Isofunctional enzymes PAD1 and UbiX catalyze formation of a novel cofactor required by ferulic acid decarboxylase and 4-hydroxy-3-polyprenylbenzoic acid decarboxylase

Abstract

The decarboxylation of antimicrobial aromatic acids such as phenylacrylic acid (cinnamic acid) and ferulic acid by yeast requires two enzymes described as phenylacrylic acid decarboxylase (PAD1) and ferulic acid decarboxylase (FDC). These enzymes are of interest for various biotechnological applications, such as the production of chemical feedstocks from lignin under mild conditions. However, the specific role of each protein in catalyzing the decarboxylation reaction remains unknown. To examine this, we have overexpressed and purified both PAD1 and FDC from E. coli. We demonstrate that PAD1 is a flavin mononucleotide (FMN)-containing protein. However, it does not function as a decarboxylase. Rather, PAD1 catalyzes the formation of a novel, diffusible cofactor required by FDC for decarboxylase activity. Coexpression of FDC and PAD1 results in the production of FDC with high levels cofactor bound. Holo-FDC catalyzes the decarboxylation of phenylacrylic acid, coumaric acid and ferulic acid with apparent kcat ranging from 1.4-4.6 s(-1). The UV-visible and mass spectra of the cofactor indicate that it appears to be a novel, modified form of reduced FMN; however, its instability precluded determination of its structure. The E. coli enzymes UbiX and UbiD are related by sequence to PAD1 and FDC respectively and are involved in the decarboxylation of 4-hydroxy-3-octaprenylbenzoic acid, an intermediate in ubiquinone biosynthesis. We found that endogenous UbiX can also activate FDC. This implies that the same cofactor is required for decarboxylation of 4-hydroxy-3-polyprenylbenzoic acid by UbiD and suggests a wider role for this cofactor in metabolism.

Figure 1.

Decarboxylation reactions catalyzed by PAD1/FDC in yeast and the related decarboxylation catalyzed by UbiX/UbiD in bacteria as part of ubiquinone biosynthesis.

Figure 2.

Purification and initial characterization of FDC. (A) SDS-PAGE and native PAGE analysis of purified FDC. (B) LC-ESI-MS analysis of purified FDC. (C) pH dependence of cinnamic acid decarboxylase activity; for assay details, see the main text. The buffers used were pH 6.0–8.0, 50 mM potassium phosphate buffer, 100 mM NaCl and pH 8.0–9.0, 50 mM Tris/Cl, 100 mM NaCl.

Figure 3.

Effect of dialysis and addition of E. coli BL21 cell lysate on FDC activity. The activity of FDC as purified from E. coli BL21 is arbitrarily assigned as 100% and corresponds to a specific activity of ~0.54 μmol styrene·min⁻¹·mg⁻¹ enzyme.

Figure 4.

Role of tPAD1 in activation of apo-FDC purified from E. coli BL21/ΔubiX. Incubation of inactive, apo-FDC with E. coli BL21/ΔubiX cell lysates and tPAD1 activates the enzyme. The activity of FDC as purified from E. coli BL21 is arbitrarily assigned as 100% (column 1 Figure 3).

Figure 5.

Purification and initial characterization of tPAD1. (A) SDS-PAGE analysis of purified tPAD1. (B) ESI-MS of purified tPAD1. (C) UV–visible spectrum of tPAD1 indicative of bound flavin cofactor(s). LS-MS of flavin cofactor isolated from tPAD1, demonstrating the protein contains FMN.

Figure 6.

Interaction of tPAD1 and FDC is not required for activation of FDC. 1 μM FDC in buffer containing 6.7 mM cinnamic acid was separated from the other components of the experiment by a 3.5 kDa cutoff dialysis membrane, and the production of styrene was monitored as a function of time. The components added to the other side of the membrane were (○) E. coli BL21/ΔubiX cell lysate; (▲) E. coli BL21 cell lysate; (■) purified tPAD1, 10 μM; (●) E. coli BL21/ΔubiX cell lysate and tPAD1, 10 μM.

Figure 7.

UV–visible spectrum of holo-FDC. The spectrum is suggestive of a reduced flavin-like chromophore.

Figure 8.

High resolution of ESI-MS spectrum of the novel cofactor for FDC. The less abundant peaks at (m+1)/z = 643.2162 and (m+1)/z = 625.2054 most likely represent the loss of one and two water molecules from the most abundant species with (m+1)/z = 661.2279. Inset: the same spectrum showing a wider spectral window.