unknown, at this time, if the cefD gene encoding isopenicillin epimerase is linked to any of these two clusters. Methionine stimulates cephalosporin biosynthesis in cultures of three different strains of A. chrysogenum. Methionine increases the levels of enzymes (isopenicillin N synthase and deacetylcephalosporin C acetyltransferase) expressed from genes (pcbC and cefG respectively) which are separated in the two different clusters of cephalosporin biosynthesis genes. This result suggests that both clusters of genes have regulatory elements which are activated by methionine. Methionine-supplemented cells showed higher levels of transcripts of the pcbAB, pcbC, cefEF genes and to a lesser extent of cefG than cells grown in absence of methionine. The levels of the cefG transcript were very low as compared to those of pcbAB, pcbC and cefEF.(ABSTRACT TRUNCATED AT 400 WORDS)

The requirement for subunit interaction in the production of Penicillium chrysogenum acyl-coenzyme A: isopenicillin N acyltransferase in Escherichia coli

Subunit interaction in the formation of active acyl-coenzyme A: isopenicillin N acyltransferase (AT) has been investigated. Various AT derivatives were produced from altered Penicillium chrysogenum penDE genes placed in Escherichia coli expression systems. The regions of penDE encoding the a (11 kDa) and β (29 kDa) AT subunits were separated at the DNA level by linker insertion at the region encoding Gly 102/Cys 103. Synthesis of AT from the resulting two-cistron mRNA resulted in active α,β-heterodimeric recombinant AT (reAT), containing subunits of 11 and 29 kDa (similar to wild-type AT). Complete separation of the a and β subunits was performed by placing the region of penDE encoding each subunit on different plasmids. Production of either subunit in the absence of the other did not form active reAT. However, cotransformation of E. coli with two plasmids, each encoding a different AT subunit, produced reAT having acyl-coenzyme A: 6-aminopenicillanic acid (acyl-CoA: 6-APA) AT activity. Mutation of penDE replacing Thr 105 with Asn resulted in inactive and uncleaved reAT. Coexpression of this mutant penDE with a penDE derivative encoding the β subunit in E. coli produced acyl-CoA: 6-APA AT activity. These results suggest that the formation of reAT involves cooperative folding events between the subunits. In vitro transcription/translation was used to determine the origin of the AT hydrolase activity that cleaves the 40-kDa precursor polypeptide. The appearance of a 29-kDa protein (and presumably the corresponding 11-kDa protein, although not observable) from the 40-kDa in vitro translated protein provides further evidence that AT hydrolysis is an autocatalytic event.

The isopenicillin-N acyltransferase of Penicillium chrysogenum has isopenicillin-N amidohydrolase, 6-aminopenicillanic acid acyltransferase and penicillin amidase activities, all of which are encoded by the single penDE gene.

The isopenicillin-N acyltransferase of Penicillium chrysogenum catalyzes the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin-N acyltransferase copurified with the acyl-CoA:6-aminopenicillanic acid (6-APA) acyltransferase activity which transfers an acyl residue from acyl-CoA derivatives (e.g. phenylacetyl-CoA, phenoxyacetyl-CoA) to 6-APA. Other thioesters of phenylacetic acid were also used as substrates. An amino acid sequence similar to that of the active site of thioesterases was found in the isopenicillin-N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenylacetyl thioesters. Purified isopenicillin-N acyltransferase also showed isopenicillin-N amidohydrolase, penicillin transacylase and penicillin amidase activities. The isopenicillin-N amidohydrolase (releasing 6-APA) showed a much lower specific activity than the isopenicillin-N acyltransferase of the same enzyme preparation, suggesting that in the isopenicillin-N acyltransferase reaction the 6-APA is not released and is directly converted into benzylpenicillin. Penicillin transacylase exchanged side chains between two hydrophobic penicillin molecules; or between one penicillin molecule and 6-APA. The penicillin amidase activity is probably the reverse of the biosynthetic acyl-CoA:6-APA acyltransferase. Four P. chrysogenum mutants deficient in acyl-CoA:6-APA acyltransferase lacked the other four related activities. Transformation of these mutants with the penDE gene restored all five enzyme activities.

Synthesis of 3-furylmethylpenicillin using an enzymatic procedure

3-Furylmethylpenicillin was synthesized in vitro from 3-furylacetic acid, 6-aminopenicillanic acid (6-APA), CoA, ATP and Mg2+. The reaction was catalyzed in two steps by the enzymes phenyl-acetyl-CoA ligase (PCL) from Pseudomonas putida and acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum. PCL catalyzes the activation of 3-furylacetic acid to 3-furylacetyl-CoA (3-F-CoA) and AT acylates the amino group of 6-APA with the 3-furylacetyl moiety of 3-F-CoA, releasing CoA and 3-furylmethylpenicillin.

Synthesis of 3-furylmethylpenicillin using an enzymatic procedure

3-Furylmethylpenicillin was synthesized in vitro from 3-furylacetic acid, 6-aminopenicillanic acid (6-APA), CoA, ATP and Mg2+. The reaction was catalyzed in two steps by the enzymes phenyl-acetyl-CoA ligase (PCL) from Pseudomonas putida and acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum. PCL catalyzes the activation of 3-furylacetic acid to 3-furylacetyl-CoA (3-F-CoA) and AT acylates the amino group of 6-APA with the 3-furylacetyl moiety of 3-F-CoA, releasing CoA and 3-furylmethylpenicillin.

Acyl-CoA: 6-APA acyltransferase from Penicillium chrysogenum. Studies on its hydrolytic activity.

Acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum Wis 54-1255 catalyzes the hydrolysis of different acyl-CoA derivatives generating, in the absence of 6-APA, free acid and CoA. The hydrolytic efficiency of AT is highest for acyl-CoA variants in which the acyl-moiety is higher than six carbon atoms. The maximal rate of catalysis was achieved in 50 mM Tris-HCl buffer, pH 8.5 at 35 degrees C. Unlike the AT activity, the acylase activity has a different optimum temperature and substrate specificity and dithiothreitol is not required for the reaction.

Design of an enzymatic hybrid system: a useful strategy for the biosynthesis of benzypenicillin in vitro

A hybrid (prokaryotic-eukaryotic) enzyme system leading to the production of benzypenicillin has been developed. In vitro synthesis of penicillin G was achieved by incubating 6-aminopenicillanic acid, CoA, phenylacetic acid, homogenously pure phenylacetyl-CoA ligase (PA-CoA ligase) from Pseudomonas putida and acyl-CoA:6-APA acyltransferase (AT) from Penicillium chrysogenum. Benzylpenicillin was also obtained when AT was coupled with PA-CoA ligase and isopenicillin N-synthetase (IPNS). This is the first description of an in vitro assay that, using enzymes of different microbial origin, mimics the three last enzymatic steps leading to the biosynthesis of penicillin G in P. chrysogenum.

Design of an enzymatic hybrid system: a useful strategy for the biosynthesis of benzypenicillin in vitro

A hybrid (prokaryotic-eukaryotic) enzyme system leading to the production of benzylpenicillin has been developed. In vitro synthesis of penicillin G was achieved by incubating 6-aminopenicillanic acid, CoA, phenylacetic acid, homogeneously pure phenylacetyl-CoA ligase (PA-CoA ligase) from Pseudomonas putida and acyl-CoA:6-APA acyltransferase (AT) from Penicillium chrysogenum. Benzylpenicillin was also obtained when AT was coupled with PA-CoA ligase and isopenicillin N-synthetase (IPNS). This is the first description of an in vitro assay that, using enzymes of different microbial origin, mimics the three last enzymatic steps leading to the biosynthesis of penicillin G in P. chrysogenum.

V. Biosynthesis of benzylpenicillin (G), phenoxymethylpenicillin (V) and octanoylpenicillin (K) from glutathione S-derivatives.

"In vitro" synthesis of benzylpenicillin and phenoxymethylpenicillin has been carried out by direct N-acylation of 6-aminopenicillanic acid (6-APA) with S-phenylacetyl- and (S-phenoxyacetyl)glutathione. The reactions were catalyzed by the enzyme acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum and in both cases the synthesis of antibiotics was enhanced by CoA. Penicillin K, a natural penicillin, was also synthesized "in vitro" by incubating (S-octanoyl)glutathione, 6-APA and AT, but in this case the formation of antibiotic required the presence of CoA. Furthermore, benzylpenicillin was obtained from (S-phenylacetyl)cysteinylglycine and 6-APA, suggesting that some intermediates of the gamma-glutamyl cycle are directly involved in the biosynthesis of penicillins. To explain "in vivo" formation of this beta-lactam antibiotic, a biosynthetic pathway which includes several glutathione-S-derivatives and a non-enzymatic reaction, is proposed.

Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum

The isopenicillin N synthase (pcbC) and acyl-CoA:6-APA acyltransferase (penDE) genes of Penicillium chrysogenum were located in a 19.5-kb DNA fragment that had been previously cloned in phage vector EMBL3. This 19.5-kb DNA fragment was mapped with several endonucleases, and the pcbC and penDE genes were located by hybridization with probes corresponding to internal fragments of each gene. A low penicillin producing strain (P. chrysogenum Wis 54-1255) and two high producing strains (AS-P-78 and P2) showed hybridizing fragments of identical sizes in their chromosomes. Dot-blot hybridization of serial dilutions of the total DNA of the three strains showed that the intensity of all the hybridizing bands was much higher in strains AS-P-78 and P2 than in Wis 54-1255. Hybridization of total DNA digestions with probes corresponding to fragments which mapped upstream or downstream of the pcbC-penDE region revealed that a fragment of at least 35 kb DNA has been amplified 9 to 14 fold in the high penicillin producing strains. The amplified region did not include the previously cloned pyrG gene that encodes OMP-decarboxylase, an enzyme involved in pyrimidine biosynthesis.

Enzymatic synthesis of penicillins.

Different penicillins (phenylacetyl, 2-hydroxyphenylacetyl, 4-hydroxyphenylacetyl, phenoxyacetyl and 2-thiopheneacetylpenicillin) have been synthesized "in vitro" by direct N-acylation of 6-aminopenicillanic acid (6-APA) with the acyl group of several acyl-CoA derivatives. The enzyme that catalyzes these reactions, acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum, was purified to homogeneity (374-fold) and its amino acid composition is given. This protein accepts as substrates several aliphatic acids and different aromatic acids with the only requirement that an acetyl-CoA moiety must be present in the substrate molecule. Shortening or lengthening of the acyl moiety prevents the 6-APA-N-acylation reaction. The presence of an amino group in the alpha-position of the acetyl group does not allow this molecule to be used as substrate. However, different substitutions in the phenyl group (hydroxylation of the carbons 2 and 4) or its replacement by another aromatic ring (thiophene) were accepted with varying reactions rates in the acylation reaction when a 176-fold purified acyltransferase was employed. The homogeneity pure enzyme accepts as substrate thiophene acetyl-CoA but it did not 2-hydroxyphenyl and 4-hydroxyphenylacetyl-CoA. The presence of an oxygen atom between the aromatic and the acetyl moieties did not affect the catalysis.

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

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.