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Structure of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: comparison of the Mn2+∗2-phosphoglycolate and the Pb2+∗2-Phosphoenolpyruvate complexes and implications for catalysis1

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Abstract

The crystal structure of the phenylalanine-regulated 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) from Escherichia coli in complex with Mn2+ and the substrate analog, 2-phosphoglycolate (PGL), was determined by molecular replacement using X-ray diffraction data to 2.0 Å resolution. DAHPS∗Mn∗PGL crystallizes in space group C2 (a=210.4 Å, b=53.2 Å, c=149.4 Å, β=116.1 °) with its four (β/α)8 barrel subunits related by non-crystallographic 222 symmetry. The refinement was carried out without non-crystallographic symmetry restraints and yielded agreement factors of R=20.9 % and Rfree=23.9 %. Mn2+, the most efficient metal activator, is coordinated by the same four side-chains (Cys61, His268, Glu302 and Asp326) as is the poorly activating Pb2+. A fifth ligand is a well-defined water molecule, which is within hydrogen bonding distance to an essential lysine residue (Lys97). The distorted octahedral coordination sphere of the metal is completed by PGL, which replaces the substrate, 2-phosphoenolpyruvate (PEP), in the active site. However, unlike PEP in the Pb∗PEP complex, PGL binds the Mn2+via one of its carboxylate oxygen atoms. A model of the active site is discussed in which PEP binds in the same orientation as does PGL in the DAHPS∗Mn∗PGL structure and the phosphate of E4P is tethered at the site of a bound sulfate anion. The re face of E4P can be positioned to interact with the si face of PEP with only small movement of the protein.

Introduction

DAHPS (3-deoxy-d-arabino-heptulosonate-7-phosphate synthase) is the initial enzyme in the biosynthesis of aromatic compounds in plants and microorganisms. It catalyzes the aldol-like, stereospecific condensation of 2-phosphoenol-pyruvate (PEP) and d-erythrose-4-phosphate (E4P) to 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) (Figure 1). DAHP is then converted by the subsequent enzymes of the pathway to chorismate, the common branch point precursor for the three aromatic amino acid residues and most other aromatic end products (reviewed by Hermann, 1995). In Escherichia coli there are three DAHPS isoforms, one specifically feedback-inhibited by l-phenylalanine, one by l-tryptophan, and one by l-tyrosine. All three isozymes require a divalent cation for activity (Stephens & Bauerle, 1991). A number of divalent metal ions activate DAHPS in vitro with efficiencies varying over a 20-fold range as follows: Mn2+ > Fe2+ ≈ Cd2+ > Co2+>Cu2+≈Ni2+ ≈ Zn2+ ⪢ Ca2+>Pb2+. The activating metal in vivo appears to be Fe2+Stephens and Bauerle 1991, Ray and Bauerle 1991.

The crystal structure of the phenylalanine-regulated DAHPS isozyme complexed with PEP and Pb2+ (DAHPS(Phe)∗Pb∗PEP) was recently determined by MAD phasing from the Pb2+ by our group (Shumilin et al., 1999). It is a homotetramer consisting of four (β/α)8 barrel subunits related by non-crystallographic 222 symmetry. Both the metal and PEP are bound at the active site of the enzyme at the C-terminal end of the barrel. The coordination of PEP and Pb2+ involves the interaction between the 6 s2 lone pair of electrons of the lead and the conjugated π system of PEP (Shimoni-Livny et al., 1998).

One of the goals here is to determine whether the role of the metal ion required by the enzyme is structural or catalytic in nature. For this purpose we have crystallized DAHPS(Phe) in complex with Mn2+, the most active metal in kinetic experiments, and with the non-reacting substrate analog, 2-phosphoglycolate (PGL), shown in Figure 1. We report here the results of our crystal structure determination to 2.0 Å resolution. Based on an analysis of packing, we propose an explanation for the lack of isomorphism observed in several of our DAHPS derivatives. The higher-level resolution of the data allowed us to detect some differences in the protein main-chain relative to DAHPS∗Pb∗PEP, to trace one of the formerly undefined loops, and to inspect the active site for solvent sites that might be involved in the catalytic reaction. Assuming that PEP has an orientation similar to that of PGL and that the phosphate group of E4P is tethered to the site of a bound sulfate anion, we found that E4P can be oriented so that the si face of PEP is in proper position to attack the re face of E4P, in accordance with the established stereochemisty (Floss et al., 1972).

Section snippets

Structure solution and model quality

The structure of DAHPS∗Mn∗PGL was solved by molecular replacement using subunit C of the DAHPS∗Pb∗PEP complex (Shumilin et al., 1999) as the search model. The rotation function search yielded straightforward solutions for all four subunits, the correlation coefficients of which, however, differ considerably (Table 1). In the initial electron density maps, areas corresponding to active site residues, ligands, and loop region 313–317 in subunit B, not contained in the search model are clearly

Purification and crystallization

DAHPS was purified in the prescence of EDTA as described by Shumilin et al. (1995) and then dialyzed against Chelexed 1.0 mM 1,3-bis[tris(hydroxy-methyl)methylamino]propane (BTP) at pH 7.0. The enzyme was crystallized at room temperature using the hanging drop vapor diffusion method. All solutions used for crystallization, except for MnSO4 and DAHPS were treated with Chelex-100 to remove metals. Crystals grew in 10 μl drops containing 0.20 mM DAHPS subunit (7.5 mg/ml), 0.37 mM MnSO4, 4.2 mM

Acknowledgements

We very much appreciate Dr Gatti’s sending us coordinates of KDOPS prior to publication. Liang Tong helped us in use and interpretation of rotation functions. Support was provided by NSF (MCB-9723633) to R.H.K and by NIH (GM-35889) to R.H.B.

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