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Title: Structure of Arabidopsis Dehydroquinate Dhydratase-Shikimate Dehydrogeanse and Implications for Metabolic Channeling in the Shikimate Pathway

Abstract

The bifunctional enzyme dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) catalyzes the dehydration of dehydroquinate to dehydroshikimate and the reduction of dehydroshikimate to shikimate in the shikimate pathway. We report the first crystal structure of Arabidopsis DHQ-SDH with shikimate bound at the SDH site and tartrate at the DHQ site. The interactions observed in the DHQ-tartrate complex reveal a conserved mode for substrate binding between the plant and microbial DHQ dehydratase family of enzymes. The SDH-shikimate complex provides the first direct evidence of the role of active site residues in the catalytic mechanism. Site-directed mutagenesis and mechanistic analysis revealed that Asp 423 and Lys 385 are key catalytic groups and Ser 336 is a key binding group. The arrangement of the two functional domains reveals that the control of metabolic flux through the shikimate pathway is achieved by increasing the effective concentration of dehydroshikimate through the proximity of the two sites.

Authors:
;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
914078
Report Number(s):
BNL-78646-2007-JA
Journal ID: ISSN 0006-2960; BICHAW; TRN: US0801525
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 45
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; 43 PARTICLE ACCELERATORS; ARABIDOPSIS; CHANNELING; CRYSTAL STRUCTURE; DEHYDRATION; ENZYMES; FUNCTIONALS; MUTAGENESIS; RESIDUES; SUBSTRATES; TARTRATES; NSLS; national synchrotron light source

Citation Formats

Singh,S., and Christendat, D.. Structure of Arabidopsis Dehydroquinate Dhydratase-Shikimate Dehydrogeanse and Implications for Metabolic Channeling in the Shikimate Pathway. United States: N. p., 2006. Web. doi:10.1021/bi060366+.
Singh,S., & Christendat, D.. Structure of Arabidopsis Dehydroquinate Dhydratase-Shikimate Dehydrogeanse and Implications for Metabolic Channeling in the Shikimate Pathway. United States. doi:10.1021/bi060366+.
Singh,S., and Christendat, D.. Sun . "Structure of Arabidopsis Dehydroquinate Dhydratase-Shikimate Dehydrogeanse and Implications for Metabolic Channeling in the Shikimate Pathway". United States. doi:10.1021/bi060366+.
@article{osti_914078,
title = {Structure of Arabidopsis Dehydroquinate Dhydratase-Shikimate Dehydrogeanse and Implications for Metabolic Channeling in the Shikimate Pathway},
author = {Singh,S. and Christendat, D.},
abstractNote = {The bifunctional enzyme dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) catalyzes the dehydration of dehydroquinate to dehydroshikimate and the reduction of dehydroshikimate to shikimate in the shikimate pathway. We report the first crystal structure of Arabidopsis DHQ-SDH with shikimate bound at the SDH site and tartrate at the DHQ site. The interactions observed in the DHQ-tartrate complex reveal a conserved mode for substrate binding between the plant and microbial DHQ dehydratase family of enzymes. The SDH-shikimate complex provides the first direct evidence of the role of active site residues in the catalytic mechanism. Site-directed mutagenesis and mechanistic analysis revealed that Asp 423 and Lys 385 are key catalytic groups and Ser 336 is a key binding group. The arrangement of the two functional domains reveals that the control of metabolic flux through the shikimate pathway is achieved by increasing the effective concentration of dehydroshikimate through the proximity of the two sites.},
doi = {10.1021/bi060366+},
journal = {Biochemistry},
number = ,
volume = 45,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analoguemore » of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form {pi}-stacking interactions with the catalytic Tyr{sup 24} have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19-24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.« less
  • The expression of plant shikimate kinase (SK; EC 2.7.1.71), an intermediate step in the shikimate pathway to aromatic amino acid biosynthesis, is induced under specific conditions of environmental stress and developmental requirements in an isoform-specific manner. Despite their important physiological role, experimental structures of plant SKs have not been determined and the biochemical nature of plant SK regulation is unknown. The Arabidopsis thaliana genome encodes two SKs, AtSK1 and AtSK2. We demonstrate that AtSK2 is highly unstable and becomes inactivated at 37 C whereas the heat-induced isoform, AtSK1, is thermostable and fully active under identical conditions at this temperature. Wemore » determined the crystal structure of AtSK2, the first SK structure from the plant kingdom, and conducted biophysical characterizations of both AtSK1 and AtSK2 towards understanding this mechanism of thermal regulation. The crystal structure of AtSK2 is generally conserved with bacterial SKs with the addition of a putative regulatory phosphorylation motif forming part of the adenosine triphosphate binding site. The heat-induced isoform, AtSK1, forms a homodimer in solution, the formation of which facilitates its relative thermostability compared to AtSK2. In silico analyses identified AtSK1 site variants that may contribute to AtSK1 stability. Our findings suggest that AtSK1 performs a unique function under heat stress conditions where AtSK2 could become inactivated. We discuss these findings in the context of regulating metabolic flux to competing downstream pathways through SK-mediated control of steady state concentrations of shikimate.« less