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An endogenous factor enhances ferulic acid decarboxylation catalyzed by phenolic acid decarboxylase from Candida guilliermondii.

ABSTRACT: The gene for a eukaryotic phenolic acid decarboxylase of Candida guilliermondii was cloned, sequenced, and expressed in Escherichia coli for the first time. The structural gene contained an open reading frame of 504 bp, corresponding to 168 amino acids with a calculated molecular mass of 19,828 Da. The deduced amino sequence exhibited low similarity to those of functional phenolic acid decarboxylases previously reported from bacteria with 25-39% identity and to those of PAD1 and FDC1 proteins from Saccharomyces cerevisiae with less than 14% identity. The C. guilliermondii phenolic acid decarboxylase converted the main substrates ferulic acid and p-coumaric acid to the respective corresponding products. Surprisingly, the ultrafiltrate (Mr 10,000-cut-off) of the cell-free extract of C. guilliermondii remarkably activated the ferulic acid decarboxylation by the purified enzyme, whereas it was almost without effect on the p-coumaric acid decarboxylation. Gel-filtration chromatography of the ultrafiltrate suggested that an endogenous amino thiol-like compound with a molecular weight greater than Mr 1,400 was responsible for the activation.

SUBMITTER: Huang HK

PROVIDER: S-EPMC3402150 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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An endogenous factor enhances ferulic acid decarboxylation catalyzed by phenolic acid decarboxylase from Candida guilliermondii.

Huang Hui-Kai HK Chen Li-Fan LF Tokashiki Masamichi M Ozawa Tadahiro T Taira Toki T Ito Susumu S

AMB Express 20120104 1

The gene for a eukaryotic phenolic acid decarboxylase of Candida guilliermondii was cloned, sequenced, and expressed in Escherichia coli for the first time. The structural gene contained an open reading frame of 504 bp, corresponding to 168 amino acids with a calculated molecular mass of 19,828 Da. The deduced amino sequence exhibited low similarity to those of functional phenolic acid decarboxylases previously reported from bacteria with 25-39% identity and to those of PAD1 and FDC1 proteins fr ...[more]

PMID: 22217315

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Project description:The interest towards ferulic acid decarboxylase (FDC), piqued by the enzyme's unique 1,3-dipolar cycloaddition mechanism and its atypic prFMN cofactor, provided several applications of the FDC mediated decarboxylations, such as the synthesis of styrenes, or its diverse derivatives, including 1,3-butadiene and the enzymatic activation of C-H bonds through the reverse carboligation reactions. While rational design-based protein engineering was successfully employed for tailoring FDC towards diverse substrates of interest, the lack of high-throughput FDC-activity assay hinders its directed evolution-based protein engineering. Herein we report a toolbox, useful for the directed evolution based and/or structure-guided protein engineering of FDC, which was validated representatively on the well described FDC, originary from Saccharomyces cerevisiae (ScFDC). Accordingly, the developed fluorescent plate-assay allows in premiere the FDC-activity screens of a mutant library in a high-throughput manner. Moreover, using the plate-assay for the activity screens of a rationally designed 23-membered ScFDC variant library against a substrate panel comprising of 16, diversely substituted cinnamic acids, revealed several variants of improved activity. The superior catalytic properties of the hits revealed by the plate-assay, were also supported by the conversion values from their analytical scale biotransformations. The computational results further endorsed the experimental findings, showing inactive binding poses of several non-transformed substrate analogues within the active site of the wild-type ScFDC, but favorable ones within the catalytic site of the variants of improved activity. The results highlight several 'hot-spot' residues involved in substrate specificity modulation of FDC, such as I189, I330, F397, I398 or Q192, of which mutations to sterically less demanding residues increased the volume of the active site, thus facilitated proper binding and increased conversions of diverse non-natural substrates. Upon revealing which mutations improve the FDC activity towards specific substrate analogues, we also provide key for the rational substrate-tailoring of FDC.

Dataset Information

An endogenous factor enhances ferulic acid decarboxylation catalyzed by phenolic acid decarboxylase from Candida guilliermondii.

Publications

An endogenous factor enhances ferulic acid decarboxylation catalyzed by phenolic acid decarboxylase from Candida guilliermondii.

Similar Datasets

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