Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU.

The biosynthesis of iron sulfur (Fe-S) clusters in Bacillus subtilis is mediated by a SUF-type gene cluster, consisting of the cysteine desulfurase SufS, the scaffold protein SufU, and the putative chaperone complex SufB/SufC/SufD. Here, we present the high-resolution crystal structure of the SufS homodimer in its product-bound state (i.e., in complex with pyrodoxal-5'-phosphate, alanine, Cys361-persulfide). By performing hydrogen/deuterium exchange (H/DX) experiments, we characterized the interaction of SufS with SufU and demonstrate that SufU induces an opening of the active site pocket of SufS. Recent data indicate that frataxin could be involved in Fe-S cluster biosynthesis by facilitating iron incorporation. H/DX experiments show that frataxin indeed interacts with the SufS/SufU complex at the active site. Our findings deepen the current understanding of Fe-S cluster biosynthesis, a complex yet essential process, in the model organism B. subtilis.

Molecular insights into frataxin-mediated iron supply for heme biosynthesis in Bacillus subtilis.

Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in Bacillus subtilis and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme b biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen-deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the in vitro-generated heme b co-factor upon Fra-mediated iron transfer was confirmed by using the B. subtilis nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme b co-factor, hence representing a key player in the iron-dependent physiology of B. subtilis.

Chemoenzymatic synthesis of novel C-ribosylated naphthoquinones.

The biological activity of many natural products is dependent on the presence of carbohydrate units, which are usually attached via an O-glycosidic linkage by glycosyltransferases. Recently, an exceptional C-ribosylation event was discovered in the biosynthesis of the polyketide antibiotic alnumycin A. The two-step process involves initial attachment of d-ribose-5-phosphate to the polyaromatic aglycone by the C-glycosynthase AlnA and subsequent dephosphorylation by AlnB, an enzyme of the haloacid dehalogenase family. Here, we tested 23 unnatural substrates to probe the C-ribosylation reaction. The chemoenzymatic synthesis of C-ribosylated juglone, 7-methyl juglone, monomethyl naphthazarin, 8-chloro-7-methyl juglone, and 9-hydroxy-1,4-anthraquinone revealed the importance of a 1,4-quinoid system with an adjacent phenolic ring in order for reaction to occur. To further rationalize the molecular basis for reactivity, factors governing substrate recognition were investigated by NMR binding experiments. Additionally, the suitability of substrates for nucleophilic substitution was assessed by molecular modeling using density functional theory (DFT) calculations.

Discovery of a two-component monooxygenase SnoaW/SnoaL2 involved in nogalamycin biosynthesis.

Nogalamycin is an anthracycline polyketide antibiotic that contains two deoxysugars, at positions C-1 and C-7. Previous biosynthetic studies conducted in vivo affiliated snoaL2 with an unusual C-1 hydroxylation reaction, but in vitro activity was not established. Here, we demonstrate that inactivation of either snoaL2 or snoaW resulted in accumulation of two nonhydroxylated metabolites, nogalamycinone and a novel anthracycline 3',4'-demethoxy-nogalose-nogalamycinone. The C-1 hydroxylation activity was successfully reconstructed in vitro in the presence of the two enzymes, NAD(P)H and the substrates. Based on relative reaction efficiencies, 3',4'-demethoxy-nogalose-nogalamycinone was identified as the likely natural substrate. A biosynthetic model was established where the atypical short-chain alcohol dehydrogenase SnoaW reduces the anthraquinone to a dihydroquinone using NADPH, which enables activation of oxygen and formation of a hydroperoxy intermediate. Finally, protonation of the intermediate by SnoaL2 yields the 1-hydroxylated product.

Formation of 5-Hydroxy-3-methoxy-1,4-naphthoquinone and 8-Hydroxy-4-methoxy-1,2-naphthoquinone from Juglone.

From the treatment of 5-hydroxy-1,4-naphthoquinone (juglone) with acetic anhydride and H2SO4 followed subsequently by treatment with methanolic HCl, 5-hydroxy-3-methoxy-1,4-naphthoquinone (3-methoxy juglone) and 8-hydroxy-4-methoxy-1,2-naphthoquinone were obtained as products rather than the anticipated product 2,5-dihydroxy-1,4-naphthoquinone (2-hydroxy juglone). The reaction and the identification of the products are discussed in terms of NMR and DFT calculations.