Journal of Molecular Biology
Mechanism of Substrate Recognition and PLP-induced Conformational Changes in LL-Diaminopimelate Aminotransferase from Arabidopsis thaliana
Introduction
Lysine biosynthesis in plants and bacteria occurs via the diaminopimelate (DAP) pathway, and has been investigated extensively because its inhibition represents an attractive target for antibiotic or herbicide development.1, 2, 3 Mammals lack the enzymes of this biosynthetic route and require L-lysine in their diet. Considerable research has focussed on the development of lysine-rich food crops because this substance is often the limiting amino acid in the human diet.4
The DAP pathway (Fig. 1) to lysine begins with the condensation of aspartate semialdehyde with pyruvate to give dihydrodipicolinic acid (DHDP), which is then reduced to tetrahydrodipicolinic acid (THDP). THDP is converted to LL-Diaminopimelate (LL-DAP), which after epimerization to meso-DAP undergoes a decarboxylation at the D-stereocenter to produce L-lysine. In most bacteria, the conversion of THDP to LL-DAP occurs via a three-step reaction sequence involving N-succinylation (or N-acetylation) of THDP, followed by transamination and desuccinylation to provide LL-DAP. For many years it was assumed that plants used a similar approach; however, Leustek and co-workers discovered recently that in higher plants and in Chlamydia the conversion of THDP to LL-DAP proceeds directly without N-acylated intermediates through transamination by LL-Diaminopimelate aminotransferase (LL-DAP-AT). This enzyme uses L-glutamate as the source of the amino group (Fig. 2).3, 5, 6, 7, 8
Recently, two variants of LL-DAP-AT have been discovered (DapL1 and DapL2) that differ significantly in sequence.7 LL-DAP-AT enzymes from plants and Chlamydia belong to the DapL1 variant of LL-DAP-AT and share approximately 50% amino acid sequence identity.7, 8 The DapL2 variant is primarily found in Archaea and shares approximately 30% amino acid sequence identity with the DapL1 variant.7, 8
Our previous analysis of the crystal structure of LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT) revealed that the enzyme is a homodimer9 and belongs to the type I fold family of PLP-dependent aminotransferases (the aspartate aminotransferase (AspAT) family).9, 10, 11 In particular, it closely resembles subgroup Ib aminotransferases, such as Thermus thermophilus HB8 aspartate aminotransferase (1BJW).12 Each subunit consists of a large domain (LD) and a small domain (SD). Both domains belong to the α-β class of protein fold; the LD and the SD fold into an α-β-α sandwich and an α-β complex, respectively. Because of the functional and structural similarities with those of AspAT, the kinetic mechanism of LL-DAP-AT is thought to resemble that of AspAT (ping-pong bi-bi mechanism). Despite the similarity in folding, the actual modes of binding of LL-DAP and L-Glu remained unknown. Modelling of these substrates into the active site of AtDAP-AT suggested that Glu97∗, Asn309∗ and Lys129 (Amino acid designators followed by an asterisk (∗) indicate that the residues originate in the other subunit of the dimer) may be positioned for the specific recognition of the distal carboxylate groups of L-Glu and LL-DAP, and for the stereospecific recognition of the Cɛ amino group of LL-DAP.9 However, AspATs undergo a conformational change upon substrate binding,12 and it seemed possible that AtDAP-AT could also undergo active site reorganization upon exposure to substrates. Here, in order to assist in the understanding of substrate recognition and catalysis by AtDAP-AT, we have determined the crystal structures of LL-DAP-AT from A. thaliana in complex with two analogues of the external aldimines, N-(5′-phosphopyridoxyl)-L-glutamate (PLP-Glu) and N-(5′-phosphopyridoxyl)-LL-Diaminopimelate (PLP-DAP) (Fig. 3), in which the imine bond between the substrate and the cofactor has been reduced. A reduced PLP-Glu analogue has been used in the study of AspAT structures with a mimic of the cofactor–substrate complex in the active site.13 We report the crystal structures of the asparagine and glutamine variants of the active site lysine, K270N and K270Q, with the bound substrate–cofactor complexes. In contrast to the native enzyme complexes having the reduced analogues, these variant enzymes contain the unreduced external aldimine of PLP with L-Glu and LL-DAP in the active site. Together with an apo-AtDAP-AT structure, the results provide new insights into the mechanism of substrate/cofactor binding and the associated conformational changes in the enzyme.
Section snippets
Structure of AtDAP-AT in complex with reduced PLP-Glu
The reduced PLP-Glu analogue was synthesized by treatment of a mixture of PLP and L-glutamate with sodium borohydride as described.14 Introduction of this analogue into the AtDAP-AT active site was accomplished by first removing PLP from the native enzyme with phenylhydrazine followed by dialysis against a buffer containing PLP-Glu. Crystallization of the enzyme containing the reduced complex was achieved by the hanging-drop vapour-diffusion method.
The structure of AtDAP-AT with reduced PLP-Glu
Conclusion
In this study, we have determined the crystal structures of AtDAP-AT in complex with two reduced substrate analogues, PLP-Glu and PLP-DAP. The structures of these complexes have revealed a novel mechanism employed by AtDAP-AT to accommodate two different substrates in the active site without the need for major conformational changes in the enzyme. Two well conserved tyrosine residues (Tyr37 and Tyr152) and Lys129 are used for binding the distal carboxylate group of both Glu and LL-DAP. A single
Crystallization and data collection of AtDAP-AT
Solutions of the purified AtDAP-AT with the bound PLP-DAP or PLP-Glu aldimine analogues were concentrated to 10 mg/mL and dialyzed against 100 mM NaCl, 20 mM Hepes pH 7.6, and 1 mM DTT. These AtDAP-AT complexes were then crystallized in 45% (NH4)2SO4(w/v), 0.1 M Hepes pH 7.5, 3% PEG400 by the hanging-drop vapour-diffusion method. Within two weeks, X-ray diffraction-quality crystals of AtDAP-AT appeared in a drop containing 1 μL of the protein solution and 1 μL of the crystallizing agent. The
Acknowledgements
We thank Dr Jonathan C. Parrish (Alberta Synchrotron Institute, ASI) and Dr James Holden (Advanced Light Source, ALS) for the data collection at beamline 8.3.1 of the ALS at the Lawrence Berkeley Laboratory. The ALS is operated by the Department of Energy and supported by the National Institutes of Health, the National Science Foundation, the University of California and the Henry Wheeler Foundation. The ASI synchrotron access program is supported by the Alberta Science and Research Authority
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