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Title: Crystal Structure of 1-Deoxy-D-xylulose 5-Phosphate Synthase, A Crucial Enzyme for Isoprenoids Biosynthesis

Abstract

Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi, and animals. In contrast, IPP is synthesized by a mevalonate-independent pathway in most bacteria, algae, and plant plastids. 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on DXS, from Escherichia coli and Deinococcus radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II, and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, DXS has a novel arrangement of these domains as compared with the other enzymes, such that the active site of DXS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the dimer. The coenzyme TPP is mostly buried in the complex, but the C-2 atom of its thiazolium ring is exposed to a pocket that is themore » substrate-binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930405
Report Number(s):
BNL-81132-2008-JA
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US200904%%685
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 282
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; ALGAE; ANIMALS; ANTIBIOTICS; ATOMS; BACTERIA; BIOSYNTHESIS; CATALYSIS; COENZYMES; CRYSTAL STRUCTURE; ENZYMES; ESCHERICHIA COLI; FUNGI; HERBICIDES; MUTAGENESIS; PRECURSOR; PYROPHOSPHATES; RESIDUES; SYNTHESIS; THIAMINE; national synchrotron light source

Citation Formats

Xiang,S., Usunow, G., Busch, G., and Tong, L. Crystal Structure of 1-Deoxy-D-xylulose 5-Phosphate Synthase, A Crucial Enzyme for Isoprenoids Biosynthesis. United States: N. p., 2007. Web. doi:10.1074/jbc.M610235200.
Xiang,S., Usunow, G., Busch, G., & Tong, L. Crystal Structure of 1-Deoxy-D-xylulose 5-Phosphate Synthase, A Crucial Enzyme for Isoprenoids Biosynthesis. United States. doi:10.1074/jbc.M610235200.
Xiang,S., Usunow, G., Busch, G., and Tong, L. Mon . "Crystal Structure of 1-Deoxy-D-xylulose 5-Phosphate Synthase, A Crucial Enzyme for Isoprenoids Biosynthesis". United States. doi:10.1074/jbc.M610235200.
@article{osti_930405,
title = {Crystal Structure of 1-Deoxy-D-xylulose 5-Phosphate Synthase, A Crucial Enzyme for Isoprenoids Biosynthesis},
author = {Xiang,S. and Usunow, G. and Busch, G. and Tong, L.},
abstractNote = {Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi, and animals. In contrast, IPP is synthesized by a mevalonate-independent pathway in most bacteria, algae, and plant plastids. 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on DXS, from Escherichia coli and Deinococcus radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II, and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, DXS has a novel arrangement of these domains as compared with the other enzymes, such that the active site of DXS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the dimer. The coenzyme TPP is mostly buried in the complex, but the C-2 atom of its thiazolium ring is exposed to a pocket that is the substrate-binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies.},
doi = {10.1074/jbc.M610235200},
journal = {Journal of Biological Chemistry},
number = ,
volume = 282,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg{sup 2+}, NADPH and fosmidomycin was determined at 2.2 Å resolution. The structure showed a well defined loop conformation at the active site of DXR. The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg{sup 2+}, NADPH and fosmidomycin was solved at 2.2 Å resolution. DXR is the key enzyme in the 2-C-methyl-d-erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed,more » indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg{sup 2+} was shifted by NADPH in the active site.« less
  • 1l-myo-inositol 1-phosphate (MIP) synthase catalyzes the conversion of D-glucose 6-phosphate to 1l-myo-inositol 1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, enolization, intramolecular aldol cyclization, and reduction. Here we present the structure of MIP synthase in complex with NAD{sup +} and a high-affinity inhibitor, 2-deoxy-D-glucitol 6-(E)-vinylhomophosphonate. This structure reveals interactions between the enzyme active site residues and the inhibitor that are significantly different from that proposed for 2-deoxy-D-glucitol 6-phosphate in the previously published structure of MIP synthase-NAD{sup +}-2-deoxy-D-glucitol 6-phosphate. There are several other conformational changes in NAD{sup +} and the enzyme activemore » site as well. Based on the new structural data, we propose a new and completely different mechanism for MIP synthase.« less
  • Isoprenoids are made by all free-living organisms and range from essential metabolites like sterols and quinones to more complex compounds like pinene and rubber. They are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages tomore » isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.« less
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