Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum.

Carlos García-Estrada,Marco A Van Den Berg,Inmaculada Vaca,Juan Francisco Martín,Roel Al Bovenberg,Ricardo V Ullán

doi:10.1186/1471-2180-9-104

Carlos García-Estrada, Marco A Van Den Berg + Show 4 more

Open Access

https://doi.org/10.1186/1471-2180-9-104

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Abstract

BackgroundPenicillium chrysogenum converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the penDE gene. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene. We have termed this gene ial because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the ial gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway.ResultsThe IAL contains motifs characteristic of the IAT such as the processing site, but lacks the peroxisomal targeting sequence ARL. Null ial mutants and overexpressing strains indicated that IAL lacks acyltransferase (penicillin biosynthetic) and amidohydrolase (6-APA forming) activities in vivo. When the canonical ARL motif (leading to peroxisomal targeting) was added to the C-terminus of the IAL protein (IALARL) by site-directed mutagenesis, no penicillin biosynthetic activity was detected. Since the IAT is only active after an accurate self-processing of the preprotein into α and β subunits, self-processing of the IAL was tested in Escherichia coli. Overexpression experiments and SDS-PAGE analysis revealed that IAL is also self-processed in two subunits, but despite the correct processing, the enzyme remained inactive in vitro.ConclusionNo activity related to the penicillin biosynthesis was detected for the IAL. Sequence comparison among the P. chrysogenum IAL, the A. nidulans IAL homologue and the IAT, revealed that the lack of enzyme activity seems to be due to an alteration of the essential Ser309 in the thioesterase active site. Homologues of the ial gene have been found in many other ascomycetes, including non-penicillin producers. Our data suggest that like in A. nidulans, the ial and penDE genes might have been formed from a single ancestral gene that became duplicated during evolution, although a separate evolutive origin for the ial and penDE genes, is also discussed.

Highlights

Penicillium chrysogenum converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the penDE gene
They are produced by different microorganisms, including filamentous fungi such as Penicillium chrysogenum and Aspergillus nidulans. These ascomycetes synthesize hydrophobic penicillins using three amino acids as precursors; L-α-aminoadipic acid, L-cysteine and L-valine to form the tripeptide δ (L-α-aminoadipyl)-Lcysteinyl-D-valine (ACV) by the multienzyme ACV synthetase (ACVS), which is encoded by the pcbAB gene. This tripeptide is converted into isopenicillin N (IPN), a reaction catalyzed by the IPN synthase (IPNS) encoded by the pcbC gene [1]
Characterization of the ial gene in P. chrysogenum, which encodes a protein (IAL) with high similarity to IAT The genome of P. chrysogenum Wis54-1255 contains a gene (Pc13g09140) that was initially described as paralogue of the penicillin biosynthetic penDE gene [27]

Summary

Introduction

Penicillium chrysogenum converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the penDE gene. They are produced by different microorganisms, including filamentous fungi such as Penicillium chrysogenum and Aspergillus nidulans These ascomycetes synthesize hydrophobic penicillins using three amino acids as precursors; L-α-aminoadipic acid, L-cysteine and L-valine to form the tripeptide δ (L-α-aminoadipyl)-Lcysteinyl-D-valine (ACV) by the multienzyme ACV synthetase (ACVS), which is encoded by the pcbAB gene. This tripeptide is converted into isopenicillin N (IPN), a reaction catalyzed by the IPN synthase (IPNS) encoded by the pcbC gene [1]. In the last step of the penicillin pathway, the L-α-aminoadipyl side chain of IPN is substituted by aromatic acyl side chains to form hydrophobic penicillins This reaction is catalysed by the isopenicillin N acyltransferase (IAT), encoded by the penDE gene [2,3]. Previous activation of the aromatic acid by a specific arylCoA ligase is required [4,5]

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