A New Decarboxylase: A Mechanistic Characterization of PrFMN Decarboxylase FDC1

[摘要] The large number of inexpensive carboxylic acids found in nature has spurred research to convert carboxylic acids to an assortment of other functional groups for a range of purposes.To accomplish this, researchers employ a variety of strategies ranging from heat and heavy metals to biocatalysts.Ferulic acid decarboxylase (FDC1) from Saccharomyces cerevisiae is a member of the UbiD family of decarboxylase enzymes.The UbiD family of proteins consists of prenylated flavin (PrFMN)-dependent enzymes that catalyze the reversible decarboxylation of a wide variety of aromatic carboxylic acids.The UbiD family attracted interest as a biocatalyst to produce chemical feedstocks from renewable sources and as a potential target for antimicrobial research.The UbiD enzyme, FDC1, and its associated protein partner, PAD1, were identified as being responsible for the detoxification of aromatic carboxylic acids in fungi. How PAD1/FDC1 work together to facilitate the decarboxylation of toxic aromatic acids remains an open question.The genes encoding PAD1 and FDC1 were independently expressed in E. coli with the PAD1 homolog UbiX knocked out.Independently, FDC1 and PAD1 were unable to decarboxylate phenylacrylic acids.Moreover, PAD1 failed to decarboxylate any of the phenylacrylic acids by itself.In contrast FDC1 was able to recover its decarboxylase activity upon the addition of PAD1 or its homolog UbiX, indicating FDC1 was solely responsible for the decarboxylase activity.Co-expression of FDC1 and PAD1 coupled with the sole purification of FDC1 exhibited spectral characteristics of a reduced flavin in an aerobic system.This increased the kcat by 8-fold.The holo-FDC1 crystal structure (PDB 4ZAC) provided insight into the decarboxylation mechanism of FDC1.FDC1 was proposed to perform a unique and controversial 1,3-dipolar cycloaddition mechanism. To address the controversial nature of the proposed mechanism KIE and a Hammett analysis were utilized to ascertain whether FDC1 undergoes the proposed mechanism or a completely different mechanism.The solvent isotope effects, normal secondary isotope effects, and the negative slope of the Hammett analysis are consist with the rate-determining step being the breakdown of the PrFMN-product adduct through a non-concerted cyclo-elimination reaction, and provides evidence in favor of the novel 1,3-dipolarcycloaddition mechanism.The proposed mechanism involves the formation of a novel pentacyclic intermediate through a novel 1,3-dipolar cycloaddition mechanism between PrFMN and the β -γ double bond of the substrate, which serves to activate the substrate towards decarboxylation.In order to trap this hypothesized intermediate, a mechanism-based inhibitor 2-fluoro-2-nitro-vinylbenzene (FNVB) was used to trap the putative cyclo-addition intermediate.Upon incubating FNVB with FDC1, there was a red-shift in the flavin spectrum which is reminiscent of an uncharged N5, C4α dialkyl flavin adduct and is consistent with a 1,3-dipolar cycloadduct.Finally by pushing the equilibrium of the FDC1 reaction to favor the formation of product-PrFMN adducts:7dstyrene-PrFMN and styrene-PrFMN intermediates were isolated and characterized by Tandem Native Mass Spectrometry.FDC1 decarboxylates phenylacrylic acids by forming a novel 1,3-dipolar cycloaddition adduct, followed by a Grob fragmentation, protonation of the intermediate, re-cyclization between the PrFMN and the product, then finally product release.This work lays the groundwork for developing new biocatalysts from these UbiD decarboxylases and the development of a new class of antifungal or antibiotics drugs. 

[发布机构] University of Michigan

[效力级别] PrFMN

[关键词] FDC1 Decarboxylase;PrFMN;Enzymatic 1;3 Dipolar Cycloaddition;Chemistry;Science;Chemistry