Journal of Molecular Biology
Volume 384, Issue 5, 31 December 2008, Pages 1314-1329
Journal home page for Journal of Molecular Biology

Mechanism of Substrate Recognition and PLP-induced Conformational Changes in LL-Diaminopimelate Aminotransferase from Arabidopsis thaliana

https://doi.org/10.1016/j.jmb.2008.10.022Get rights and content

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 α-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 Cɛ 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.

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

References (31)

  • HudsonA.O. et al.

    An LL-diaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants

    Plant Physiol.

    (2006)
  • McCoyA.J. et al.

    L,L-Diaminopimelate aminotransferase, a trans-kingdom enzyme shared by Chlamydia and plants for synthesis of diaminopimelate/lysine

    Proc. Natl Acad. Sci. USA

    (2006)
  • HudsonA.O. et al.

    Biochemical and phylogenetic characterization of a novel diaminopimelate biosynthesis pathway in prokaryotes identifies a diverged form of LL-diaminopimelate aminotransferase

    J. Bacteriol.

    (2008)
  • EliotA.C. et al.

    Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations

    Annu. Rev. Biochem.

    (2004)
  • MehtaP.K. et al.

    Aminotransferases: demonstration of homology and division into evolutionary subgroups

    Eur. J. Biochem.

    (1993)
  • Cited by (20)

    • Imine chemistry in plant metabolism

      2021, Current Opinion in Plant Biology
      Citation Excerpt :

      ALD1 belongs to the family of PLP-dependent type I aminotransferases and is most closely related to the conserved l,l-diaminopimelate-aminotransferase (DAP-AT) (EC 2.6.1.83) required for l-lysine biosynthesis. ALD1 and DAP-AT share a high degree of overall structural similarity but bear different features at their active sites, which are responsible for the distinct biochemical functions of the two enzymes [29,30]. It is probable that ALD1 evolved from a progenitor DAP-AT through gene duplication followed by neofunctionalization.

    View all citing articles on Scopus
    View full text