Jessica L. Vey

Structural Basis for Glycyl Radical Formation By Pyruvate Formate-Lyase Activating Enzyme

Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G734 of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G734. Our structures provide fundamental insights into the interactions between the activase and the G734 loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G734 of pyruvate formate-lyase.

Structure and mechanism of ORF36, an amino sugar oxidizing enzyme in everninomicin biosynthesis .

Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent Gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitro sugar, l-evernitrose, analogues of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically generated thymidine diphosphate (TDP)-l-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five-enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-l-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single-oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of (18)O(2) establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 Å resolution X-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-CoA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36.

Crystallographic insights into the structure-activity relationships of diazaborine enoyl-ACP reductase inhibitors.

Enoyl-ACP reductase, the last enzyme of the fatty-acid biosynthetic pathway, is the molecular target for several successful antibiotics such as the tuberculosis therapeutic isoniazid. It is currently under investigation as a narrow-spectrum antibiotic target for the treatment of several types of bacterial infections. The diazaborine family is a group of boron heterocycle-based synthetic antibacterial inhibitors known to target enoyl-ACP reductase. Development of this class of molecules has thus far focused solely on the sulfonyl-containing versions. Here, the requirement for the sulfonyl group in the diazaborine scaffold was investigated by examining several recently characterized enoyl-ACP reductase inhibitors that lack the sulfonyl group and exhibit additional variability in substitutions, size and flexibility. Biochemical studies are reported showing the inhibition of Escherichia coli enoyl-ACP reductase by four diazaborines, and the crystal structures of two of the inhibitors bound to E. coli enoyl-ACP reductase solved to 2.07 and 2.11 Å resolution are reported. The results show that the sulfonyl group can be replaced with an amide or thioamide without disruption of the mode of inhibition of the molecule.

Initial investigations of C4a-(hydro)peroxyflavin intermediate formation by dibenzothiophene monooxygenase

Dibenzothiophene monooxygenase is the initiating enzyme in the Rhodococcus 4S biodesulfurization pathway. A member of the Class D flavin monooxygenases, it uses FMN to activate molecular oxygen for oxygenation of the substrate, dibenzothiophene. Here, we have used stopped-flow spectrophotometry to show that DszC forms a peroxyflavin intermediate in the absence of substrate. Mutagenesis of Ser163 and His391 to Ala appears to decrease the binding affinity for reduced FMN and eliminates the enzymes ability to stabilize the peroxyflavin intermediate.

Structure of DnmZ, a nitrososynthase in the Streptomyces peucetius anthracycline biosynthetic pathway.

The anthracyclines are a class of highly effective natural product chemotherapeutics and are used to treat a range of cancers, including leukemia. The toxicity of the anthracyclines has stimulated efforts to further diversify the scaffold of the natural product, which has led to renewed interest in the biosynthetic pathway responsible for the formation and modification of this family of molecules. DnmZ is an N-hydroxylating flavin monooxygenase (a nitrososynthase) that catalyzes the oxidation of the exocyclic amine of the sugar nucleotide dTDP-L-epi-vancosamine to its nitroso form. Its specific role in the anthracycline biosynthetic pathway involves the synthesis of the seven-carbon acetal moiety attached to C4 of L-daunosamine observed in the anthracycline baumycin. Here, X-ray crystallography was used to elucidate the three-dimensional structure of DnmZ. Two crystal structures of DnmZ were yielded: that of the enzyme alone, solved to 3.00 Å resolution, and that of the enzyme in complex with thymidine diphosphate, the nucleotide carrier portion of the substrate, solved to 2.74 Å resolution. These models add insights into the structural features involved in substrate specificity and conformational changes involved in thymidine diphosphate binding by the nitrososynthases.