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Robust, objective, and repeatable approaches that define flow thresholds for fish passage across critical natural barriers such as riffles, rapids, and waterfalls are required for determining environmental flow strategies. These approaches also provide an opportunity to garner community sector backing for environmental flow releases from dams in support of tangible environmental beneficiaries—native fish. This paper outlines the results of a two‐dimensional hydraulic modelling approach to fish passage assessment for Australian bass (Percalates novemaculeata) that was used to inform the development of an environmental flow regime downstream of Warrragamba Dam, NSW, Australia. Flow rates of ≥500 MLd−1 were found to facilitate depth‐limited upstream passage through a 20‐km river reach that contained 19 natural passage barriers to adult Australian bass up to 400–450 mm in length. Ideal passage conditions were determined at flow rates of ≥1,000 MLd−1. Juvenile bass passage was found to be inhibited by high velocities at flow rates >250 MLd−1, with flows of 100–250 MLd−1 providing ideal conditions for juvenile passage. Fish length, body depth, and caudal fin depth data, as used in this study for Australian bass, provided more precise fish passage depth thresholds. Precision in fish passage assessments is important as each centimetre of additional flow depth influences cost–benefit analyses of environmental flow releases versus consumptive water uses. Although hydraulic modelling and field‐based approaches to fish passage assessment are well established, there is currently a lack of published data on native Australian freshwater fish length, body depth, and caudal fin depth data for use in fish passage assessments and for inclusion in "fish‐friendly" government policy initiatives. [ABSTRACT FROM AUTHOR]

Enzym‐vermittelte Kaskadenreaktionen sind weit verbreitet. Um einen Vergleich mit den mechanistischen Kategorisierungen der komplementären Kaskadenreaktionen in der organischen Synthese zu ermöglichen sowie um die gemeinsamen Grundlagen aufzuzeigen, erörtern wir hier vier Arten von Enzymkaskadenreaktionen: vermittelt durch nucleophile, elektrophile, pericyclische und radikalische Reaktionen. Zwei Subtypen von Enzymen, die radikalische Kaskaden erzeugen, befinden sich an den entgegengesetzten Enden der Abundanzskala von Sauerstoff: Eisen‐basierte Enzyme nutzen O2, um hochvalente Eisen‐Oxo‐Spezies zu erzeugen, die nichtaktivierte C‐H‐Bindungen in Substraten homolytisch spalten und Gerüstumlagerungen einleiten. Am anaeroben Ende spalten Enzyme reversibel S‐Adenosylmethionin (SAM) unter Bildung des 5′‐Desoxyadenosyl‐Radikals als starkes Oxidans, um die homolytische Spaltung von C‐H‐Bindungen in gebundenen Substraten auszulösen. Die letztgenannten Enzyme werden als Radikal‐SAM‐Enzyme bezeichnet. Die erstgenannten Enzyme kategorisieren wir als "verhinderte Oxygenasen". [ABSTRACT FROM AUTHOR]

Enzyme‐mediated cascade reactions are widespread in biosynthesis. To facilitate comparison with the mechanistic categorizations of cascade reactions by synthetic chemists and delineate the common underlying chemistry, we discuss four types of enzymatic cascade reactions: those involving nucleophilic, electrophilic, pericyclic, and radical reactions. Two subtypes of enzymes that generate radical cascades exist at opposite ends of the oxygen abundance spectrum. Iron‐based enzymes use O2 to generate high valent iron‐oxo species to homolyze unactivated C−H bonds in substrates to initiate skeletal rearrangements. At anaerobic end, enzymes reversibly cleave S‐adenosylmethionine (SAM) to generate the 5′‐deoxyadenosyl radical as a powerful oxidant to initiate C−H bond homolysis in bound substrates. The latter enzymes are termed radical SAM enzymes. We categorize the former as "thwarted oxygenases". [ABSTRACT FROM AUTHOR]

The term chemical biology emerged about 25 years ago and encompasses a set of research inquiries at the intersections of chemistry and biology. Before chemical biology there was biological chemistry for 100 years or more, but the traverse from one to the other has not just been a switching of noun and adjective. Over the past quarter century chemists, many from organic synthetic lineages, have become convinced that the open systems of biology have become appropriate venues to bring chemical thinking for library design, screening, and molecular scaffold optimization. Whereas biological chemistry may be described as the universe of chemistry that happens in nature, chemical biologists often bring new, unnatural molecular scaffolds to decipher the logics of biology. That seems a limiting definition and I prefer the mantra: think chemically, act biologically. [ABSTRACT FROM AUTHOR]

Frei vorkommende nichtproteinogene Aminosäuren sind schon lange wegen ihrer antimetabolen Eigenschaften bekannt. Entdeckt werden sie meist aufgrund der Reaktivität gegenüber der katalytischen Wirkung der Zielenzyme. Führt man sie regiospezifisch in biogene Peptide und Proteine ein, kann es möglich werden, eine neue Ära der Medizinalchemie an der Grenze zwischen kleinen und großen Wirkstoffen einzuleiten. Außerdem eröffnet die ortsspezifische Funktionalisierung von Proteinen besonders attraktive Strategien für die posttranslationale Proteinmodifizierung. Auch bieten viele der Aminosäuren, die von der Natur nicht für den Einbau in Proteine ausgewählt wurden, reiche architektonische Möglichkeiten bei Einführung in ribosomal hergestellte Polypeptide. Dieser Aufsatz fasst die Biosynthesewege zu den wichtigsten Klassen nichtkanonischer Bausteine und deren Stoffwechsellogik zusammen, die in nichtribosomalen Peptidgerüsten und nichtribosomalen Peptid ‐ Polyketid ‐ Hybriden resultieren. Mikroorganismen haben die bemerkenswerte Fähigkeit, Aminosäuregerüste aufzubauen, die nicht in Proteine eingebaut werden. Dieser Aufsatz fasst die Biosynthesewege und die Stoffwechsellogik der wichtigsten Klassen der nichtkanonischen Aminosäuren zusammen, die letztendlich ihren Weg in nichtribosomale Peptide und in hybride nichtribosomale Peptid ‐ /Polyketidgerüste finden. [ABSTRACT FROM AUTHOR]

Freestanding nonproteinogenic amino acids have long been recognized for their antimetabolite properties and tendency to be uncovered to reactive functionalities by the catalytic action of target enzymes. By installing them regiospecifically into biogenic peptides and proteins, it may be possible to usher a new era at the interface between small molecule and large molecule medicinal chemistry. Site-selective protein functionalization offers uniquely attractive strategies for posttranslational modification of proteins. Last, but not least, many of the amino acids not selected by nature for protein incorporation offer rich architectural possibilities in the context of ribosomally derived polypeptides. This Review summarizes the biosynthetic routes to and metabolic logic for the major classes of the noncanonical amino acid building blocks that end up in both nonribosomal peptide frameworks and in hybrid nonribosomal peptide-polyketide scaffolds. [ABSTRACT FROM AUTHOR]

The 39-kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a ‘Trojan horse’ antibiotic. The approximately 260-residue C-terminal region of MccB is homologous to ubiquitin-like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB-catalysed C-terminal MccA-acyl-adenylation is reminiscent of the E1-catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C-terminal di-glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C-terminal Asn. Furthermore, after an intramolecular rearrangement of MccA-acyl-adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate-linked analogue of acyl-adenylated aspartic acid. We report six-crystal structures of MccB in apo, substrate-, intermediate-, and inhibitor-bound forms. Structural and kinetic analyses reveal a novel-peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic-active site for catalysing dual adenosine triphosphate-consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two-step activation for generating the peptidyl-antibiotic MccC7. [ABSTRACT FROM AUTHOR]

Microcin B17 (MccB17) is a ribosomally encoded DNA-gyrase inhibitor. Ribosomally encoded antibiotics are derived from precursors containing an N-terminal leader, which is removed during maturation, and a C-terminal structural peptide. PreMccB17, the translational product of mcbA, is modified into proMccB17 by the action of three enzymes, McbB, McbC, and McbD. A chromosomally encoded peptidase then converts proMccB17 into MccB17. The role of McbB, McbC, and McbD is to convert glycine, cysteine, and serine residues present in preMccB17 into four thiazole and four oxazole rings. Using a modification-specific antibody rather than antimicrobial activity, we show that the 26-amino-acid N-terminal leader of preMccB17 is essential for the conversion of preMccB17 into proMccB17. Neither a preMccB17 peptide lacking the leader nor a preMccB17-β-galactosidase fusion lacking the leader are post-translationally modified. [ABSTRACT FROM AUTHOR]

The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of D-3-hydroxybutyryl-CoA for poly(D-3hydroxybutyrate) biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/ mg). The enzyme is an α4 homotetramer of four 25-kDa subunits. It has a Km of 2 µM and a kcat/Km of 1.8 × 108 M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyI-CoA above 10 µM. K is 10-10 M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride-equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101-107], we identified a 28-residue stretch (residues 3-31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3-oxo-acyl-CoA analogs and analogs lacking the adenosine 3',5'-bisphosphate moiety of CoA. Only the straight-chain C5 analog (3-oxo-propionyl-CoA) was found to be an alternative substrate (40%) for the reductase. [ABSTRACT FROM AUTHOR]

We have examined the biosynthesis and accumulation of cyanobacterial sunscreening pigment scytonemin within intertidal microbial mat communities using a combination of chemical, molecular, and phylogenetic approaches. Both laminated (layered) and nonlaminated mats contained scytonemin, with morphologically distinct mats having different cyanobacterial community compositions. Within laminated microbial mats, regions with and without scytonemin had different dominant oxygenic phototrophs, with scytonemin-producing areas consisting primarily of Lyngbya aestuarii and scytonemin-deficient areas dominated by a eukaryotic alga. The nonlaminated mat was populated by a diverse group of cyanobacteria and did not contain algae. The amplification and phylogenetic assignment of scytonemin biosynthetic gene scyC from laminated mat samples confirmed that the dominant cyanobacterium in these areas, L. aestuarii, is likely responsible for sunscreen production. This study is the first to utilize an understanding of the molecular basis of scytonemin assembly to explore its synthesis and function within natural microbial communities., Chemistry and Chemical Biology

Crystals of recombinant NovN, an O-carbamoyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in two different crystal forms. Crystal form I belonged to space group C2 and native data were collected to 2.9 Å resolution in-house. Crystal form II had I-centred orthorhombic symmetry and native data were recorded to a resolution of 2.3 Å at a synchrotron. NovN catalyses the final step in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase. [ABSTRACT FROM AUTHOR]

Crystals of recombinant NovP (subunit MW = 29 967 Da; 262 amino acids), an S-adenosyl-l-methionine-dependent O-methyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group P2, with unit-cell parameters a = 51.81, b = 46.04, c = 61.22 Å, β = 104.97°. Native data to a maximum resolution of 1.4 Å were collected from a single crystal at the synchrotron. NovP is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase. [ABSTRACT FROM AUTHOR]

Presents crystallization and preliminary X-ray studies on the putative dTDP sugar empimerase NovW from the novobiocin biosynthetic cluster of Streptomyces spheroides. Method used to study the analysis; Experimental observations of the analysis; Description about X-ray diffraction and crystal analysis.

The vancomycin class of antibiotics is regarded as the last line of defence against Gram-positive bacteria. The compounds used clinically are very complex organic molecules and are made by fermentation. The biosynthesis of these is complex and fascinating. Its study holds out the prospect of utilizing genetic engineering of the enzymes in the pathway in order to produce novel vancomycin analogues. In part, this requires detailed structural insight into substrate specificity as well as the enzyme mechanism. The crystallization of one of the enzymes in the chloroeremomycin biosynthetic pathway (a member of the vancomycin family), dTDP-3-amino-4-keto 2,3,6- trideoxy-3-C-methyl-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, is reported here. The protein is fourth in the pathway which makes a carbohydrate essential for the activity of chloroeremomycin. The crystals of EvaD diffract to 1.5 Å and have unit-cell parameters a = 98.6, b = 72.0, c = 57.1 Å with space group P21212. Data to this resolution were collected at the European Synchrotron Radiation Facility. [ABSTRACT FROM AUTHOR]

Thousands of polyketide and nonribosomal peptide natural products are assembled on multimodular enzymatic assembly lines. Natural product acyl chains grow as a series of thioester intermediates covalently tethered via phosphoantetheinyl arms to carrier protein domains, with initiation, elongation, and termination steps. Tailoring enzymes can act during chain growth on the assembly lines or in post assembly line modifications. Two on-assembly line tailoring processes that diversify natural product scaffolds are: (1) introduction of beta branching in polyketides, and (2) depsipeptide formation in nonribosomal peptides by α-hydroxy acid instead of α-amino acid monomers to make ester rather than amide bonds. The polyketide beta branching arises by convergence of isoprenoid and polyketide enzymatic assembly linee machinery while the ester linkages in NRPs such as valinomycin and cereulide arise by in situ reductions of tethered αketo acid monomers [ABSTRACT FROM AUTHOR]

Review: 43 refs. [ABSTRACT FROM AUTHOR]

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Review: 239 refs. [ABSTRACT FROM AUTHOR]

Rebeccamycin, an antitumor compound, is synthesized by L. aerocolonigenes from the precursor L-tryptophan [Biosci. Biotechnol. Biochem. (2003) 67: 127-38]. One step in the biosynthetic pathway involves the net eight-electron oxidation of chlorinated chromopyrrolic acid to generate the rebeccamycin aglycone, which is carried out by the enzymatic pair, RebP and RebC [PNAS (2005) 102: 461-6]. Substitution of RebC with StaC -- a homologue with 65% identity to RebC -- results in the production of the staurosporine aglycone in lieu of the rebeccamycin aglycone [JACS (2006) 128: 12289-98]. While both RebC and StaC are annotated as flavin monooxygenases, only RebC is able to bind flavin adenine dinucleotide [JACS (2006) 128: 12289-98]. The structure of RebC bound to flavin adenine dinucleotide (FAD) was recently solved with 1.8A resolution using phases from a samarium derivative. The structure of RebC bears much resemblance to phenol hydroxylase, a flavin-dependent monooxygenase. This suggests that RebC is also a flavin-dependent monooxygenase, which might react with a yet-unidentified product produced by RebP. Residues in the structure near FAD are potentially responsible for the differences in FAD-binding and activity found between RebC and StaC. We are using the crystal structure as a guide to investigate the properties of various RebC mutants, in hope of determining the structural basis for enzyme specificity. [ABSTRACT FROM AUTHOR]

The indolocarbazoles rebeccamycin and staurosporine, originally isolated from the actinomycetes, Lechevalieria aerocolonigenes and Streptomyces longisporoflavus, have received much attention due to their strong anticancer activities. These natural products are biosynthesized by parallel pathways, involving complex oxidative transformations and couplings to fashion the fused six-ring aglycones from a starting pair of L-tryptophan molecules. This overall 10-14 electron oxidation of two L-tryptophans (or 7-chloro-L-tryptophans) is catalyzed by only four enzymes: RebO, RebD, RebP and RebC (or their Sta homologues). RebP and StaP, in particular, are of interest due to their ability to catalyze both aryl-aryl coupling and oxidative decarboxylation, effecting a net 4-8 electron oxidation to three distinct aglycone products. Their flavin partner enzymes, RebC and StaC, direct the oxidative flux of the reaction to yield only one of these aglycone forms. We have reconstituted the complete biosynthetic pathway to the rebeccamycin and staurosporine aglycones. This work reveals some of Nature's unique mechanistic strategies in constructing natural products by oxidative pathways. [ABSTRACT FROM AUTHOR]