Photocobilins integrate B12 and bilin photochemistry for enzyme control.

Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B12) can act as a light-sensing chromophore heralded a new field of B12-photobiology. Although microbial genome analysis indicates that photoactive B12-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.

The prFMNH2-binding chaperone LpdD assists UbiD decarboxylase activation.

The UbiD enzyme family of prenylated flavin (prFMN)-dependent reversible decarboxylases is near ubiquitously present in microbes. For some UbiD family members, enzyme activation through prFMNH2 binding and subsequent oxidative maturation of the cofactor readily occurs, both in vivo in a heterologous host and through in vitro reconstitution. However, isolation of the active holo-enzyme has proven intractable for others, notably the canonical Escherichia coli UbiD. We show that E. coli heterologous expression of the small protein LpdD-associated with the UbiD-like gallate decarboxylase LpdC from Lactobacillus plantarum-unexpectedly leads to 3,4-dihydroxybenzoic acid decarboxylation whole-cell activity. This activity was shown to be linked to endogenous E. coli ubiD expression levels. The crystal structure of the purified LpdD reveals a dimeric protein with structural similarity to the eukaryotic heterodimeric proteasome assembly chaperone Pba3/4. Solution studies demonstrate that LpdD protein specifically binds to reduced prFMN species only. The addition of the LpdD-prFMNH2 complex supports reconstitution and activation of the purified E. coli apo-UbiD in vitro, leading to modest 3,4-dihydroxybenzoic acid decarboxylation. These observations suggest that LpdD acts as a prFMNH2-binding chaperone, enabling apo-UbiD activation through enhanced prFMNH2 incorporation and subsequent oxidative maturation. Hence, while a single highly conserved flavin prenyltransferase UbiX is found associated with UbiD enzymes, our observations suggest considerable diversity in UbiD maturation, ranging from robust autocatalytic to chaperone-mediated processes. Unlocking the full (de)carboxylation scope of the UbiD-enzyme family will thus require more than UbiX coexpression.

Redox driven B12-ligand switch drives CarH photoresponse.

CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.

Efficient NADPH-dependent dehalogenation afforded by a self-sufficient reductive dehalogenase.

Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.

Structure Based Discovery of Inhibitors of CYP125 and CYP142 from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host-derived lipids such as cholesterol not only support Mtb growth, but are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5-(pyridin-4-yl)-1H-indole-2-carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure-guided approach, combined with biophysical characterization, compounds with micromolar range in-cell activity against clinically relevant drug-resistant isolates were obtained. These will incite further development of much-needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.

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.

A Vitamin B2 -Photocatalysed Approach to Methionine Analogues.

Access to new non-canonical amino acid residues is crucial for medicinal chemistry and chemical biology. Analogues of the amino acid methionine have been far less explored-despite their use in biochemistry, pharmacology and peptide bioconjugation. This is largely due to limited synthetic access. Herein, we exploit a new disconnection to access non-natural methionines through the development of a photochemical method for the radical α-C-H functionalization of sulfides with alkenes, in water, using inexpensive and commercially-available riboflavin (vitamin B2 ) as a photocatalyst. Our photochemical conditions allow the two-step synthesis of novel methionine analogues-by radical addition to unsaturated amino acid derivatives-and the chemoselective modification of peptide side-chains to yield non-natural methionine residues within small peptides. The mechanism of the bio-inspired flavin photocatalysis has been probed by experimental, DFT and TDDFT studies.

Structural insights into UbiD reversible decarboxylation.

The ubiquitous UbiX-UbiD system is associated with a wide range of microbial (de)carboxylation reactions. Recent X-ray crystallographic studies have contributed to elucidating the enigmatic mechanism underpinning the conversion of α,ß-unsaturated acids by this system. The UbiD component utilises a unique cofactor, prenylated flavin (prFMN), generated by the bespoke action of the associated UbiX flavin prenyltransferase. Structure determination of a range of UbiX/UbiD representatives has revealed a generic mode of action for both the flavin-to-prFMN metamorphosis and the (de)carboxylation. In contrast to the conserved UbiX, the UbiD superfamily is associated with a versatile substrate range. The latter is reflected in the considerable variety of UbiD quaternary structure, dynamic behaviour and active site architecture. Directed evolution of UbiD enzymes has taken advantage of this apparent malleability to generate new variants supporting in vivo hydrocarbon production. Other applications include coupling UbiD to carboxylic acid reductase to convert alkenes into α,ß-unsaturated aldehydes via enzymatic CO2 fixation.

Enzymatic N-Allylation of Primary and Secondary Amines Using Renewable Cinnamic Acids Enabled by Bacterial Reductive Aminases.

Allylic amines are a versatile class of synthetic precursors of many valuable nitrogen-containing organic compounds, including pharmaceuticals. Enzymatic allylic amination methods provide a sustainable route to these compounds but are often restricted to allylic primary amines. We report a biocatalytic system for the reductive N-allylation of primary and secondary amines, using biomass-derivable cinnamic acids. The two-step one-pot system comprises an initial carboxylate reduction step catalyzed by a carboxylic acid reductase to generate the corresponding α,ß-unsaturated aldehyde in situ. This is followed by reductive amination of the aldehyde catalyzed by a bacterial reductive aminase pIR23 or BacRedAm to yield the corresponding allylic amine. We exploited pIR23, a prototype bacterial reductive aminase, self-sufficient in catalyzing formal reductive amination of α,ß-unsaturated aldehydes with various amines, generating a broad range of secondary and tertiary amines accessed in up to 94% conversion under mild reaction conditions. Analysis of products isolated from preparative reactions demonstrated that only selective hydrogenation of the C=N bond had occurred, preserving the adjacent alkene moiety. This process represents an environmentally benign and sustainable approach for the synthesis of secondary and tertiary allylic amine frameworks, using renewable allylating reagents and avoiding harsh reaction conditions. The selectivity of the system ensures that bis-allylation of the alkylamines and (over)reduction of the alkene moiety are avoided.

Heterologous production and biophysical characterization of catabolic Nitratireductor pacificus pht-3B reductive dehalogenase.

Reductive dehalogenases provide a possible route to the biotechnological remediation of widespread anthropogenic environmental organohalide contamination. These bacterial enzymes employ cobalamin and an internal electron transfer chain of two [4Fe-4S] clusters to remove halide ions from organohalides, leaving an organic molecule more amenable to further transformations. Detailed protocols for the cloning, heterologous expression, purification, crystallization and characterization of the catabolic dehalogenase from Nitratireductor pacificus pht-3B (NpRdhA) are presented, together with insight into enzyme turnover, substrate selectivity and the use of electron paramagnetic resonance (EPR) spectroscopy as an active site probe.

Structural and biochemical characterization of the prenylated flavin mononucleotide-dependent indole-3-carboxylic acid decarboxylase.

The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide cofactor for catalysis. However, only a handful of UbiD family members have been characterized in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied. Structural insights from crystal structure determination combined with small-angle X-ray scattering measurements reveal that the enzyme likely undergoes an open-closed transition as a consequence of domain motion, an event that is likely coupled to catalysis. We also demonstrate that the indole-3-carboxylic acid decarboxylase can be coupled with carboxylic acid reductase to produce indole-3-carboxyaldehyde from indole + CO2 under ambient conditions. These insights provide further evidence for a common mode of action in the widespread UbiD enzyme family.

A new strategy for hit generation: Novel in cellulo active inhibitors of CYP121A1 from Mycobacterium tuberculosis via a combined X-ray crystallographic and phenotypic screening approach (XP screen).

There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a validation of target engagement as well as determination of in cellulo activity. The utility of this method is illustrated by way of an XP Screen against CYP121A1, a cytochrome P450 enzyme from Mycobacterium tuberculosis (Mtb) championed as a validated drug discovery target. A focused screening set was synthesized and tested by such means, with several members of the set showing promising activity against Mtb strain H37Rv. One compound was observed as an X-ray hit against CYP121A1 and showed improved activity against Mtb strain H37Rv under multiple assay conditions (pan-assay activity). Data obtained during X-ray crystallographic screening were utilized in a structure-based campaign to design a limited number of analogues (less than twenty), many of which also showed pan-assay activity against Mtb strain H37Rv. These included the benzo[b][1,4]oxazine derivative (MIC90 6.25 µM), a novel hit compound suitable as a starting point for a more involved hit to lead candidate medicinal chemistry campaign.

A guide to time-resolved structural analysis of light-activated proteins.

Dynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X-ray free electron lasers have facilitated investigations of structural dynamics in proteins with unprecedented temporal and spatial resolution. Light-activated proteins are attractive targets for time-resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light-absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light absorption, rapid chemical/small-scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enables the desired chemical and/or biological output response. Time-resolved serial femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light-activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time-resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time-resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods.

A Vitamin B2-Photocatalysed Approach to Methionine Analogues.

Access to new non-canonical amino acid residues is crucial for medicinal chemistry and chemical biology. Analogues of the amino acid methionine have been far less explored-despite their use in biochemistry, pharmacology and peptide bioconjugation. This is largely due to limited synthetic access. Herein, we exploit a new disconnection to access non-natural methionines through the development of a photochemical method for the radical α-C-H functionalization of sulfides with alkenes, in water, using inexpensive and commercially-available riboflavin (vitamin B2) as a photocatalyst. Our photochemical conditions allow the two-step synthesis of novel methionine analogues-by radical addition to unsaturated amino acid derivatives-and the chemoselective modification of peptide side-chains to yield non-natural methionine residues within small peptides. The mechanism of the bio-inspired flavin photocatalysis has been probed by experimental, DFT and TDDFT studies.

Directed evolution of prenylated FMN-dependent Fdc supports efficient in vivo isobutene production.

Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group.

UbiD domain dynamics underpins aromatic decarboxylation.

The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.

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.

Structure and Mechanism of Pseudomonas aeruginosa PA0254/HudA, a prFMN-Dependent Pyrrole-2-carboxylic Acid Decarboxylase Linked to Virulence.

The UbiD family of reversible (de)carboxylases depends on the recently discovered prenylated-FMN (prFMN) cofactor for activity. The model enzyme ferulic acid decarboxylase (Fdc1) decarboxylates unsaturated aliphatic acids via a reversible 1,3-cycloaddition process. Protein engineering has extended the Fdc1 substrate range to include (hetero)aromatic acids, although catalytic rates remain poor. This raises the question how efficient decarboxylation of (hetero)aromatic acids is achieved by other UbiD family members. Here, we show that the Pseudomonas aeruginosa virulence attenuation factor PA0254/HudA is a pyrrole-2-carboxylic acid decarboxylase. The crystal structure of the enzyme in the presence of the reversible inhibitor imidazole reveals a covalent prFMN-imidazole adduct is formed. Substrate screening reveals HudA and selected active site variants can accept a modest range of heteroaromatic compounds, including thiophene-2-carboxylic acid. Together with computational studies, our data suggests prFMN covalent catalysis occurs via electrophilic aromatic substitution and links HudA activity with the inhibitory effects of pyrrole-2-carboxylic acid on P. aeruginosa quorum sensing.

A Biological Route to Conjugated Alkenes: Microbial Production of Hepta-1,3,5-triene.

Conjugated alkenes such as dienes and polyenes have a range of applications as pharmaceutical agents and valuable building blocks in the polymer industry. Development of a renewable route to these compounds provides an alternative to fossil fuel derived production. The enzyme family of the UbiD decarboxylases offers substantial scope for alkene production, readily converting poly unsaturated acids. However, biochemical pathways producing the required substrates are poorly characterized, and UbiD-application has hitherto been limited to biological styrene production. Herein, we present a proof-of-principle study for microbial production of polyenes using a bioinspired strategy employing a polyketide synthase (PKS) in combination with a UbiD-enzyme. Deconstructing a bacterial iterative type II PKS enabled repurposing the broad-spectrum antibiotic andrimid biosynthesis pathway to access the metabolic intermediate 2,4,6-octatrienoic acid, a valuable chemical for material and pharmaceutical industry. Combination with the fungal ferulic acid decarboxylase (Fdc1) led to a biocatalytic cascade-type reaction for the production of hepta-1,3,5-triene in vivo. Our approach provides a novel route to generate unsaturated hydrocarbons and related chemicals and provides a blue-print for future development and application.

Synthetic Enzyme-Catalyzed CO2 Fixation Reactions.

In recent years, (de)carboxylases that catalyze reversible (de)carboxylation have been targeted for application as carboxylation catalysts. This has led to the development of proof-of-concept (bio)synthetic CO2 fixation routes for chemical production. However, further progress towards industrial application has been hampered by the thermodynamic constraint that accompanies fixing CO2 to organic molecules. In this Review, biocatalytic carboxylation methods are discussed with emphases on the diverse strategies devised to alleviate the inherent thermodynamic constraints and their application in synthetic CO2 -fixation cascades.