X-ray crystallography and EPR spectroscopy are used to characterize a soluble, oxygen-tolerant reductive dehalogenase from Nitratireductor pacificus pht-3B; the data suggest that the cobalt in the cobalamin cofactor ligates the halogen atom of the substrate, directly abstracting the halogen atom via an oxidative addition. Reductive dehalogenases are cobalamin-dependent enzymes that catalyse the removal of a halogen atom from organohalides, organic molecules that contain one or more halogen atoms. A large proportion of environmental pollutants are organohalides and reductive dehalogenases are responsible for the biological dehalogenation of these compounds, including polychlorinated biphenyls. In this manuscript, the authors used X-ray crystallography and EPR spectroscopy to characterize a soluble, oxygen-tolerant reductive dehalogenase, pht-3B, from Nitratireductor pacificus. Their data suggest that the cobalt in the cobalamin cofactor forms a covalent bond with the halogen atom of the substrate, directly abstracting the halogen atom via an oxidative addition. This mechanism is fundamentally different from the mechanisms of other cobalamin-containing enzymes. These findings will be of relevance to future use of reductive dehalogenases in bioremediation and biocatalysis. Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides1. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle2,3. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria4,5, with substrates including polychlorinated biphenyls or dioxins6,7. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies8,9,10,11,12. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon–cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies13, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen–cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

The Fdc1 protein from Aspergillus niger (which is homologous to the UbiD enzyme) uses a new prenylated flavin cofactor to achieve 1,3-dipolar cycloaddition chemistry and catalyse the reversible decarboxylation of aromatic carboxylic acids. A pair of manuscripts published this week [in this issue of Nature] describes detailed studies of the structure and mechanism of the enzymes UbiD and UbiX that together are responsible for non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone biosynthesis and microbial biodegradation of aromatic compounds. Mark White et al. show that UbiX is a flavin prenyltransferase, catalysing the covalent attachment of a fourth ring (the dimethylallyl group) to the N5 and C6 atoms of the three-ring riboflavin system. The unusual flavin-derived cofactor synthesized by UbiX is then used by UbiD, a decarboxylase, in the next step of the ubiquinone biosynthetic pathway. Karl Payne et al. show that the Fdc protein from Aspergillus niger (which is homologous to UbiD) uses 1,3-dipolar cycloaddition chemistry to catalyse the reversible decarboxylation of aromatic carboxylic acids. This is the first example of an enzyme-catalysed 1,3-dipolar cycloaddition reaction. The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis1,2,3 or microbial biodegradation of aromatic compounds4,5,6, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear7,8,9. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad110. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor–cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry11,12, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

Ubiquinone is an essential component of electron transfer chains found both in bacteria and in mitochondria; the bacterial enzyme UbiX involved in ubiquinone biosynthesis is a flavin prenyltransferase, and the flavin-derived cofactor synthesized by UbiX is used by the UbiD decarboxylase in the ubiquinone biosynthetic pathway. A pair of manuscripts published this week [in this issue of Nature] describes detailed studies of the structure and mechanism of the enzymes UbiD and UbiX that together are responsible for non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone biosynthesis and microbial biodegradation of aromatic compounds. Mark White et al. show that UbiX is a flavin prenyltransferase, catalysing the covalent attachment of a fourth ring (the dimethylallyl group) to the N5 and C6 atoms of the three-ring riboflavin system. The unusual flavin-derived cofactor synthesized by UbiX is then used by UbiD, a decarboxylase, in the next step of the ubiquinone biosynthetic pathway. Karl Payne et al. show that the Fdc protein from Aspergillus niger (which is homologous to UbiD) uses 1,3-dipolar cycloaddition chemistry to catalyse the reversible decarboxylation of aromatic carboxylic acids. This is the first example of an enzyme-catalysed 1,3-dipolar cycloaddition reaction. Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains1. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor2. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected3,4. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD5. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases6,7, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases8. The active site environment is dominated by π systems, which assist phosphate-C1′ bond breakage following FMN reduction, leading to formation of the N5–C1′ bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3′–C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.

The biological production of FDCA is of considerable value as a potential replacement for petrochemical derived monomers such as terephthalate, used in polyethylene terephthalate (PET) plastics. HmfF belongs to an uncharacterised branch of the prenylated flavin (prFMN) dependent UbiD-family of reversible (de)carboxylases, and is proposed to convert 2,5-furan-dicarboxylic acid (FDCA) to furoic acid in vivo. We present detailed characterisation of HmfF and demonstrate that HmfF can catalyse furoic acid carboxylation at elevated CO2 levels in vitro. We report the crystal structure of a thermophilic HmfF from Pelotomaculum thermopropionicum, revealing the active site located above the prFMN cofactor contains a furoic acid/FDCA binding site composed of residues H296-R304-R331 specific to the HmfF branch of UbiD enzymes. Variants of the latter are compromised in activity, while H296N alters the substrate preference to pyrrol compounds. Solution studies and crystal structure determination of an engineered dimeric form of the enzyme revealed an unexpected key role for a UbiD-family wide conserved Leu residue in activity. The structural insights into substrate and cofactor binding provide a template for further exploitation of HmfF in the production of FDCA plastic precursors, and improve our understanding of catalysis by members of the UbiD enzyme family.

Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1^Ubix, the cofactor confers structural stability to the enzyme. IM–MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM–MS data.