Isopenicillin N Synthase: Crystallographic Studies

Abstract Radical cyclizations are essential reactions in the biosynthesis of secondary metabolites and the chemical synthesis of societally valuable molecules. In this review, we highlight the general mechanisms utilized in biocatalytic radical cyclizations. We specifically highlight cytochrome P450 monooxygenases (P450s) involved in the biosynthesis of mycocyclosin and vancomycin, non-heme iron- and α-ketoglutarate-dependent dioxygenases (Fe/αKGDs) used in the biosynthesis of kainic acid, scopolamine, and isopenicillin N, and radical S-adenosylmethionine (SAM) enzymes that facilitate the biosynthesis of oxetanocin A, menaquinone, and F420. Beyond natural mechanisms, we also examine repurposed flavin-dependent ‘ene’-reductases (ERED) for non-natural radical cyclization. Overall, these general mechanisms underscore the opportunity for enzymes to augment and enhance the synthesis of complex molecules using radical mechanisms.

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Isopenicillin N synthase: mechanistic studies

Chemical Reviews ◽

10.1021/cr00105a001 ◽

1990 ◽

Vol 90 (7) ◽

pp. 1079-1088 ◽

Cited By ~ 146

Author(s):

Jack E. Baldwin ◽

Mark Bradley

Keyword(s):

Mechanistic Studies ◽

Isopenicillin N

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The unprocessed preprotein form IATC103S of the isopenicillin N acyltransferase is transported inside peroxisomes and regulates its self-processing

Fungal Genetics and Biology ◽

10.1016/j.fgb.2008.03.005 ◽

2008 ◽

Vol 45 (6) ◽

pp. 1043-1052 ◽

Cited By ~ 22

Author(s):

Carlos García-Estrada ◽

Inmaculada Vaca ◽

Francisco Fierro ◽

Klaas Sjollema ◽

Marten Veenhuis ◽

...

Keyword(s):

Isopenicillin N

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Structural Studies on the Reaction of Isopenicillin N Synthase with the Truncated Substrate Analogues δ-(l-α-aminoadipoyl)-l-cysteinyl-glycine and δ-(l-α-aminoadipoyl)-l-cysteinyl-d-alanine†,‡

Biochemistry ◽

10.1021/bi047478q ◽

2005 ◽

Vol 44 (17) ◽

pp. 6619-6628 ◽

Cited By ~ 25

Author(s):

Alexandra J. Long ◽

Ian J. Clifton ◽

Peter L. Roach ◽

Jack E. Baldwin ◽

Peter J. Rutledge ◽

...

Keyword(s):

Structural Studies ◽

Substrate Analogues ◽

Isopenicillin N

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An Efficient Fungal RNA-Silencing System Using the DsRed Reporter Gene

Applied and Environmental Microbiology ◽

10.1128/aem.02127-06 ◽

2006 ◽

Vol 73 (3) ◽

pp. 962-970 ◽

Cited By ~ 45

Author(s):

Danielle Janus ◽

Birgit Hoff ◽

Eckhard Hofmann ◽

Ulrich Kï¿½ck

Keyword(s):

Rna Silencing ◽

Blot Hybridization ◽

Strain Improvement ◽

Northern Hybridization ◽

Attractive Alternative ◽

Cephalosporin C ◽

Gene Encoding ◽

Dsred Protein ◽

High Level ◽

Isopenicillin N

ABSTRACT In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.

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