Download Structure and Properties of the Shikimate Pathway Type I D 3

STRUCTURE AND PROPERTIES OF THE SHIKIMATE PATHWAY TYPE ID 3-DEOXY-DARABINO-HEPTULOSONATE 7-PHOSPHATE SYNTHASE FROM PYROCOCCUS FURIOSUS:
INSIGHTS INTO THE OLIGOMEROPHILICITY OF PROTEINS
Geoffrey B. Jameson, Bryan F. Anderson, Mark L. Patchett*, Gillian E. Norris*, Linley
R. Schofield, Emily J. Parker
Institutes of Fundamental Sciences and *Molecular BioSciences, Massey University,
Palmerston North, New Zealand
3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the
condensation of phosphoenol pyruvate with erythrose 4-phosphate to make the sevencarbon phosphorylated monosaccharide DAH7P. This reaction is the first step of the
shikimate pathway for the synthesis of aromatic amino acids and other aromatic
molecules. This pathway is absent in animals, but present in bacteria, archaea and fungi.
The type I D DAH7PS from Pyrococcus furiosus, in contrast to other type I DAH7PS
including the structurally characterised type I from Escherichia coli and
Saccharomyces cerevisiae and a type I D from Thermotoga maritima, displays a wide
substrate preference for four- and five-carbon phosphorylated monosaccharides and is
missing the 60-80-residue N-terminal motif for feedback regulation by the final
aromatic amino-acid product [1].
Exclusive preference for the five-carbon
monosaccharide, arabinose 5-phosphate, is shown by the related type I K enzymes,
KDO8PS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), important for the
synthesis of cell-wall lipopolysaccharides of gram-negative bacteria.
The structure was solved, with some difficulty, by molecular replacement, using
eventually a hybrid model constructed from the DAH7PS from E. coli (21 % identity)
and the KDO8PS from E. coli (26 % identity, relative to the resultant structural
alignment). The structure was refined to R (Rfree) = 0.18 (0.23) using data to 2.60 Å
from an orthorhombic crystal, space group I222, with parameters a = 87.231, b =
110.024, c = 144.350 Å,  =  =  = 90o and Z (Z') = 16(2). The structure is a (/)8
barrel, which crystallographically appears as a tight dimer of dimers, in which >21 %
the surface area of the tetramer is buried relative to that of four isolated monomers and
each of the two main dimer interfaces buries >11 % of the surface area relative to that of
a pair of isolated monomers. However, in solution the structure is clearly dimeric.
Oligimerophilicity, in this case the preference for forming a tetramer as compared to
dimers, occurs when the surface area buried on tetramerisation exceeds that on forming
dimers: [Stetramer =: Sabcd - (Sa+Sb+Sc+Sd)] > [2Sdimer =: (Sab + Scd +Sbc + Sad) 2(Sa+Sb+Sc+Sd)], where Sabcd is the surface area of the tetramer, Sa, Sb, Sc and Sd are
surface areas of the monomers, and Sab, Scd, Sbc and Sad are surface areas of the four
dimers with the smallest surface area (i.e., largest buried area on dimerisation). In the
case of the DAH7PS from P. furiosus, Stetramer > 2Sdimer, a tetrameric disadvantage
consistent with observed solution behaviour. Tetramer/dimer preferences of other
proteins are analysed. There has been considerable recent interest in subunit contacts in
the crystalline state and the physiologically relevant association [2,3], and in other
metrics for assessing these contacts.
References
[1] - L. R. Schofield, M. L. Patchett and E. J. Parker, Prot. Expr. Purif. 34, 17-27, (2004).
[2] - R. P. Bahadur, P. Chakrabarti, F. Rodier and J. Janin, J. Mol. Biol. 336, 943-955, (2004).
[3] - I. M. A. Nooren and J. M. Thornton, EMBO J. 22, 3486-3492, (2003).