Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 293, Issue 7, Pages 2272-2287Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA117.000881
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Funding
- BBSRC [BB/K017802/1, BB/P000622/1]
- ERC [ADG_695013]
- BBSRC iCASE studentship
- Biotechnology and Biological Sciences Research Council [BB/P000622/1, BB/K017802/1, 1482350] Funding Source: researchfish
- BBSRC [BB/P000622/1, BB/K017802/1] Funding Source: UKRI
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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, prFMN(ketimine) and prFMN(iminium), have been observed. It also has been suggested that only the prFMN(iminium) 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 ofprFMN(iminium) to prFMN(ketimine) is a light-dependent process that is largely independent of the Glu(277)-Arg(173)-Glu(282) 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 prFMN(iminium) 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.
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