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

Estrada, Carlos García; Vaca, Inmaculada; Fierro, Francisco; Sjollema, Klaas; Veenhuis, Marten; Martı́n, Juan F.

doi:10.1016/j.fgb.2008.03.005

Cited by 35 publications

(32 citation statements)

References 37 publications

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“…Like other enzymes of this family, Tan could operate as an N-terminal nucleophile hydrolase, because the N-terminal cysteine residue of the mature enzyme could function as a nucleophile in catalysis of peptide bond cleavage of ␤-alanyl conjugates. This activity has also been proposed for IAT (22). Experimental evidence, however, is lacking in both cases.…”

Section: Discussionmentioning

confidence: 66%

“…Completion of processing at 25°C took about 3 h (Fig. 6), a time period that coincides roughly with the time required for recombinant P. chrysogenum IAT to be processed in E. coli (22).…”

Section: Impact Of Tan Processing Time On Its Circadian Activity Profmentioning

confidence: 66%

“…Tan Self-processing Assessment in E. coli-Self-processing time was determined in growth-competent E. coli according to Garcia-Estrada et al (22). Tan expression from pQE82L in BL21-Rosetta TM cells was induced with IPTG as described above.…”

Section: Methodsmentioning

confidence: 99%

“…Also, enzymatic activity appeared to depend on cleavage into ␣,␤-heterodimers. Because unprocessed IAT mutants were shown to exert a regulatory role on processing of wild type IAT, it was speculated that processing might require pre-protein dimer formation and self-cleavage might be an inter-molecular process (22). If Tan processing reveals the same stringent dependence on the GlyCys motif, activation of enzymatic activity might also depend on this process and maturation of activity might require a certain time period.…”

Section: Null Mutant Analysis Revealed That Amino Acid Argmentioning

confidence: 99%

“…Formation-The proposal that IAT activation depends on dimer formation as a prerequisite for an intermolecular processing step (22) led us to investigate the molecular weight of native E. coli expressed wild type and of processing mutant His 6 -Tan C122A fusion protein. Tan protein preparations were applied to a Superdex TM 200 size exclusion chromatography column.…”

Section: Native Tan Has a Protein Size Compatible With Dimermentioning

confidence: 99%

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Alternative Tasks of Drosophila Tan in Neurotransmitter Recycling Versus Cuticle Sclerotization Disclosed by Kinetic Properties

Aust

Brüsselbach

Pütz

et al. 2010

Journal of Biological Chemistry

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Upon a stimulus of light, histamine is released from Drosophila photoreceptor axonal endings. It is taken up into glia where Ebony converts it into ␤-alanyl-histamine (carcinine). Carcinine moves into photoreceptor cells and is there cleaved into ␤-alanine and histamine by Tan activity. Tan thus provides a key function in the recycling pathway of the neurotransmitter histamine. It is also involved in the process of cuticle formation. There, it cleaves ␤-alanyl-dopamine, a major component in cuticle sclerotization. Active Tan enzyme is generated by a selfprocessing proteolytic cleavage from a pre-protein at a conserved Gly-Cys sequence motif. We confirmed the dependence on the Gly-Cys motif by in vitro mutagenesis. Processing time delays the rise to full Tan activity up to 3 h behind its putative circadian RNA expression in head. To investigate its pleiotropic functions, we have expressed Tan as a His 6 fusion protein in Escherichia coli and have purified it to homogeneity. We found wild type and mutant His 6 -Tan protein co-migrating in size exclusion chromatography with a molecular weight compatible with homodimer formation. We conclude that dimer formation is preceding pre-protein processing. Drosophila tan 1 null mutant analysis revealed that amino acid Arg 217 is absolutely required for processing. Substitution of Met 256 in tan 5 , on the contrary, does not affect processing extensively but renders it prone to degradation. This also leads to a strong tan phenotype although His 6 -Tan 5 retains activity. Kinetic parameters of Tan reveal characteristic differences in K m and k cat values of carcinine and ␤-alanyl-dopamine cleavage, which conclusively illustrate the divergent tasks met by Tan.

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Section: Discussionmentioning

confidence: 66%

“…Completion of processing at 25°C took about 3 h (Fig. 6), a time period that coincides roughly with the time required for recombinant P. chrysogenum IAT to be processed in E. coli (22).…”

Section: Impact Of Tan Processing Time On Its Circadian Activity Profmentioning

confidence: 66%

Section: Methodsmentioning

confidence: 99%

Section: Null Mutant Analysis Revealed That Amino Acid Argmentioning

confidence: 99%

Section: Native Tan Has a Protein Size Compatible With Dimermentioning

confidence: 99%

See 3 more Smart Citations

Alternative Tasks of Drosophila Tan in Neurotransmitter Recycling Versus Cuticle Sclerotization Disclosed by Kinetic Properties

Aust

Brüsselbach

Pütz

et al. 2010

Journal of Biological Chemistry

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Biosynthetic concepts for the production of β‐lactam antibiotics in Penicillium chrysogenum

Weber

Bovenberg

Driessen

2011

Biotechnology Journal

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Industrial production of β-lactam antibiotics by the filamentous fungus Penicillium chrysogenum is based on successive classical strain improvement cycles. This review summarizes our current knowledge on the results of this classical strain improvement process, and discusses avenues to improve β-lactam biosynthesis and to exploit P. chrysogenum as an industrial host for the production of other antibiotics and peptide products. Genomic and transcriptional analysis of strain lineages has led to the identification of several important alterations in high-yielding strains, including the amplification of the penicillin biosynthetic gene cluster, elevated transcription of genes involved in biosynthesis of penicillin and amino acid precursors, and genes encoding microbody proliferation factors. In recent years, successful metabolic engineering and synthetic biology approaches have resulted in the redirection of the penicillin pathway towards the production of cephalosporins. This sets a new direction in industrial antibiotics productions towards more sustainable methods for the fermentative production of unnatural antibiotics and related compounds.

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Penicillins

Garcı́a-Estrada¹,

Martı́n²

2014

Fungal Biology

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

Cited by 35 publications

References 37 publications

Alternative Tasks of Drosophila Tan in Neurotransmitter Recycling Versus Cuticle Sclerotization Disclosed by Kinetic Properties

Alternative Tasks of Drosophila Tan in Neurotransmitter Recycling Versus Cuticle Sclerotization Disclosed by Kinetic Properties

Biosynthetic concepts for the production of β‐lactam antibiotics in Penicillium chrysogenum

Penicillins

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