Mechanistic Insights into Validoxylamine A 7\u27-Phosphate Synthesis by VldE Using the Structure of the Entire Product Complex

The pseudo-glycosyltransferase VldE catalyzes non-glycosidic C-N coupling between an unsaturated cyclitol and a saturated aminocyclitol with the conservation of the stereochemical configuration of the substrates to form validoxylamine A 7′-phosphate, the biosynthetic precursor of the antibiotic validamycin A. To study the molecular basis of its mechanism, the three-dimensional structures of VldE from Streptomyces hygroscopicus subsp. limoneus was determined in apo form, in complex with GDP, in complex with GDP and validoxylamine A 7′-phosphate, and in complex with GDP and trehalose. The structure of VldE with the catalytic site in both an open and closed conformation is also described. With these structures, the preferred binding of the guanine moiety by VldE, rather than the uracil moiety as seen in OtsA could be explained. The elucidation of the VldE structure in complex with the entirety of its products provides insight into the internal return mechanism by which catalysis occurs with a net retention of the stereochemical configuration of the donated cyclitol. © 2012 Cavalier et al

Structure of a Sedoheptulose 7-Phosphate Cyclase: ValA from Streptomyces hygroscopicus

Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008 which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD⁺ and Zn²⁺ cofactors. ValA has a fold and active site organization resembling the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD⁺ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis, but is accomplished by selective binding of either the α or β pyranose anomer of the substrate

Bioactive properties of streptomyces may affect the dominance of Tricholoma matsutake in shiro

Tricholoma matsutake is known to be the dominant fungal species in matsutake fruitbody neighboring (shiro) soil. To understand the mechanisms behind matsutake dominance, we studied the bacterial communities in matsutake dominant shiro soil and non-shiro soil, isolated the strains of Streptomyces from matsutake mycorrhizal root tips both from shiro soil and from the Pinus densiflora seedlings cultivated in shiro soil. Further, we investigated three Streptomyces spp. for their ability to inhibit fungal growth and Pinus densiflora seedling root elongation as well as two strains for their antifungal and antioxidative properties. Our results showed that Actinobacteria was the most abundant phylum in shiro soil. However, the differences in the Actinobacterial community composition (phylum or order level) between shiro and non-shiro soils were not significant, as indicated by PERMANOVA analyses. A genus belonging to Actinobacteria, Streptomyces, was present on the matsutake mycorrhizas, although in minority. The two antifungal assays revealed that the broths of three Streptomyces spp. had either inhibitory, neutral or promoting effects on the growth of different forest soil fungi as well as on the root elongation of the seedlings. The extracts of two strains, including one isolated from the P. densiflora seedlings, inhibited the growth of either pathogenic or ectomycorrhizal fungi. The effect depended on the medium used to cultivate the strains, but not the solvent used for the extraction. Two Streptomyces spp. showed antioxidant activity in one out of three assays used, in a ferric reducing antioxidant power assay. The observed properties seem to have several functions in matsutake shiro soil and they may contribute to the protection of the shiro area for T. matsutake dominance.Peer reviewe

Mechanistic Insights into Validoxylamine A 7’-Phosphate Synthesis by VldE Using the Structure of the Entire Product Complex

The pseudo-glycosyltransferase VldE catalyzes non-glycosidic C-N coupling between an unsaturated cyclitol and a saturated aminocyclitol with the conservation of the stereochemical configuration of the substrates to form validoxylamine A 7'-phosphate, the biosynthetic precursor of the antibiotic validamycin A. To study the molecular basis of its mechanism, the three-dimensional structures of VldE from Streptomyces hygroscopicus subsp. limoneus was determined in apo form, in complex with GDP, in complex with GDP and validoxylamine A 7'-phosphate, and in complex with GDP and trehalose. The structure of VldE with the catalytic site in both an "open'' and "closed'' conformation is also described. With these structures, the preferred binding of the guanine moiety by VldE, rather than the uracil moiety as seen in OtsA could be explained. The elucidation of the VldE structure in complex with the entirety of its products provides insight into the internal return mechanism by which catalysis occurs with a net retention of the stereochemical configuration of the donated cyclitol

De novo synthesis of a sunscreen compound in vertebrates

Ultraviolet-protective compounds, such as mycosporine-like amino acids (MAAs) and related gadusols produced by some bacteria, fungi, algae, and marine invertebrates, are critical for the survival of reef-building corals and other marine organisms exposed to high-solar irradiance. These compounds have also been found in marine fish, where their accumulation is thought to be of dietary or symbiont origin. In this study, we report the unexpected discovery that fish can synthesize gadusol de novo and that the analogous pathways are also present in amphibians, reptiles, and birds. Furthermore, we demonstrate that engineered yeast containing the fish genes can produce and secrete gadusol. The discovery of the gadusol pathway in vertebrates provides a platform for understanding its role in these animals, and the possibility of engineering yeast to efficiently produce a natural sunscreen and antioxidant presents an avenue for its large-scale production for possible use in pharmaceuticals and cosmetics.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by eLife Sciences Publications. The published article can be found at: http://elifesciences.org/Supporting information can be found at: http://elifesciences.org/content/4/e0591

Effects of carbon ion beam-induced mutagenesis for the screening of RED production-deficient mutants of Streptomyces coelicolor JCM4020.

Streptomyces lividans TK23 interacts with mycolic acid-containing bacteria (MACB), such as Tsukamurella pulmonis TP-B0596, and this direct cell contact activates its secondary metabolism (e.g., the production of undecylprodigiosin: RED). Here, we employed carbon (12C5+) ion beam-induced mutagenesis to investigate the signature of induced point mutations and further identify the gene(s) responsible for the production of secondary metabolites induced by T. pulmonis. We irradiated spores of the Streptomyces coelicolor strain JCM4020 with carbon ions to generate a mutant library. We screened the RED production-deficient mutants of S. coelicolor by mixing them with T. pulmonis TP-B0596 on agar plates, identifying the red/white phenotype of the growing colonies. Through this process, we selected 59 RED-deficient mutants from around 152,000 tested spores. We resequenced the genomes of 16 mutants and identified 44 point mutations, which revealed the signatures induced by 12C5+-irradiation. Via gene complementation experiments, we also revealed that two genes-glutamate synthase (gltB) and elongation factor G (fusA)-are responsible for the reduced production of RED

Characterization of ABC transporter which cause the delayed contact-dependent RED production in Streptomyces coelicolor

We have studied about the bacterial interaction induced production of secondary metabolites by actinomycetes. Tsukamurella pulmonis TP-B0596 (Tp) had been shown to possess ability to induce production of secondary metabolites by Streptomyces species, which are not detected or poorly produced in a mono-culture. Until now, more than 30 new compounds had been isolated from the co-culture with various actinomycetes and Tp. Object of this study is to elucidate the gene(s) which are involved in the regulation of secondary metabolism caused by bacterial interaction. Elucidation of the mechanism can lead to the fundamental understanding of bacterial interaction induced secondary metabolism, as well as application for genetic tools to discover novel bioactive natural products from untapped genes. We had employed carbon ion beams induced mutation to investigated the gene(s) responsible for the responsive production of secondary metabolism induced by Tp. Using Streptomyces coelicolor JCM4020, random spore mutant library was generated. Undecylprodigiosin (RED) production deficient mutants was screened by mixing with Tp on agar plates. Through this screening, 59 mutants from around 152,000 tested spores were obtained. The genome re-sequence and gene complementation studies were revealed that molybdopterin biosynthetic enzyme, and TetR-like transcriptional regulator are also responsible for the phenotype. We further analyzed the involvement of TetR-like transcriptional regulator by targeted gene disruption in S. coelicolor A3(2) and confirmed the delayed production of RED

KarplusPAndrewBiochemistryBiophysicsStructureSedoheptulose7-Phosphate.pdf

Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008 which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD⁺ and Zn²⁺ cofactors. ValA has a fold and active site organization resembling the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD⁺ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis, but is accomplished by selective binding of either the α or β pyranose anomer of the substrate