Sascha Keller

John Innes Centre, Norwich, England, United Kingdom

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Publications (3)11.76 Total impact

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    ABSTRACT: CloQ is an aromatic prenyltransferase from the clorobiocin biosynthetic pathway of Streptomyces roseochromogenes var. oscitans. It is involved in the synthesis of the prenylated hydroxybenzoate moiety of the antibiotic, specifically catalyzing the attachment of a dimethylallyl moiety to 4-hydroxyphenylpyruvate. Herein, we report the crystal structure of CloQ and use it as a framework for interpreting biochemical data from both wild-type and variant proteins. CloQ belongs to the aromatic prenyltransferase family, which is characterized by an unusual core fold comprising five consecutive ααββ elements that form a central 10-stranded anti-parallel β-barrel. The latter delineates a solvent-accessible cavity where substrates bind and catalysis takes place. This cavity has well-defined polar and nonpolar regions, which have distinct roles in substrate binding and facilitate a Friedel-Crafts-type mechanism. We propose that the juxtaposition of five positively charged residues in the polar region circumvents the necessity for a Mg(2+), which, by contrast, is a strict requirement for the majority of prenyltransferases characterized to date. Our structure of CloQ complexed with 4-hydroxyphenylpyruvate reveals the formation of a covalent link between the substrate and Cys215 to yield a thiohemiketal species. Through site-directed mutagenesis, we show that this link is not essential for enzyme activity in vitro. Furthermore, we demonstrate that CloQ will accept alternative substrates and, therefore, has the capacity to generate a range of prenylated compounds. Since prenylation is thought to enhance the bioactivity of many natural products, CloQ offers considerable promise as a biocatalyst for the chemoenzymatic synthesis of novel compounds with therapeutic potential.
    Journal of Molecular Biology 10/2010; 404(4):611-26. DOI:10.1016/j.jmb.2010.09.067 · 3.96 Impact Factor
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    ABSTRACT: Crystals of recombinant NovR (subunit MW = 29 924 Da; 270 amino acids), a non-haem iron oxygenase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group C2, with unit-cell parameters a = 86.69, b = 139.38, c = 100.82 A, beta = 101.18 degrees . Native data were collected to a resolution of 2.1 A from a single crystal at a synchrotron and a molecular-replacement solution was obtained using the program AMoRe. The starting phase information was very poor and did not permit model building. Phases were subsequently improved using a combination of fourfold averaging and very gradual phase extension in the program DM to yield an interpretable map. NovR belongs to a novel class of non-haem iron oxygenases that share sequence similarity with class II aldolases. It is predicted to perform two consecutive oxidative decarboxylation steps in the biosynthesis of the prenylated hydroxybenzoic acid moiety of the aminocoumarin antibiotic novobiocin.
    Acta Crystallographica Section D Biological Crystallography 01/2007; 62(Pt 12):1564-70. DOI:10.1107/S0907444906040169 · 7.23 Impact Factor
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    ABSTRACT: Crystals of recombinant CloQ (subunit MW = 35 626 Da; 324 amino acids), an aromatic prenyltransferase from Streptomyces roseochromogenes, were grown by vapour diffusion. The protein crystallizes in space group I4(1)22, with unit-cell parameters a = b = 135.19, c = 98.13 A. Native data from a single crystal were recorded to a resolution of 2.2 A in-house. Preliminary analysis of these data indicated that the asymmetric unit corresponds to a monomer, giving an estimated solvent content of 60.6%. CloQ is involved in the biosynthesis of the aminocoumarin antibiotic clorobiocin, which targets the essential bacterial enzyme DNA gyrase.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2006; 62(Pt 11):1153-5. DOI:10.1107/S1744309106042527 · 0.57 Impact Factor