Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana
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.
- Research Organization:
- Stanford Linear Accelerator Center (SLAC)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC02-76SF00515
- OSTI ID:
- 953602
- Report Number(s):
- SLAC-REPRINT-2009-281
- Journal Information:
- J. Mol. Biol 384:1314,2008, Journal Name: J. Mol. Biol 384:1314,2008 Journal Issue: 5 Vol. 384; ISSN JMOBAK; ISSN 0022-2836
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
36 MATERIALS SCIENCE
AMINO ACIDS
AMINOTRANSFERASES
ANTIBIOTICS
ARABIDOPSIS
CATALYSIS
CHAINS
CONFORMATIONAL CHANGES
CRYSTAL STRUCTURE
ENZYMES
HERBICIDES
HYDROGEN
INTERACTIONS
LYSINE
MOLECULES
MUTAGENESIS
Other
OTHER
BIO
PHOSPHATES
PLANTS
POTENTIALS
PYRIDOXAL
RESIDUES
SALTS
SUBSTRATES
TARGETS
TORSION
WATER