The novobiocin biosynthetic gene cluster from Streptomyces spheroides NCIB 11891 was cloned by using homologous deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene fragments as probes. Doublestranded sequencing of 25.6 kb revealed the presence of 23 putative open reading frames (ORFs), including the gene for novobiocin resistance, gyrB r , and at least 11 further ORFs to which a possible role in novobiocin biosynthesis could be assigned. An insertional inactivation experiment with a dNDP-glucose 4,6-dehydratase fragment resulted in abolishment of novobiocin production, since biosynthesis of the deoxysugar moiety of novobiocin was blocked. Heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase, in Streptomyces lividans TK24 further confirmed the involvement of the analyzed genes in the biosynthesis of the antibiotic.Novobiocin is produced by Streptomyces spheroides and Streptomyces niveus and belongs to the aminocoumarin antibiotics. Bacterial DNA gyrase represents the target of these coumarins (41), and novobiocin inhibits this enzyme by interaction with the N-terminal 24-kDa subdomain of the gyrB subunit (27). In addition to its antibacterial action, novobiocin shows synergistic effects with antitumor drugs such as etoposide or teniposide (37,49).Little is known about the biosynthesis of novobiocin. Structurally, it is composed of three moieties, a noviose sugar (ring C), a substituted coumarin (ring B), and a prenylated 4-hydroxybenzoic acid (ring A), and these rings are linked by glycosidic and amide bonds (Fig. 1). Radioactive feeding experiments in the 1960s and 1970s showed that noviose is directly derived from D-glucose, whereas tyrosine serves as a precursor of ring A and ring B (3, 6, 31). This was recently confirmed by a feeding experiment with [1-13 C]glucose (33) which also showed that the dimethylallyl moiety of novobiocin was formed through the nonmevalonate pathway.Molecular biological studies have been restricted to the investigation of novobiocin resistance genes (43, 52), especially gyrB r (61, 62), and the production of novobiocin-deficient mutants (19). Discovery of the genetic basis of the biosynthesis of aminocoumarin antibiotics could provide a useful tool for drug development. "Combinatorial biosynthesis," the interchange of genes involved in antibiotic biosynthesis between different microorganisms or the creation of hybrid genes and, consequently, proteins with new enzymatic properties, allows the production of modified or even novel antibiotics (23). In the past, much effort has been undertaken in the manipulation of the biosynthesis of polyketide antibiotics (25,42,56), and recently, progress has also been made in the construction of hybrid peptide synthetase genes (55, 59). The discovery of gene clusters for other types of secondary metabolites can offer additional possibilities for combinatorial biosynthesis.Here we report on the identification of the novobiocin biosynthetic gene cluster from S. spheroides NCIB 11891. The gene...

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The aminocoumarin antibiotic coumermycin A 1 produced by Streptomyces rishiriensis DSM 40489 contains two amide bonds. The biosynthetic gene cluster of coumermycin contains a putative amide synthetase gene, couL, encoding a protein of 529 amino acids. CouL was overexpressed as hexahistidine fusion protein in Escherichia coli and purified by metal affinity chromatography, resulting in a nearly homogenous protein. CouL catalysed the formation of both amide bonds of coumermycin A 1 , i.e. between the central 3-methylpyrrole-2,4-dicarboxylic acid and two aminocoumarin moieties. Gel exclusion chromatography showed that the enzyme is active as a monomer. The activity was strictly dependent on the presence of ATP and Mn 2+ or Mg 2+. The apparent K m values were determined as 26 lM for the 3-methylpyrrole-2,4-dicarboxylic acid and 44 lM for the aminocoumarin moiety, respectively. Several analogues of the pyrrole dicarboxylic acid were accepted as substrates. In contrast, pyridine carboxylic acids were not accepted. 3-Dimethylallyl-4-hydroxybenzoic acid, the acyl component in novobiocin biosynthesis, was well accepted, despite its structural difference from the genuine acyl substrate of CouL.

During the development of the central nervous system (CNS), the correct wiring of outgrowing neurites is mediated by antagonistic mechanisms. Aberrant growth is prevented by repulsive factors such as semaphorins. Expression of the ligands Sema3A and -3E and the receptors neuropilin Npn-1, -2a and -2b in the chick visual system were analyzed by RT-PCR. Whereas Sema3A and its major receptor Npn-1 were abundant, Sema3E and Npn-2 isoform expression was highly restricted and developmentally regulated. Peak expression occurred during retinal axon innervation of the tectum. Functional in vitro assays with recombinant proteins revealed a topography-specific growth cone collapsing activity of Sema3A for tectal axons. Interestingly, whereas tectal axons collapsed in a topographic-specific manner only in the presence of Sema3A, retinal axons responded only to Sema3E. The collapsing activity was intracellularly mediated by cGMP. For a detailed analysis of neuronal responses to sempahorins, time lapse video recording was performed. When tectal and retinal axons were pre-exposed to brain-derived neurotrophic factor (BDNF), a protective effect was evident only in the case of retinal axons. Our results suggest a molecular mechanism whereby ingrowth of retinal axons into the tectum can be regulated by Sema3E/BDNF modulation without disturbing tectal axon growth out of the tectum mediated by Sema3A.

A dimethylallyl diphosphate:4‐hydroxyphenylpyruvate dimethylallyl transferase from the novobiocin producer Streptomyces spheroides DSM 40292 was identified, partially purified and characterized. The enzyme was soluble, and specific for 4‐hydroxyphenylpyruvate and dimethylallyl diphosphate as substrates. It is most likely involved in the biosynthesis of the prenylated 4‐hydroxybenzoate moiety of the aminocoumarin antibiotic novobiocin.