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Title: Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana

Abstract

LL-Diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal phosphate (PLP)-dependent enzyme in the lysine biosynthetic pathways of plants and Chlamydia, is a potential target for the development of herbicides or antibiotics. This homodimeric enzyme converts L-tetrahydrodipicolinic acid (THDP) directly to LL-DAP using L-glutamate as the source of the amino group. Earlier, we described the 3D structures of native and malate-bound LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT). Seven additional crystal structures of AtDAP-AT and its variants are reported here as part of an investigation into the mechanism of substrate recognition and catalysis. Two structures are of AtDAP-AT with reduced external aldimine analogues: N-(5'-phosphopyridoxyl)-L-glutamate (PLP-Glu) and N-(5'-phosphopyridoxyl)- LL-Diaminopimelate (PLP-DAP) bound in the active site. Surprisingly, they reveal that both L-glutamate and LL-DAP are recognized in a very similar fashion by the same sets of amino acid residues; both molecules adopt twisted V-shaped conformations. With both substrates, the {alpha}-carboxylates are bound in a salt bridge with Arg404, whereas the distal carboxylates are recognized via hydrogen bonds to the well-conserved side chains of Tyr37, Tyr125 and Lys129. The distal C{sup {var_epsilon}} amino group of LL-DAP is specifically recognized by several non-covalent interactions with residues from the other subunit (Asn309*, Tyr94*, Gly95*, and Glu97* (Amino acid designators followed bymore » an asterisk (*) indicate that the residues originate in the other subunit of the dimer)) and by three bound water molecules. Two catalytically inactive variants of AtDAP-AT were created via site-directed mutagenesis of the active site lysine (K270N and K270Q). The structures of these variants permitted the observation of the unreduced external aldimines of PLP with L-glutamate and with LL-DAP in the active site, and revealed differences in the torsion angle about the PLP-substrate bond. Lastly, an apo-AtDAP-AT structure missing PLP revealed details of conformational changes induced by PLP binding and substrate entry into the active site.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Stanford Linear Accelerator Center (SLAC)
Sponsoring Org.:
USDOE
OSTI Identifier:
953602
Report Number(s):
SLAC-REPRINT-2009-281
Journal ID: ISSN 0022-2836; JMOBAK; TRN: US201002%%1430
DOE Contract Number:  
AC02-76SF00515
Resource Type:
Journal Article
Journal Name:
J. Mol. Biol 384:1314,2008
Additional Journal Information:
Journal Volume: 384; Journal Issue: 5; Journal ID: ISSN 0022-2836
Country of Publication:
United States
Language:
English
Subject:
08 HYDROGEN; 36 MATERIALS SCIENCE; AMINO ACIDS; AMINOTRANSFERASES; ANTIBIOTICS; ARABIDOPSIS; CATALYSIS; CHAINS; CONFORMATIONAL CHANGES; CRYSTAL STRUCTURE; ENZYMES; HERBICIDES; HYDROGEN; INTERACTIONS; LYSINE; MOLECULES; MUTAGENESIS; PHOSPHATES; PLANTS; POTENTIALS; PYRIDOXAL; RESIDUES; SALTS; SUBSTRATES; TARGETS; TORSION; WATER; Other,OTHER, BIO

Citation Formats

Watanabe, N, Clay, M D, Belkum, M J van, Cherney, M M, Vederas, J C, and James, M N.G. Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana. United States: N. p., 2009. Web.
Watanabe, N, Clay, M D, Belkum, M J van, Cherney, M M, Vederas, J C, & James, M N.G. Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana. United States.
Watanabe, N, Clay, M D, Belkum, M J van, Cherney, M M, Vederas, J C, and James, M N.G. Tue . "Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana". United States.
@article{osti_953602,
title = {Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana},
author = {Watanabe, N and Clay, M D and Belkum, M J van and Cherney, M M and Vederas, J C and James, M N.G.},
abstractNote = {LL-Diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal phosphate (PLP)-dependent enzyme in the lysine biosynthetic pathways of plants and Chlamydia, is a potential target for the development of herbicides or antibiotics. This homodimeric enzyme converts L-tetrahydrodipicolinic acid (THDP) directly to LL-DAP using L-glutamate as the source of the amino group. Earlier, we described the 3D structures of native and malate-bound LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT). Seven additional crystal structures of AtDAP-AT and its variants are reported here as part of an investigation into the mechanism of substrate recognition and catalysis. Two structures are of AtDAP-AT with reduced external aldimine analogues: N-(5'-phosphopyridoxyl)-L-glutamate (PLP-Glu) and N-(5'-phosphopyridoxyl)- LL-Diaminopimelate (PLP-DAP) bound in the active site. Surprisingly, they reveal that both L-glutamate and LL-DAP are recognized in a very similar fashion by the same sets of amino acid residues; both molecules adopt twisted V-shaped conformations. With both substrates, the {alpha}-carboxylates are bound in a salt bridge with Arg404, whereas the distal carboxylates are recognized via hydrogen bonds to the well-conserved side chains of Tyr37, Tyr125 and Lys129. The distal C{sup {var_epsilon}} amino group of LL-DAP is specifically recognized by several non-covalent interactions with residues from the other subunit (Asn309*, Tyr94*, Gly95*, and Glu97* (Amino acid designators followed by an asterisk (*) indicate that the residues originate in the other subunit of the dimer)) and by three bound water molecules. Two catalytically inactive variants of AtDAP-AT were created via site-directed mutagenesis of the active site lysine (K270N and K270Q). The structures of these variants permitted the observation of the unreduced external aldimines of PLP with L-glutamate and with LL-DAP in the active site, and revealed differences in the torsion angle about the PLP-substrate bond. Lastly, an apo-AtDAP-AT structure missing PLP revealed details of conformational changes induced by PLP binding and substrate entry into the active site.},
doi = {},
journal = {J. Mol. Biol 384:1314,2008},
issn = {0022-2836},
number = 5,
volume = 384,
place = {United States},
year = {2009},
month = {5}
}