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.

The utilization of CO₂ as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme‐catalyzed para‐carboxylation of catechols, employing 3,4‐dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMN^iminium species. This study reports on the in vitro reconstitution and activation of a prFMN‐dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN‐associated 1,3‐dipolar cycloadditions in related enzymes.

The activity of the reversible decarboxylase enzyme Fdc1 is dependent on prenylated FMN (prFMN), a recently discovered cofactor. The oxidized prFMN supports a 1,3-dipolar cycloaddition mechanism that underpins reversible decarboxylation. Fdc1 is a distinct member of the UbiD family of enzymes, with the canonical UbiD catalyzing the (de)carboxylation of para-hydroxybenzoic acid-type substrates. Here we show that the Escherichia coli UbiD enzyme, which is implicated in ubiquinone biosynthesis, cannot be isolated in an active holoenzyme form despite the fact active holoFdc1 is readily obtained. Formation of holoUbiD requires reconstitution in vitro of the apoUbiD with reduced prFMN. Furthermore, although the Fdc1 apoenzyme can be readily reconstituted and activated, in vitro oxidation to the mature prFMN cofactor stalls at formation of a radical prFMN species in holoUbiD. Further oxidative maturation in vitro occurs only at alkaline pH, suggesting a proton-coupled electron transfer precedes formation of the fully oxidized prFMN. Crystal structures of holoUbiD reveal a relatively open active site potentially occluded from solvent through domain motion. The presence of a prFMN sulfite-adduct in one of the UbiD crystal structures confirms oxidative maturation does occur at ambient pH on a slow time scale. Activity could not be detected for a range of putative para-hydroxybenzoic acid substrates tested. However, the lack of an obvious hydrophobic binding pocket for the octaprenyl tail of the proposed ubiquinone precursor substrate does suggest UbiD might act on a non-prenylated precursor. Our data reveals an unexpected variation occurs in domain mobility, prFMN binding, and maturation by the UbiD enzyme family.

Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers.