首页|Motifs in the C-terminal region of the Penicillium chrysogenum ACV synthetase are essential for valine epimerization and processivity of tripeptide formation

Motifs in the C-terminal region of the Penicillium chrysogenum ACV synthetase are essential for valine epimerization and processivity of tripeptide formation

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  • Elsevier
The first step in the penicillin biosynthetic pathway is the non-ribosomal condensation of L-α-aminoadipic acid, L-cysteine and L-valine into the tripeptide δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV). This reaction is catalysed by the multienzyme ACV synthetase (ACVS), which is encoded in the filamentous fungus Penicillium chrysogenum by the pcbAB gene. This enzyme contains at least ten catalytic domains. The precise role of the C-terminal domain of this multidomain NRPS still remains obscure. The C-terminal region of ACVS bears the epimerase and the thioesterase domains and may be involved in the epimerization of LLL-ACV to LLD-ACV and in the hydrolysis of the thioester bond. In this work, the conserved motifs ~(3371)EGHGRE~(3376) (located in the putative epimerase domain) and ~(3629)GWSFG~(3633) (located in the thioesterase domain) were changed by site-directed-mutagenesis to LGFGLL and GWAFG, respectively. In addition, the whole thioesterase domain (230 amino acids) and the different parts of this domain were deleted. The activity of these mutant enzymes was assessed in vivo by two different procedures: i) through the quantification of bisACV produced by the fungus and ii) by quantifying the benzylpenicillin production using tailored strains of P. chrysogenum, which lack the pcbAB gene, as host strains. All indicated mutant enzymes showed lower or null activity than the control strain confirming that E3371, H3373, R3375 and E3376 belong to the epimerase active centre. Different fragments included in the C-terminal region of ACVS control thioester hydrolysis. Overexpression of the sequence encoding the ACVS integrated thioesterase domain as a separate (stand-alone) transcriptional unit complemented mutants lacking the integrated thioesterase domain, although with low ACV releasing activity, suggesting that the stand-alone thio-esterease interacts with the other ACVS domains.

non-ribosomal peptide synthetasespenicillin biosynthesisL-Valine epimeraseACV syntethasepenicillin

Xiaobin Wu、Carlos Garcia-Estrada、Inmaculada Vaca、Juan-Francisco Martin

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Instituto de Biotecnologia (INBIOTEC), Parque Cientifico de Leon, Av. Real, 1, 24006 Leon, Spain

Instituto de Biotecnologia (INBIOTEC), Parque Cientifico de Leon, Av. Real, 1, 24006 Leon, Spain,Departamento de Quimica, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, fiunoa, Santiago de Chile, Chile

Instituto de Biotecnologia (INBIOTEC), Parque Cientifico de Leon, Av. Real, 1, 24006 Leon, Spain,Area de Microbiologia, Departamento de Biologja Molecular, Facultad de CC. Bioldgicas y Ambientales, Universidad de Leon, Campus de Vegazana s/n, 24071 Leon, Spain

2012

10.1016/j.biochi.2011.08.002

Biochimie

Biochimie

SCI
ISSN：0300-9084
年,卷(期)：2012.94(2)
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