Potent Inhibition of DXP Synthase by a -Diaryl Bisubstrate Analog.

New antimicrobial strategies are needed to address pathogen resistance to currently used antibiotics. Bacterial central metabolism is a promising target space for the development of agents that selectively target bacterial pathogens. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to DXP, which is required for synthesis of essential vitamins and isoprenoids in bacterial pathogens.

Mechanistic and Structural Insights into a Divergent PLP-Dependent l-Enduracididine Cyclase from a Toxic Cyanobacterium.

Cyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cyanobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure.

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium.

Cyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cya-nobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure.

Structural Basis of Stereospecific Vanadium-Dependent Haloperoxidase Family Enzymes in Napyradiomycin Biosynthesis.

Vanadium-dependent haloperoxidases (VHPOs) from bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products.

Ancient plant-like terpene biosynthesis in corals.

Octocorals are major contributors of terpenoid chemical diversity in the ocean. Natural products from other sessile marine animals are primarily biosynthesized by symbiotic microbes rather than by the host. Here, we challenge this long-standing paradigm by describing a monophyletic lineage of animal-encoded terpene cyclases (TCs) ubiquitous in octocorals.

Enzymatic assembly of the salinosporamide γ-lactam-β-lactone anticancer warhead.

The marine microbial natural product salinosporamide A (marizomib) is a potent proteasome inhibitor currently in clinical trials for the treatment of brain cancer. Salinosporamide A is characterized by a complex and densely functionalized γ-lactam-β-lactone bicyclic warhead, the assembly of which has long remained a biosynthetic mystery. Here, we report an enzymatic route to the salinosporamide core catalyzed by a standalone ketosynthase (KS), SalC.

XFEL serial crystallography reveals the room temperature structure of methyl-coenzyme M reductase.

Methyl-Coenzyme M Reductase (MCR) catalyzes the biosynthesis of methane in methanogenic archaea, using a catalytic Ni-centered Cofactor F430 in its active site. It also catalyzes the reverse reaction, that is, the anaerobic activation and oxidation, including the cleavage of the CH bond in methane. Because methanogenesis is the major source of methane on earth, understanding the reaction mechanism of this enzyme can have massive implications in global energy balances.

Structural basis for non-radical catalysis by TsrM, a radical SAM methylase.

Tryptophan 2C methyltransferase (TsrM) methylates C2 of the indole ring of L-tryptophan during biosynthesis of the quinaldic acid moiety of thiostrepton. TsrM is annotated as a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5'-deoxyadenosyl 5'-radical intermediate, a hallmark of radical SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae, which are the first structures of a cobalamin-dependent radical SAM methylase.

X-ray crystallography-based structural elucidation of enzyme-bound intermediates along the 1-deoxy-d-xylulose 5-phosphate synthase reaction coordinate.

1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) uses thiamine diphosphate (ThDP) to convert pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) into 1-deoxy-d-xylulose 5-phosphate (DXP), an essential bacterial metabolite. DXP is not utilized by humans; hence, DXPS has been an attractive antibacterial target. Here, we investigate DXPS from (DXPS), showing that it has similar kinetic parameters and (54 ± 3 and 11 ± 1 μm, respectively) and comparable catalytic activity ( = 45 ± 2 min) with previously studied bacterial DXPS enzymes and employing it to obtain missing structural data on this enzyme family.

A reverse TCA cycle 2-oxoacid:ferredoxin oxidoreductase that makes C-C bonds from CO.

2-oxoglutarate:ferredoxin oxidoreductase (OGOR) is a thiamine pyrophosphate (TPP) and [4Fe-4S] cluster-dependent enzyme from the reductive tricarboxylic acid (rTCA) cycle that fixes CO to succinyl-CoA, forming 2-oxoglutarate and CoA. Here we report an OGOR from the rTCA cycle of MC-1, along with all three potential ferredoxin (Fd) redox partners. We demonstrate OGOR operates bidirectionally (both CO-fixing and 2-oxoglutarate oxidizing), and that only one Fd (Fd1) supports efficient catalysis.

Disruption of an oligomeric interface prevents allosteric inhibition of class Ia ribonucleotide reductase.

Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an α subunit that houses the active site and allosteric regulatory sites and a β subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact αβ state allowing for radical transfer from β to α RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity.

Binding site for coenzyme A revealed in the structure of pyruvate:ferredoxin oxidoreductase from .

Pyruvate:ferredoxin oxidoreductase (PFOR) is a microbial enzyme that uses thiamine pyrophosphate (TPP), three [4Fe-4S] clusters, and coenzyme A (CoA) in the reversible oxidation of pyruvate to generate acetyl-CoA and carbon dioxide. The two electrons that are generated as a result of pyruvate decarboxylation are used in the reduction of low potential ferredoxins, which provide reducing equivalents for central metabolism, including the Wood-Ljungdahl pathway. PFOR is a member of the 2-oxoacid:ferredoxin oxidoreductase (OFOR) superfamily, which plays major roles in both microbial redox reactions and carbon dioxide fixation.

A structural phylogeny for understanding 2-oxoacid oxidoreductase function.

2-Oxoacid:ferredoxin oxidoreductases (OFORs) are essential enzymes in microbial one-carbon metabolism. They use thiamine pyrophosphate to reversibly cleave carbon-carbon bonds, generating low potential (∼-500mV) electrons. Crystallographic analysis of a recently discovered OFOR, an oxalate oxidoreductase (OOR), has provided a second view of OFOR architecture and active site composition.

Waltzing around cofactors.

The metallocofactor involved in fixing nitrogen is not a rigid scaffold, as was previously thought.

Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli.

Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively.

One-carbon chemistry of oxalate oxidoreductase captured by X-ray crystallography.

Thiamine pyrophosphate (TPP)-dependent oxalate oxidoreductase (OOR) metabolizes oxalate, generating two molecules of CO2 and two low-potential electrons, thus providing both the carbon and reducing equivalents for operation of the Wood-Ljungdahl pathway of acetogenesis. Here we present structures of OOR in which two different reaction intermediate bound states have been trapped: the covalent adducts between TPP and oxalate and between TPP and CO2. These structures, along with the previously determined structure of substrate-free OOR, allow us to visualize how active site rearrangements can drive catalysis.

Structural basis for gene regulation by a B12-dependent photoreceptor.

Photoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin B12 derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure.