Abstract
We have solved the 2.5-A crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, an enzyme involved in the mevalonate-independent 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis. The structure reveals that the enzyme is present as a homodimer. Each monomer displays a V-like shape and is composed of an amino-terminal dinucleotide binding domain, a connective domain, and a carboxyl-terminal four-helix bundle domain. The connective domain is responsible for dimerization and harbors most of the active site. The strictly conserved acidic residues Asp(150), Glu(152), Glu(231), and Glu(234) are clustered at the putative active site and are probably involved in the binding of divalent cations mandatory for enzyme activity. The connective and four-helix bundle domains show significant mobility upon superposition of the dinucleotide binding domains of the three conformational states present in the asymmetric unit of the crystal. A still more pronounced flexibility is observed for a loop spanning residues 186 to 216, which adopts two completely different conformations within the three protein conformers. A possible involvement of this loop in an induced fit during substrate binding is discussed.
Highlights
Isoprenoids, formed by the condensation of varying numbers of isopentenyl diphosphate (IPP)1 units, constitute a major class of both primary and secondary metabolites including, for example, the ubiquitous sterols as well as dolichols, plastochinones, carotenoids, the prenyl side chains of chlorophylls, and ubiquinones [1]
E. coli DOXP reductoisomerase, which is present as a dimer in the present crystal structure, is composed of three domains: an amino-terminal dinucleotide binding domain, a carboxylterminal four-helix bundle, and a connective domain that mediates the formation of the dimer
The latter comprises a cluster of strictly conserved acidic amino acids that in the present apo structure form a set of salt bridges to adjacent basic residues which are, interestingly enough, invariant. This is strongly reminiscent of the situation in acetohydroxy acid isomeroreductase, which catalyzes a similar reaction to DOXP reductoisomerase
Summary
Molecular Cloning, Overexpression, and Purification—The E. coli dxr gene (GenBankTM accession number AB01300) encoding DOXP reductoisomerase was PCR-amplified from genomic E. coli K12 DNA using the primers Ecdxrfor (5Ј-GCGGATCCATGAAGCAACTCACCATTCTG3Ј) and Ecdxrrev (5Ј-CCGGAAGCTTTCAGCTTGCGAGACGCATCA3Ј), purchased from Interactiva (Ulm, Germany). The amplified gene was inserted into the pQE9 overexpression vector (Qiagen, Hilden, Germany) via a BamHI and a HindIII restriction site introduced by the primers (restriction sites are underlined) This resulted in a dxr gene in which the encoded protein was endowed with an amino-terminal His6tag separated from the original amino-terminal methionine by a glycine and a serine residue. The enzyme eluted with 50 mM imidazole in buffer A and was rebuffered in 20 mM Tris-HCl, pH 8.0, using a prepacked PD10 Sephadex G-25 gel filtration column (Amersham Biosciences, Inc.). Final purification was carried out by gel filtration using an XK 16/60 Superdex 75 column equilibrated with 20 mM Tris-HCl, pH 8.0, 40 mM NaCl. The protein was concentrated for crystallization to 15 mg mlϪ1 using a YM-30 ultrafiltration unit (Millipore, Eschborn, Germany).
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