Probing the role of protein conformational changes in the mechanism of prenylated-FMN-dependent phenazine-1-carboxylic acid decarboxylase.

Phenazine-1-carboxylic acid decarboxylase (PhdA) is a prenylated-FMN-dependent (prFMN) enzyme belonging to the UbiD family of decarboxylases. Many UbiD-like enzymes catalyze (de)carboxylation reactions on aromatic rings and conjugated double bonds and are potentially valuable industrial catalysts. We have investigated the mechanism of PhdA using a slow turnover substrate, 2,3-dimethylquinoxaline-5-carboxylic acid (DQCA). Detailed analysis of the pH dependence and solvent deuterium isotope effects associated with the reaction uncovered unusual kinetic behavior. At low substrate concentrations, a substantial inverse solvent isotope effect (SIE) is observed on Vmax/KM of ∼ 0.5 when reaction rates of DQCA in H2O and D2O are compared. Under the same conditions, a normal SIE of 4.15 is measured by internal competition for proton transfer to the product. These apparently contradictory results indicate that the SIE values report on different steps in the mechanism. A proton inventory analysis of the reaction under Vmax/KM and Vmax conditions points to a "medium effect" as the source of the inverse SIE. Molecular dynamics simulations of the effect of D2O on PhdA structure support that D2O reduces the conformational lability of the enzyme and results in a more compact structure, akin to the active, "closed" conformer observed in crystal structures of some UbiD-like enzymes. Consistent with the simulations, PhdA was found to be more stable in D2O and to bind DQCA more tightly, leading to the observed rate enhancement under Vmax/KM conditions.

The UbiX-UbiD system: The biosynthesis and use of prenylated flavin (prFMN).

The UbiX-UbiD system consists of the flavin prenyltransferase UbiX that produces prenylated FMN that serves as the cofactor for the (de)carboxylase UbiD. Recent developments have provided structural insights into the mechanism of both enzymes, detailing unusual chemistry in each case. The proposed reversible 1,3-dipolar cycloaddition between the cofactor and substrate serves as a model to explain many of the key UbiD family features. However, considerable variation exists in the many branches of the UbiD family tree.

Prenylated flavins: structures and mechanisms.

The UbiX/UbiD system is widespread in microbes and responsible for the reversible decarboxylation of unsaturated carboxylic acids. The UbiD enzyme catalyzes this unusual reaction using a prenylated flavin (prFMN) as cofactor, the latter formed by the flavin prenyltransferase UbiX. A detailed picture of the biochemistry of flavin prenylation, oxidative maturation, and covalent catalysis underpinning reversible decarboxylation is emerging. This reveals the prFMN cofactor can undergo a wide range of transformations, complemented by considerable UbiD-variability. These provide a blueprint for biotechnological applications aimed at producing hydrocarbons or aromatic C-H activation through carboxylation.

Unravelling interactions between active site residues and DMAP in the initial steps of prenylated flavin mononucleotide biosynthesis catalyzed by PaUbiX.

BACKGROUND: Prenylated flavin mononucleotide (prFMN) is a recently discovered, heavily modified flavin compound. It is the only known cofactor that enables enzymatic 1,3-dipolar cycloaddition reactions. It is produced by enzymes from the UbiX family, from flavin mononucleotide and either dimethylallyl mono- or diphosphate. prFMN biosynthesis is currently reported to be initiated by protonation of the substrate by Glu140. METHODS: Computational chemistry methods are applied herein - Constant pH MD, classical MD simulations, and QM cluster optimizations. RESULTS: Glu140 competes for a single proton with Lys129 prior to prFMN biosynthesis, but it is the latter that adopted a protonated state. Once the prenyl-FMN adduct is formed, Glu140 occurs in a protonated state far more often, while the occupancy of protonated Lys129 does not change. Lys129, Glu140, and Arg122 seem to play a key role in either stabilizing or protonating DMAP phosphate group within the PaUbiX active site throughout initial steps of prFMN biosynthesis. CONCLUSIONS: The role of Lys129 in the functioning of PaUbiX is reported for the first time. Glu140 is unlikely to act as a proton donor in prFMN biosynthesis. Instead, Lys129 and Arg122 fulfil this role. Glu140 still plays a role in contributing to hydrogen-bond network. This behavior is most likely conserved throughout the UbiX family due to the structural similarity of the active sites of those proteins. SIGNIFICANCE: Mechanistic insights into a crucial biochemical process, the biosynthesis of prFMN, are provided. This study, although purely computational, extends and perfectly complements the knowledge obtained in classical laboratory experiments.

The In Vitro Production of prFMN for Reconstitution of UbiD Enzymes.

Prenylated flavin (prFMN) is a modified FMN cofactor, the isoalloxazine is extended by an additional six membered nonaromatic ring. The modification confers azomethine ylide characteristics on the oxidised prFMN, allowing it to support the reversible nonoxidative decarboxylation of unsaturated acids by the UbiD family of decarboxylases. In absence of a chemical synthesis route for prFMN, enzymatic production by the flavin prenyltransferase, UbiX, is required for in vitro reconstitution of prFMN-dependent enzymes. Here we provide an overview of the methods for producing prFMN in vitro using the flavin prenyltransferase UbiX, and the subsequent reconstitution and activation of UbiD enzymes.

Biochemistry of prenylated-FMN enzymes.

The reversible (de)carboxylation of unsaturated carboxylic acids is carried out by the UbiX-UbiD system, ubiquitously present in microbes. The biochemical basis of this challenging reaction has recently been uncovered by the discovery of the UbiD cofactor, prenylated FMN (prFMN). This heavily modified flavin is synthesized by the flavin prenyltransferase UbiX, which catalyzes the non-metal dependent prenyl transfer from dimethylallyl(pyro)phosphate (DMAP(P)) to the flavin N5 and C6 positions, creating a fourth non-aromatic ring. Following prenylation, prFMN undergoes oxidative maturation to form the iminium species required for UbiD activity. prFMNiminium acts as a prostethic group and is bound via metal ion mediated interactions between UbiD and the prFMNiminium phosphate moiety. The modified isoalloxazine ring is place adjacent to the E(D)-R-E UbiD signature sequent motif. The fungal ferulic acid decarboxylase Fdc from Aspergillus niger has emerged as a UbiD-model system, and has yielded atomic level insight into the prFMNiminium mediated (de)carboxylation. A wealth of data now supports a mechanism reliant on reversible 1,3 dipolar cycloaddition between substrate and cofactor for this enzyme. This poses the intriguing question whether a similar mechanism is used by all UbiD enzymes, especially those that act as carboxylases on inherently more difficult substrates such as phenylphosphate or benzene/naphthalene. Indeed, considerable variability in terms of oligomerization, domain motion and active site structure is now reported for the UbiD family.

Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes.

The recent discovery of the prenylated FMN (prFMN) cofactor has led to a renewed interest in the prFMN-dependent UbiD family of enzymes. The latter catalyses the reversible decarboxylation of alpha-beta unsaturated carboxylic acids and features widely in microbial metabolism. The flavin prenyltransferase UbiX synthesizes prFMN from reduced FMN and phosphorylated dimethylallyl precursors. Oxidative maturation of the resulting prFMNreduced species to the active prFMNiminium form is required for UbiD activity. Heterologous production of active holo-UbiD requires co-expression of UbiX, but the levels of prFMN incorporation and oxidative maturation appear variable. Detailed protocols and strategies for in vitro reconstitution and oxidative maturation of UbiD are presented that can yield an alternative source of active holo-UbiD for biochemical studies.

Prenylated FMN: Biosynthesis, purification, and Fdc1 activation.

Prenylated flavin mononucleotide (prFMN) is a recently discovered flavin cofactor produced by the UbiX family of FMN prenyltransferases, and is required for the activity of UbiD-like reversible decarboxylases. The latter enzymes are known to be involved in ubiquinone biosynthesis and biotransformation of lignin, aromatic compounds, and unsaturated aliphatic acids. However, exploration of uncharacterized UbiD proteins for biotechnological applications is hindered by our limited knowledge about the biochemistry of prFMN and prFMN-dependent enzymes. Here, we describe experimental protocols and considerations for the biosynthesis of prFMN in vivo and in vitro, in addition to cofactor extraction and application for activation of UbiD proteins.

Biosynthesis and Activity of Prenylated FMN Cofactors.

Prenylated flavin mononucleotide (prFMN) is a recently discovered cofactor required by the UbiD family of reversible decarboxylases involved in ubiquinone biosynthesis, biological decomposition of lignin, and biotransformation of aromatic compounds. This cofactor is synthesized by UbiX-like prenyltransferases catalyzing the transfer of the dimethylallyl moiety of dimethylallyl-monophosphate (DMAP) to FMN. The origin of DMAP for prFMN biosynthesis and the biochemical properties of free prFMN are unknown. We show that in Escherichia coli cells, DMAP can be produced by phosphorylating prenol using ThiM or dephosphorylating DMAPP using Nudix hydrolases. We produced 14 active prenyltransferases whose properties enabled the purification and characterization of protein-free forms of prFMN. In vitro assays revealed that the UbiD-like ferulate decarboxylase (Fdc1) can be activated by free prFMNiminium or C2'-hydroxylated prFMNiminium under both oxidized and reduced conditions. These insights into the biosynthesis and properties of prFMN will facilitate further elucidation of the biochemical diversity of reversible UbiD (de)carboxylases.