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Structure and mechanism of the magnesium-independent aromatic prenyltransferase CloQ from the clorobiocin biosynthetic pathway
DOI:
10.1016/j.jmb.2010.09.067
PMID:
20946900
Authors:
Ute
Metzger
(John Innes Centre; University of Tuebingen)
,
Sascha
Keller
(John Innes Centre)
,
Clare
Stevenson
(John Innes Centre)
,
Lutz
Heide
(Universität Tübingen)
,
David M.
Lawson
(John Innes Centre)
Co-authored by industrial partner:
No
Type:
Journal Paper
Journal:
Journal Of Molecular Biology
State:
Published (Approved)
Published:
October 2010
Diamond Proposal Number(s):
1219
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 ???? elements that form a central 10-stranded anti-parallel ?-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 Mg2+, 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 Keywords: Biosynthetic; Coenzymes; Crystallography; X-Ray; Dimethylallyltranstransferase; Magnesium; Models; Molecular; Mutagenesis; Site-Directed; Mutant; Novobiocin; Phenylpyruvic; Protein; Tertiary; Streptomyces
Diamond Keywords: Bacteria; Enzymes
Subject Areas:
Biology and Bio-materials,
Chemistry
Instruments:
I02-Macromolecular Crystallography
Added On:
18/10/2010 09:26
Discipline Tags:
Biotechnology
Biochemistry
Chemistry
Structural biology
Engineering & Technology
Life Sciences & Biotech
Technical Tags:
Diffraction
Macromolecular Crystallography (MX)