Step Of Penicillin Biosynthesis Research Articles

Pexophagy modes during penicillin biosynthesis in Penicillium rubens P2-32-T.

Pexophagy is a peroxisome degradation process. The last two steps of penicillin biosynthesis in Penicillium rubens are carried out in peroxisomes. These organelles proliferate in large numbers during this process, so that after the penicillin secretion, their removal is essential as a regulatory mechanism. In this work, two pexophagy modes are described for the high-penicillin producing strain P. rubens P2-32-T, by transmission electron microscopy (TEM) on 24- and 48-h cultures (when maximum penicillin production is achieved). The obtained images show peroxisome phagocytosis by vacuoles in three different ways: macropexophagy, micropexophagy, and a new proposed model: unipexophagy.

Structural characterization of an isopenicillin N synthase family oxygenase from Pseudomonas aeruginosa PAO1

Isopenicillin N synthase (IPNS) is a nonheme-Fe2+-dependent enzyme that mediates a key step in penicillin biosynthesis. It catalyses the conversion of the tripeptide δ-(l-α-aminoadipoyl)-l-cysteine-d-valine (ACV) to isopenicillin N, which is a key precursor to β-lactam antibiotics. The pa4191 gene in Pseudomonas aeruginosa PAO1 has provisionally been annotated as a member of the IPNS family. In this work, we report the crystal structure of PA4191 from P. aeruginosa (PaIPNS hereafter). The 1.65 Å resolution PaIPNS structure forms a jelly roll fold and is confirmed to be a member of the IPNS family based on structural homology. A metal centre within the jelly roll consists of the strictly conserved His201, Asp203 and His257 residues. MicroScale Thermophoresis binding analysis confirms that PaIPNS is a metal-binding protein with a strong preference for iron, but that it does not bind the tripeptide ACV. Structural comparison of PaIPNS with a previously reported IPNS-ACV complex structure reveals a restricted binding pocket that is unable to accommodate ACV.

The crystal structure of isopenicillin N synthase with a dipeptide substrate analogue

Isopenicillin N synthase (IPNS) converts its linear tripeptide substrate δ-l-α-aminoadipoyl-l-cysteinyl-d-valine (ACV) to bicyclic isopenicillin N (IPN), the key step in penicillin biosynthesis. Solution-phase incubation experiments have shown that IPNS will accept and oxidise a diverse array of substrate analogues, including tripeptides that incorporate l-homocysteine as their second residue, and tripeptides with truncated side-chains at the third amino acid such as δ-l-α-aminoadipoyl-l-cysteinyl-d-α-aminobutyrate (ACAb), δ-l-α-aminoadipoyl-l-cysteinyl-d-alanine (ACA) and δ-l-α-aminoadipoyl-l-cysteinyl-glycine (ACG). However IPNS does not react with dipeptide substrates. To probe this selectivity we have crystallised the enzyme with the dipeptide δ-l-α-aminoadipoyl-l-homocysteine (AhC) and solved a crystal structure for the IPNS:Fe(II):AhC complex to 1.40Å resolution. This structure reveals an unexpected mode of peptide binding at the IPNS active site, in which the homocysteinyl thiolate does not bind to iron. Instead the primary mode of binding sees the homocysteinyl carboxylate coordinated to the metal, while its side-chain is oriented into the region of the active site normally occupied by the benzyl group of protein residue Phe211.

The transport of phenylacetic acid across the peroxisomal membrane is mediated by the PaaT protein in Penicillium chrysogenum

Penicillium chrysogenum, an industrial microorganism used worldwide for penicillin production, is an excellent model to study the biochemistry and the cell biology of enzymes involved in the synthesis of secondary metabolites. The well-known peroxisomal location of the last two steps of penicillin biosynthesis (phenylacetyl-CoA ligase and isopenicillin N acyltransferase) requires the import into the peroxisomes of the intermediate isopenicillin N and the precursors phenylacetic acid and coenzyme A. The mechanisms for the molecular transport of these precursors are still poorly understood. In this work, a search was made, in the genome of P. chrysogenum, in order to find a Major Facilitator Superfamily (MFS) membrane protein homologous to CefT of Acremonium chrysogenum, which is known to confer resistance to phenylacetic acid. The paaT gene was found to encode a MFS membrane protein containing 12 transmembrane spanners and one Pex19p-binding domain for Pex19-mediated targeting to peroxisomal membranes. RNA interference-mediated silencing of the paaT gene caused a clear reduction of benzylpenicillin secretion and increased the sensitivity of P. chrysogenum to the penicillin precursor phenylacetic acid. The opposite behavior was found when paaT was overexpressed from the glutamate dehydrogenase promoter that increases phenylacetic acid resistance and penicillin production. Localization studies by fluorescent laser scanning microscopy using PaaT-DsRed and EGFP-SKL fluorescent fusion proteins clearly showed that the protein was located in the peroxisomal membrane. The results suggested that PaaT is involved in penicillin production, most likely through the translocation of side-chain precursors (phenylacetic acid and phenoxyacetic acid) from the cytosol to the peroxisomal lumen across the peroxisomal membrane of P. chrysogenum.

The crystal structure of isopenicillin N synthase with δ-( l-α-aminoadipoyl)- l-cysteinyl- d-methionine reveals thioether coordination to iron

Isopenicillin N synthase (IPNS) catalyses cyclization of δ-( l-α-aminoadipoyl)- l-cysteinyl- d-valine (ACV) to isopenicillin N (IPN), the central step in penicillin biosynthesis. Previous studies have shown that IPNS turns over a wide range of substrate analogues in which the valine residue of its natural substrate is replaced with other amino acids. IPNS accepts and oxidizes numerous substrates that bear hydrocarbon sidechains in this position, however the enzyme is less tolerant of analogues presenting polar functionality in place of the valinyl isopropyl group. We report a new ACV analogue δ-( l-α-aminoadipoyl)- l-cysteinyl- d-methionine (ACM), which incorporates a thioether in place of the valinyl sidechain. ACM has been synthesized using solution phase methods and crystallized with IPNS. A crystal structure has been elucidated for the IPNS:Fe(II):ACM complex at 1.40 Å resolution. This structure reveals that ACM binds in the IPNS active site such that the sulfur atom of the methionine thioether binds to iron in the oxygen binding site at a distance of 2.57 Å. The sulfur of the cysteinyl thiolate sits 2.36 Å from the metal.

Homology modeling of the structure of acyl coA:isopenicillin N-acyltransferase (IAT) from Penicillium chrysogenum. IAT interaction studies with isopenicillin-N, combining molecular dynamics simulations and docking

In the last step of penicillin biosynthesis, acyl-CoA:isopenicillin N acyltransferase (IAT) (E.C. 2.3.1.164) catalyzes the conversion of isopenicillin N (IPN) to penicillin G. IAT substitutes the α-aminoadipic acid side chain of IPN by a phenylacetic acid phenolate group (from phenylacetyl-CoA). Having a three-dimensional (3D) structure of IAT helps to determine the steps involved in side chain exchange by identifying the atomic details of substrate recognition. We predicted the IAT 3-D structure (α- and β-subunits), as well as the manner of IPN and phenylacetyl-CoA bind to the mature enzyme (β-subunit). The 3D IAT prediction was achieved by homology modeling and molecular docking in different snapshots, and refined by molecular dynamic simulations. Our model can reasonably interpret the results of a number of experiments, where key residues for IAT processing as well as strictly conserved residues most probably involved with enzymatic activity were mutated. Based on the results of docking studies, energies associated with the complexes, and binding constants calculated, we identified a site located in the region generated by β1, β2 and β5 strands, which forms part of the central structure of β-subunit, as the potential binding site of IPN. The site comprises the amino acid residues Cys103, Asp121, Phe122, Phe123, Ala168, Leu169, His170, Gln172, Phe212, Arg241, Leu262, Asp264, Arg302, Ser309, and Arg310. Through hydrogen bonds, the IPN binding site establishes interactions with Cys103, Leu169, Gln172, Asp264 and Arg310. Our model is also validated by a recently revealed crystal structure of the mature enzyme.

Regulation and compartmentalization of β‐lactam biosynthesis

SummaryPenicillins and cephalosporins are β‐lactam antibiotics widely used in human medicine. The biosynthesis of these compounds starts by the condensation of the amino acids l‐α‐aminoadipic acid, l‐cysteine and l‐valine to form the tripeptide δ‐l‐α‐aminoadipyl‐l‐cysteinyl‐d‐valine catalysed by the non‐ribosomal peptide ‘ACV synthetase’. Subsequently, this tripeptide is cyclized to isopenicillin N that in Penicillium is converted to hydrophobic penicillins, e.g. benzylpenicillin. In Acremonium and in streptomycetes, isopenicillin N is later isomerized to penicillin N and finally converted to cephalosporin. Expression of genes of the penicillin (pcbAB, pcbC, pendDE) and cephalosporin clusters (pcbAB, pcbC, cefD1, cefD2, cefEF, cefG) is controlled by pleitropic regulators including LaeA, a methylase involved in heterochromatin rearrangement. The enzymes catalysing the last two steps of penicillin biosynthesis (phenylacetyl‐CoA ligase and isopenicillin N acyltransferase) are located in microbodies, as shown by immunoelectron microscopy and microbodies proteome analyses. Similarly, the Acremonium two‐component CefD1–CefD2 epimerization system is also located in microbodies. This compartmentalization implies intracellular transport of isopenicillin N (in the penicillin pathway) or isopenicillin N and penicillin N in the cephalosporin route. Two transporters of the MFS family cefT and cefM are involved in transport of intermediates and/or secretion of cephalosporins. However, there is no known transporter of benzylpenicillin despite its large production in industrial strains.

Structural Studies on the Reaction of Isopenicillin N Synthase with a Sterically Demanding Depsipeptide Substrate Analogue

Isopenicillin N synthase (IPNS) is a nonheme iron(II)-dependent oxidase that catalyses the central step in penicillin biosynthesis, conversion of the tripeptide delta-L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN). This report describes mechanistic studies using the analogue delta-(L-alpha-aminoadipoyl)-(3S-methyl)-L-cysteine D-alpha-hydroxyisovaleryl ester (A(S)mCOV), designed to intercept the catalytic cycle at an early stage. A(S)mCOV incorporates two modifications from the natural substrate: the second and third residues are joined by an ester, so this analogue lacks the key amide of ACV and cannot form a beta-lactam; and the cysteinyl residue is substituted at its beta-carbon, bearing a (3S)-methyl group. It was anticipated that this methyl group will impinge directly on the site in which the co-substrate dioxygen binds. The novel depsipeptide A(S)mCOV was prepared in 13 steps and crystallised with IPNS anaerobically. The 1.65 A structure of the IPNS-Fe(II)-A(S)mCOV complex reveals that the additional beta-methyl group is not oriented directly into the oxygen binding site, but does increase steric demand in the active site and increases disorder in the position of the isovaleryl side chain. Crystals of IPNS-Fe(II)-A(S)mCOV were incubated with high-pressure oxygen gas, driving substrate turnover to a single product, an ene-thiol/C-hydroxylated depsipeptide. A mechanism is proposed for the reaction of A(S)mCOV with IPNS, linking this result to previous crystallographic studies with related depsipeptides and solution-phase experiments with cysteine-methylated tripeptides. This result demonstrates that a (3S)-methyl group at the substrate cysteinyl beta-carbon is not in itself a block to IPNS activity as previously proposed, and sheds further light on the steric complexities of IPNS catalysis.

δ-( l-α-Aminoadipyl)- l-cysteinyl- d-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by δ-( l-α-aminoadipyl)- l-cysteinyl- d-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids l-α-aminoadipate, l-cysteine, and l-valine into the tripeptide ACV. ACV synthetase has previously been localized to the vacuole where it is thought to utilize amino acids from the vacuolar pools. We localized ACV synthetase by subcellular fractionation and immuno-electron microscopy under conditions that prevented proteolysis and found it to co-localize with isopenicillin N synthetase in the cytosol, while acyltransferase localizes in microbodies. These data imply that the key enzymatic steps in penicillin biosynthesis are confined to only two compartments, i.e., the cytosol and microbody.

Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes.

Penicillin antibiotics are all produced from fermentation-derived penicillins because their chemical synthesis is not commercially viable. The key step in penicillin biosynthesis, in which both the beta-lactam and thiazolidine rings of the nucleus are created, is mediated by isopenicillin N synthase (IPNS), which binds ferrous iron and uses dioxygen as a cosubstrate. In a unique enzymatic step, with no chemical precedent, IPNS catalyses the transfer of four hydrogen atoms from its tripeptide substrate to dioxygen forming, in a single reaction, the complete bicyclic nucleus of the penicillins. We now report the structure of IPNS complexed with manganese, which reveals the active site is unusually buried within a 'jelly-roll' motif and lined by hydrophobic residues, and suggest how this structure permits the process of penicillin formation. Sequence analyses indicate IPNS, 1-aminocyclopropane-1-carboxylic acid oxidase and many of the 2-oxo-acid-dependent oxygenases contain a conserved jelly-roll motif, forming a new structural family of enzymes.

Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum.

The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular locations. The enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase was found to be associated with membranes or small organelles. The next enzyme isopenicillin N-synthetase appeared to be a cytosolic enzyme. The enzyme which is involved in the last step of penicillin biosynthesis, acyltransferase, was located in organelles with a diameter of 200-800 nm. These organelles, most probably, are microbodies. A positive correlation was found between the capacity for penicillin production and the number of organelles per cell when comparing different P. chrysogenum strains.

The multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421,073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases.

The nucleotide sequence of the Penicillium chrysogenum Oli13 acvA gene encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, which performs the first step in penicillin biosynthesis, has been determined. The acvA gene contains an open reading frame of 11,238 bp encoding a protein of 3746 amino acids with a predicted mol. wt of 421,073 dalton. Three domains within the protein of approximately 570 amino acids have between 38% and 43% identity with each other and share similarity with two antibiotic peptide synthetases from Bacillus brevis as well as two other enzymes capable of performing ATP-pyrophosphate exchange reactions. The acvA gene is located close to the pcbC gene encoding isopenicillin N synthetase, the enzyme for the second step of beta-lactam biosynthesis, and is transcribed in the opposite orientation to it. The intergenic region of 1107 bp from which the acvA and pcbC genes are divergently transcribed has also been sequenced.