Orthosomycins are oligosaccharide antibiotics that include avilamycin, everninomicin, and hygromycin B and are hallmarked by a rigidifying interglycosidic spirocyclic ortho-δ-lactone (orthoester) linkage between at least one pair of carbohydrates. A subset of orthosomycins additionally contain a carbohydrate capped by a methylenedioxy bridge. The orthoester linkage is necessary for antibiotic activity but rarely observed in natural products. Orthoester linkage and methylenedioxy bridge biosynthesis require similar oxidative cyclizations adjacent to a sugar ring. We have identified a conserved group of nonheme iron, α-ketoglutarate-dependent oxygenases likely responsible for this chemistry. High-resolution crystal structures of the EvdO1 and EvdO2 oxygenases of everninomicin biosynthesis, the AviO1 oxygenase of avilamycin biosynthesis, and HygX of hygromycin B biosynthesis show how these enzymes accommodate large substrates, a challenge that requires a variation in metal coordination in HygX. Excitingly, the ternary complex of HygX with cosubstrate α-ketoglutarate and putative product hygromycin B identified an orientation of one glycosidic linkage of hygromycin B consistent with metal-catalyzed hydrogen atom abstraction from substrate. These structural results are complemented by gene disruption of the oxygenases evdO1 and evdMO1 from the everninomicin biosynthetic cluster, which demonstrate that functional oxygenase activity is critical for antibiotic production. Our data therefore support a role for these enzymes in the production of key features of the orthosomycin antibiotics.antibiotic biosynthesis | oxidative cyclization | crystal structure | nonheme iron α-ketoglutarate-dependent oxygenases

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.Complex II superfamily enzymes provide a crucial link between oxidoreduction reactions in the membrane bilayer and in the soluble milieu (1). During aerobic respiration in Escherichia coli, the complex II enzyme succinate:ubiquinone oxidoreductase oxidizes succinate and reduces ubiquinone. In bacterial anaerobic respiration with fumarate as the terminal electron acceptor, the reaction proceeds in the opposite direction, and the complex II homolog menaquinol:fumarate oxidoreductase (QFR) 4 oxidizes menaquinol and transfers the electrons to fumarate. Complex II superfamily members contain either three or four polypeptide chains (Fig. 1A): two soluble subunits (the flavoprotein, FrdA, and the iron protein, FrdB, in E. coli QFR) and either one or two integral membrane subunits (FrdC and FrdD in the E. coli QFR). Although there are significant differences in the integral membrane subunits across the family, complex II enzymes all share a high percentage of sequence identity in the soluble subunits, including the flavoprotein, where the kinetically challenging oxidoreduction of fumarate and succinate takes place (1).Numerous molecules, including metabolic intermediates and ingested toxins, structurally resemble succinate and fumarate. Some of these are excluded from the active site, whereas others act as inhibitors (Fig. 1B). Several of these come from normal respiratory processes. For example, oxaloacetate is a Krebs cycle intermediate and acts as a tight, slow

Background: G proteins couple receptor binding to nucleotide release via an allosteric network. Results: Mutation of allosteric sites of G␣ i1 stabilizes a transient signaling conformation and may highlight an allosteric connection between receptor and nucleotide. Conclusion: The P-loop interacts with Switch I in the K345L variant of G␣ i1 . Significance: G protein signaling is critical for numerous cellular functions, and GDP release is the rate-limiting step of the cycle.

Background: Different quinone substrates are used by complex II. Results: Structural and kinetic analyses show that two arginine residues modulate the enzyme interaction with different quinones. Conclusion: Specific arginines compensate for each other in proton transfer during quinone oxidoreduction in the complex II homolog fumarate reductase. Significance: Plasticity in quinone binding may be important for bioenergetic transformations.

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