Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent

The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes.National Center for Research Resources (U.S.) (P41RR012408)National Institute of General Medical Sciences (U.S.) (P41GM103473

Mechanism of Assembly of the Dimanganese-Tyrosyl Radical Cofactor of Class Ib Ribonucleotide Reductase: Enzymatic Generation of Superoxide Is Required for Tyrosine Oxidation via a Mn(III)Mn(IV) Intermediate

Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y•) generated by oxidation of a reduced dinuclear metal cluster. The Fe[superscript III][subscript 2]-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe[superscript II][subscript 2]-NrdB, O[subscript 2], and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn[superscript III][subscript 2]-Y• cofactor in their NrdF subunit. Mn[superscript II][subscript 2]-NrdF does not react with O[subscript 2], but it binds the reduced form of a conserved flavodoxin-like protein, NrdI[subscript hq], which, in the presence of O[subscript 2], reacts to form the Mn[superscript III][subscript 2]-Y• cofactor. Here we investigate the mechanism of assembly of the Mn[superscript III][subscript 2]-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn[superscript II][subscript 2]-NrdF, NrdI[subscript hq], and O[subscript 2] has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI[subscript hq] reduces O[subscript 2] to O[subscript 2]•– (40–48 s[superscript –1], 0.6 mM O[subscript 2]), the O[subscript 2]•– channels to and reacts with Mn[superscript II][subscript 2]-NrdF to form a Mn[superscript III]Mn[superscript IV] intermediate (2.2 ± 0.4 s[superscript –1]), and the Mn[superscript III]Mn[superscript IV] species oxidizes tyrosine to Y• (0.08–0.15 s[superscript –1]). Controlled production of O[subscript 2]•– by NrdI[subscript hq] during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn[superscript II][subscript 2] cluster with O[subscript 2] and satisfies the requirement for an “extra” reducing equivalent in Y• generation.National Institutes of Health (U.S.) (Grant GM81393)United States. Dept. of Defense (National Defense Science and Engineering Graduate (NDSEG) Fellowships

Ny E6 Østfold grense - Vestby og ny dobbeltsporet jernbane Smørbekk - Rustad. Beregninger og vurderinger av vannforurensninger

To vassdrag vil kunne bli direkte berørt av forurensninger fra den planlagte utbygging. Dette er Kambovassdraget som munner ut i sjøen Kambo, og Hølenvassdraget som munner ut ved Son. Vassdragene vil kunne bli betydelig påvirket i anleggsfasen, mens i vegens og jernbanenes driftfase vil påvirkningene bli moderate. Bekkelukkinger kan komme til å få negative innvirkninger på oppgangs- og gyteforhold for sjøørret. Det er registrert 21 fjellbrønner og en kilde langs traseèn. Av disse ligger 3 brønner i traseèn og må erstattes. For ytterligere 2 brønner og 1 kilde er det stor risiko for endret kapasitet eller vannkvalitet. Av 16 foreslåtte massedeponier frarådes ett benyttet. For de ulike påvirkningene er det foreslått avbøtende tiltak

Demonstrasjonsprosjekt for implementering av EUs Vanndirektiv i Vansjø-Hobøl. Fase 2: Skisse til veiledere for karakteriseringsoppgavene i 2004, samt forslag til overvåkingsprogram

Årsliste 2003Rapporten presenterer skisse til veiledning for karakterisering og overvåking av vannforekomster i hht. kravene i EUs Rammedirektiv for Vann. Metoder og kriterier for inndeling og typifisering av vannforekomster, samt for naturfaglig vurdering av risiko for dårlig status er foreslått. Metoder og datagrunnlag som er nødvendig for økonomisk karakterisering av vannbrukere og kostnadsdekking for vanntjenester er også presentert. Bruk av metodene er vist for Vansjø-Hobøl-vassdraget. Direktivets krav til overvåking av vannforekomster er sammenstilt, og forslag til overvåkingsprogram i tråd med disse kravene er foreslått for Vansjø-Hobøl-vassdraget. Ressursbehovet for gjennomføring av karakterisering og overvåking er estimert for dette vassdraget og forsøkt oppskalert til nasjonalt nivå.Direktoratsgruppen for Implementering av EUs Rammedirektiv for Van

Bacillus subtilis Class Ib Ribonucleotide Reductase Is a Dimanganese(III)-Tyrosyl Radical Enzyme

Bacillus subtilis class Ib ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, providing the building blocks for DNA replication and repair. It is composed of two proteins: α (NrdE) and β (NrdF). β contains the metallo-cofactor, essential for the initiation of the reduction process. The RNR genes are organized within the nrdI-nrdE-nrdF-ymaB operon. Each protein has been cloned, expressed, and purified from Escherichia coli. As isolated, recombinant NrdF (rNrdF) contained a diferric-tyrosyl radical [Fe(III)[subscript 2-]Y[superscript•] cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O[subscript 2]. Apo-rNrdF loaded using 4 Mn(II)/β[subscript 2], O[subscript 2], and reduced NrdI (a flavodoxin) can form a dimanganese(III)-Y[superscript•] [Mn(III)[subscript 2-]Y[superscript•]] cofactor. In the presence of rNrdE, ATP, and CDP, Mn(III)[subscript 2-]Y[superscript•] and Fe(III)[subscript 2-]Y[superscript•] rNrdF generate dCDP at rates of 132 and 10 nmol min[superscript –1] mg[superscript –1], respectively (both normalized for 1 Y[superscript•]/β[subscript 2]). To determine the endogenous cofactor of NrdF in B. subtilis, the entire operon was placed behind a Pspank(hy) promoter and integrated into the B. subtilis genome at the amyE site. All four genes were induced in cells grown in Luria-Bertani medium, with levels of NrdE and NrdF elevated 35-fold relative to that of the wild-type strain. NrdE and NrdF were copurified in a 1:1 ratio from this engineered B. subtilis. The visible, EPR, and atomic absorption spectra of the purified NrdENrdF complex (eNrdF) exhibited characteristics of a Mn(III)[subscript 2-]Y[superscript•] center with 2 Mn/β[subscript 2] and 0.5 Y[superscript•]/β[subscript 2] and an activity of 318–363 nmol min[superscript –1] mg[superscript –1] (normalized for 1 Y[superscript•]/β[subscript 2]). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)[subscript 2]-Y[superscript•] enzyme.National Institutes of Health (U.S.) (Grant number GM81393

Modelled salmon lice dispersion and infestation patterns in a sub-arctic fjord

Salmon lice infestation is a major challenge for the aquaculture industry in Norway, threatening wild salmonid populations and causing welfare problems for farmed salmon. Lice dispersion and infestation patterns are simulated by combining a high-resolution hydrodynamic model for the Norwegian coast and fjords with an individual-based model for salmon lice. We here present results from Altafjorden, a sub-arctic fjord with large stocks of wild salmonids, where the inner part is protected as a National Salmon Fjord. The outer part of the fjord hosts several fish farms, and our simulations demonstrate how ocean currents can disperse lice between farms as well as into the protected part of the fjord. The relative contributions from the farms in the different parts of the fjord depends on their locations relative to the currents and circulation patterns in the fjord. Knowledge of how the highly variable water currents disperse salmon lice within fjord systems is necessary for managing farm locations and production quotas, if the goal is to minimize infestation pressure on wild salmonids and between fish farms.publishedVersio

The Dimanganese(II) Site of Bacillus subtilis Class Ib Ribonucleotide Reductase

Class Ib ribonucleotide reductases (RNRs) use a dimanganese-tyrosyl radical cofactor, Mn[III over 2]-Y[superscript •], in their homodimeric NrdF (β2) subunit to initiate reduction of ribonucleotides to deoxyribonucleotides. The structure of the Mn[II over 2] form of NrdF is an important component in understanding O[subscript 2]-mediated formation of the active metallocofactor, a subject of much interest because a unique flavodoxin, NrdI, is required for cofactor assembly. Biochemical studies and sequence alignments suggest that NrdF and NrdI proteins diverge into three phylogenetically distinct groups. The only crystal structure to date of a NrdF with a fully ordered and occupied dimanganese site is that of Escherichia coli Mn[II over 2]-NrdF, prototypical of the enzymes from actinobacteria and proteobacteria. Here we report the 1.9 Å resolution crystal structure of Bacillus subtilis Mn[II over 2]-NrdF, representative of the enzymes from a second group, from Bacillus and Staphylococcus. The structures of the metal clusters in the β2 dimer are distinct from those observed in E. coli Mn[II over 2]-NrdF. These differences illustrate the key role that solvent molecules and protein residues in the second coordination sphere of the Mn[II over 2] cluster play in determining conformations of carboxylate residues at the metal sites and demonstrate that diverse coordination geometries are capable of serving as starting points for Mn[III over 2]-Y[superscript •] cofactor assembly in class Ib RNRs.United States. Dept. of Energy (Contract DE-AC02-06CH11357)Michigan Economic Development Corporation (Michigan Technology Tri-Corridor Grant 085P1000817)National Cancer Institute (U.S.) (Y1-CO-1020)National Institute of General Medical Sciences (U.S.) (Y1-GM-1104

Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2

Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) play an important role in the conversion of recalcitrant polysaccharides, but their mode of action has remained largely enigmatic. It is generally believed that catalysis by LPMOs requires molecular oxygen and a reductant that delivers two electrons per catalytic cycle. Using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show here that H2O2, rather than O-2, is the preferred co-substrate of LPMOs. By controlling H(2)O2 supply, stable reaction kinetics are achieved, the LPMOs work in the absence of O-2, and the reductant is consumed in priming rather than in stoichiometric amounts. The use of H2O2 by a monocopper enzyme that is otherwise cofactor-free offers new perspectives regarding the mode of action of copper enzymes. Furthermore, these findings have implications for the enzymatic conversion of biomass in Nature and in industrial biorefining