Structure and Mechanism of Ferulic Acid Decarboxylase (FDC1) from Saccharomyces cerevisiae

Abstract

The nonoxidative decarboxylation of aromatic acids occurs in a range of microbes and is of interest for bioprocessing and metabolic engineering. Although phenolic acid decarboxylases provide useful tools for bioindustrial applications, the molecular bases for how these enzymes function are only beginning to be examined. Here we present the 2.35-Å-resolution X-ray crystal structure of the ferulic acid decarboxylase (FDC1; UbiD) from Saccharomyces cerevisiae. FDC1 shares structural similarity with the UbiD family of enzymes that are involved in ubiquinone biosynthesis. The position of 4-vinylphenol, the product of p-coumaric acid decarboxylation, in the structure identifies a large hydrophobic cavity as the active site. Differences in the ␤2e-␣5 loop of chains in the crystal structure suggest that the conformational flexibility of this loop allows access to the active site. The structure also implicates Glu285 as the general base in the nonoxidative decarboxylation reaction catalyzed by FDC1. Biochemical analysis showed a loss of enzymatic activity in the E285A mutant. Modeling of 3-methoxy-4-hydroxy-5-decaprenylbenzoate, a partial structure of the physiological UbiD substrate, in the binding site suggests that an ϳ30-Å-long pocket adjacent to the catalytic site may accommodate the isoprenoid tail of the substrate needed for ubiquinone biosynthesis in yeast. The three-dimensional structure of yeast FDC1 provides a template for guiding protein engineering studies aimed at optimizing the efficiency of aromatic acid decarboxylation reactions in bioindustrial applications.T he chemical production of benzenoids, such as styrene, from petroleum provides a wide range of building blocks for use in paints, dyes, plastics, and synthetic pharmaceuticals. Current styrene production involves the energy-intensive dehydrogenation of petroleum-derived ethylbenzene and yields more than 30 million tons of material each year (1). As an alternative to petroleumbased synthesis, research into finding renewable sources of benzenoid compounds in nature has focused attention on multiple plant and microbial pathways. Substituted cinnamic acids are abundant molecules in plant lignin polymers and can provide feedstocks for microbial bioprocessing methods aimed at yielding value-added products (2, 3). Degradation of lignin releases ferulic and p-coumaric acids that can be converted to 4-vinylguaicol (4-ethenyl-2-methoxyphenol) and 4-vinylphenol (4-ethenylphenol), respectively, and other hydroxycinnamates can be metabolized to vanillin as a natural flavoring in foods, beverages, and other products (4, 5). Related aromatic compounds are also natural components in wine and other fermented beverages and food (6-8). Similarly, other production routes for catechol and styrene, using microbes that metabolize benzenoid molecules by nonoxidative decarboxylation, have also been explored recently (8-10). For example, overexpression of phenylalanine ammonia lyase from Arabidopsis thaliana and of ferulic acid decarboxylase (FDC) from Saccharomyces cerevis...

Enzymes involved in the anaerobic degradation of ortho‐phthalate by the nitrate‐reducing bacterium Azoarcus sp. strain PA01

“…Phylogenetic analysis of the putative o ‐phthalyl‐CoA decarboxylase revealed that PhtDa (PA01_00217) was affiliated with UbiD‐like decarboxylases (Bhuiya et al , ) that are known to decarboxylate, for example 3‐polyprenyl‐4‐hydroxybenzoate in Escherichia coli (Baba et al ., ). UbiD‐like decarboxylases are commonly found in bacteria (Jacewicz et al ., ) and are involved in ubiquinone biosynthesis (Zhang and Javor, ).…”

The Impact of Simple Phenolic Compounds on Beer Aroma and Flavor

“…These include species which represent contaminants in a brewing environment, but also species and strains utilized in brewing. Many strains of brewer's yeast (Saccharomyces cerevisiae), as well as Brettanomyces sp., Lactobacillus, and Pediococcus are PAD+ [50][51][52][53][54]. These non-Saccharomyces genera of microbes are utilized in traditional sour beers of Belgium, and increasingly in other "wild" and sour beer styles popular in the craft brewing industry [36,37,[39][40][41]50].…”

“…This "phenolic off-flavor" character requires two enzymes, a phenylacrylic acid decarboxylase (PAD1) and a ferulic acid decarboxylase (FDC1). Somewhat confusingly, the PAD1 enzyme is not a decarboxylase, but catalyzes the synthesis of an FMN-related co-factor required for the function of FDC1 [52,55]. FDC1 enzyme catalyzes the decarboxylation of cinnamic acid and derivatives.…”

“…FDC1 enzyme catalyzes the decarboxylation of cinnamic acid and derivatives. Among the well-characterized microbial decarboxylase enzymes, the Saccharomyces FDC1 is of the few that can use cinnamic acid as a substrate yielding styrene as a product; others are only functional on hydroxycinnamic acids [52].…”

“…cerevisiae Pad1 and Fdc1 genes are analogous to the UbiX and UbiD genes of E. coli, respectively, involved in the ubiquinone biosynthetic pathway [53,55]. The recently determined 3D structure of the full-length 503 amino acid FDC1 protein reveals a cinnamic acid substrate binding site that can be modeled to accept the much larger substrate predicted for a role in the ubiquinone pathway [52]. Pad1/Fdc1 double knockout yeast are not deficient in ubiquinone biosynthesis however, suggesting the possible presence of additional genes for this biosynthetic pathway [55].…”