1. Attaining Sustainable Services from Ecosystems through Trade-off Scenarios - ASSETS
1Centre for Biological Sciences, University of Southampton, SO17 1BJ, UK, Conservation International, Chancellor Colleage Malawi, Bilbao, CIAT
Our Overall Goal is to explicitly quantify the linkages between the natural ecosystem services that affect – and are affected by – food security and nutritional health for the rural poor at the forest-agricultural interface.
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Twelve Pdx1 proteins assemble into the active enzyme complex as a bi-layered hexameric ring. The monomeric Pdx1 protein has a (ba)8-barrel (TIM) fold.
Pdx1 catalyses synthesis of PLP from ribose 5-phosphate and glyceraldehyde 3-phosphate in a multi-step reaction. The substrate ammonia, in vivo supplied by the interacting Pdx2 subunit, can be supplied by addition of ammonium salts. The reaction scheme shown was postulated on the basis of combined use of spectroscopic and NMR methods (Hanes et al. 2008).
After addition of the substrates ribose 5-phosphate and ammonia, Pdx1 accumulates the reaction intermediate known as I320. The enzyme forms a covalent Schiff base linkage between intermediate and a Pdx1 lysine side chain in the active site. The enzyme-intermediate complex displays an absorption peak at 320nm.
Three different structures have been postulated for the structure of this intermediate, leading to proposals of different reaction mechanisms. From the work carried out at the ESRF we can now propose possible structures of I320 and respective reaction mechanisms leading to I320 formation.
Identifying the nature of the I320 intermediate
Mechanisms of I320 formation proposed by Hanes et al. (left) and Raschle et al. (right).
The PLP intermediate
The Z3 intermediate is formed when crystals of the Pdx1 enzyme are soaked with PLP. Data obtained from the ESRF support a covalent intermediate with the enzyme. The complex displays an absorption peak at 410nm (blue curve), while surrounding mother liquor displays an absorption maximum of 395 nm which comes from free PLP (re`d curve). A previous x-ray study (Zhang et al 2010) had suggested that PLP may bind to the enzyme through non-covalent interactions, however we see evidence for a covalent intermediate from the spectrum and structure determination.
UV/Vis absorption spectrum of a Pdx1 crystal that was allowed to accumulate I320 by addition of ribose 5-phospahte and ammonia.
At present, the molecular structure of I320 is not clear. The experiments carried out here with wild type enzymes and with variant proteins that are inhibited in I320 formation will help to resolve these issues, using x-ray diffraction data, structure determination, and online UV-Vis spectroscopy experiments.
Aims of future work
PLP synthase is ideal for linked crystallographic and spectroscopic studies, as it is possible to follow catalytic reactions in the crystal, due to this being a slow enzyme, which forms several stable and chromophoric intermediates. However, the current results are hardly satisfying as many crystals need to be tested, overlapping densities from different intermediates are observed, and data need to be selected from hundreds of experiments.
Micro-focus x-ray sources give distinct advantages over conventional larger beams with respect to probing smaller crystals or small regions of larger crystals. Micro-focus techniques are to be used to study enzyme complexes of PLP synthase, with two fundamental experiments, both requiring online spectroscopy:
scanning across crystals, solving structures from smaller portions of a larger crystal to investigate whether islands homogenous with respect to intermediate formation exist;
using small crystals and multi-crystal merging, while optimising the merge with respect to the use data from crystals that show similar intermediates.
UV/Vis absorption spectrum derived from exposure of a PLP soaked Pdx1 crystal (blue) and from surrounding mother liquor (red). The 2F0-Fc density map at 1.7 Å was obtained after refinement of the structure in which only the phosphate was included as ligand.
References
Derrer B, Windeisen V, Guédez Rodríguez G, Seidler J, Gengenbacher M, Lehmann WD, Rippe K, Sinning I, Tews I, Kappes B. “Defining the structural requirements for ribose 5-phosphate-binding and intersubunit cross-talk of the malarial pyridoxal 5-phosphate synthase.” FEBS Lett. 584 (2010),
Guédez G, Hipp K, Windeisen V, Derrer B, Gengenbacher M, Böttcher B, Sinning I, Kappes B, Tews I. “Assembly of the eukaryotic PLP-synthase complex from Plasmodium and activation of the Pdx1 enzyme.” Structure. 20 (2012),
Hanes JW, Keresztes I, Begley TP. “13C NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase.” Nat Chem Biol. 4 (2008),
Raschle T, Arigoni D, Brunisholz R, Rechsteiner H, Amrhein N, Fitzpatrick TB. “Reaction mechanism of pyridoxal 5'-phosphate synthase. Detection of an enzyme-bound chromophoric intermediate.” J Biol Chem 282 (2007),
Strohmeier M, Raschle T, Mazurkiewicz J, Rippe K, Sinning I, Fitzpatrick TB, Tews I. “Structure of a bacterial pyridoxal 5'-phosphate synthase complex.” Proc Natl Acad Sci U S A. 103 (2006),
Zhang X, Teng YB, Liu JP, He YX, Zhou K, Chen Y, Zhou CZ. “Structural insights into the catalytic mechanism of the yeast pyridoxal 5-phosphate synthase Snz1.” Biochem J 432 (2010),
The authors would like to thank Martin Flot, Antoine Royant and Martin Weik for their help and access to facilities at the ESRF.