Engineered enzymes for enantioselective nucleophilic aromatic substitutions.

Nucleophilic aromatic substitutions (SAr) are among the most widely used processes in the pharmaceutical and agrochemical industries, allowing convergent assembly of complex molecules through C-C and C-X (X = O, N, S) bond formation. SAr reactions are typically carried out using forcing conditions, involving polar aprotic solvents, stoichiometric bases and elevated temperatures, which do not allow for control over reaction selectivity. Despite the importance of SAr chemistry, there are only a handful of selective catalytic methods reported that rely on small organic hydrogen-bonding or phase-transfer catalysts.

Fragment-based development of small molecule inhibitors targeting cholesterol metabolism.

() is the world's most deadly infectious pathogen and new drugs are urgently required to combat the emergence of multi- (MDR) and extensively- (XDR) drug resistant strains. The bacterium specifically upregulates sterol uptake pathways in infected macrophages and the metabolism of host-derived cholesterol is essential for long-term survival Here, we report the development of antitubercular small molecules that inhibit the cholesterol oxidases CYP125 and CYP142, which catalyze the initial step of cholesterol metabolism. An efficient biophysical fragment screen was used to characterize the structure-activity relationships of CYP125 and CYP142, and identify a non-azole small molecule that can bind to the heme cofactor of both enzymes.

Expression and purification of Mycobacterium tuberculosis F-dependent glucose-6-phosphate dehydrogenase enzyme using Escherichia coli.

Since their discovery in Mycobacterium tuberculosis (Mtb), F-dependent enzymes have been identified as both important drug targets and potential industrial biocatalysts, including for bioremediation of otherwise recalcitrant substrates. Mtb-FGD1, utilizes glucose 6-phosphate (G6P) as an electron donor for the reduction of F. Current expression systems for Mtb-FGD1 use Mycobacterium smegmatis as host, because of the tendency for it to form inclusion bodies in E.

SignatureFinder enables sequence mining to identify cobalamin-dependent photoreceptor proteins.

Photoreceptors control cellular processes in response to light. Most photoreceptors sense blue or red light, but the recent discovery of the cobalamin-dependent photoreceptor, CarH, has expanded the wavelength range of photoreception to other regions of the electromagnetic spectrum to include the green light region. Further identification of cobalamin-dependent green light-sensitive photoreceptors has been hampered owing to poor annotation of the light responsiveness of cobalamin-binding domains (CBDs) in public databases.

Methods to study prFMN-UbiD mediated (de)carboxylation.

The microbial UbiX-UbiD system facilitates the reversible (de)carboxylation of alpha, beta-unsaturated carboxylic acids, including aromatic compounds. The direct C-H carboxylation presents an attractive method for functionalisation and carbon capture but is difficult to achieve under mild conditions. Hence, UbiD-mediated Csp2-H activation can serve as a versatile tool for developing new biocatalytic routes to transform aryl or alkene compounds and carbon dioxide into valuable commodity chemicals.

Photocobilins integrate B and bilin photochemistry for enzyme control.

Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B) can act as a light-sensing chromophore heralded a new field of B-photobiology. Although microbial genome analysis indicates that photoactive B-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking.

The prFMNH-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 prFMNH 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.

Redox driven B-ligand switch drives CarH photoresponse.

CarH is a coenzyme B-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation.

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.

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.

A Vitamin B-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.

A Vitamin B -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.

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,β-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.

Enzymatic -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 -allylation of primary and secondary amines, using biomass-derivable cinnamic acids.

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.

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.

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.

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.

Structure and Mechanism of 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.

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.

Synthetic Enzyme-Catalyzed CO 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 CO fixation routes for chemical production. However, further progress towards industrial application has been hampered by the thermodynamic constraint that accompanies fixing CO to organic molecules.

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.