Abstract
The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide (prFMN) as cofactor, bound adjacent to a conserved Glu–Arg–Glu/Asp ionic network in the enzyme's active site. It is proposed that UbiD activation requires oxidative maturation of the cofactor, for which two distinct isomers, prFMNketimine and prFMNiminium, have been observed. It also has been suggested that only the prFMNiminium form is relevant to catalysis, which requires transient cycloaddition between substrate and cofactor. Using Aspergillus niger Fdc1 as a model system, we reveal that isomerization of prFMNiminium to prFMNketimine is a light-dependent process that is largely independent of the Glu277–Arg173–Glu282 network and accompanied by irreversible loss of activity. On the other hand, efficient catalysis was highly dependent on an intact Glu–Arg–Glu network, as only Glu → Asp substitutions retain activity. Surprisingly, oxidative maturation to form the prFMNiminium species is severely affected only for the R173A variant. In summary, the unusual irreversible isomerization of prFMN is light-dependent and probably proceeds via high-energy intermediates but is independent of the Glu–Arg–Glu network. Our results from mutagenesis, crystallographic, spectroscopic, and kinetic experiments indicate a clear role for the Glu–Arg–Glu network in both catalysis and oxidative maturation.
Highlights
The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide as cofactor, bound adjacent to a conserved Glu–Arg–Glu/Asp ionic network in the enzyme’s active site
The UbiD family contains a highly conserved RXnEX4(E/D) sequence motif, which is believed to play a role in catalysis
Despite the fact that a mechanism for catalysis has been proposed for the prFMNketimine (7), indirect evidence supports the hypothesis that prFMNiminium is the catalytically active form of the cofactor (7, 10, 11)
Summary
The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide (prFMN) as cofactor, bound adjacent to a conserved Glu–Arg–Glu/Asp ionic network in the enzyme’s active site. Atomic resolution crystal structures of Aspergillus niger Fdc, a UbiD-type (de)carboxylase, revealed two forms of the oxidized cofactor These correspond to an isoalloxazine N5-iminium form (prFMNiminium) and the isomeric N5-secondary ketimine form (prFMNketimine), the latter having a very distinct ring structure derived from the isoalloxazine ring system (Fig. 1A). Preliminary studies of R173A, E277Q, and E282Q variants suggested that all are catalytically inactive and possess altered UV-visible spectra (7) This implies the role of this network could be 2-fold: first to facilitate oxidative maturation of the cofactor to the iminium form (Fig. 1A) and second to act as a key acid-base during catalysis (Fig. 1B). In addition to the high-resolution crystal structures, mass spectrometric, spectroscopic, kinetic, and hydrogen/deuterium exchange studies of six Fdc variants provide clear evidence for the role of the Glu–Arg–Glu motif in both oxidative maturation and catalysis
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