Photon Factory Activity Report 2014 #32 (2015) B

AR-NW3A/2014G189

Structure-function analysis of enzymes related to myo-inositol phosphorylation
Masahiro Fujihashi*, Ryuhei Nagata, Kunio Miki
Department of Chemistry, Graduate School of Science, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan

1 Introduction
Archaea has many metabolic pathways that differ frombacterial and eukaryotic ones. For example, T.
kodakarensis is known to synthesize 3-phosphoglyceratefrom AMP via ribose-1,5-phosphate.
TK2285 is an enzyme from T. kodakarensis that
phosphorylates myo-inositol [1]. Only two myo-inositol
kinases have been identified so far, and little is known
about these enzymes. Especially, which of the sixhydroxy groups of myo-inositol is phosphorylated has notbeen identified, because the mirror symmetry of myoinositol makes the identification of phosphorylatedhydroxy group by HPLC or NMR difficult. Thus, we
attempted to determine the crystal structures of TK2285
protein in complexed with substrates and with products.
2 Experiment
Crystals of the substrates-complex were obtained using
AMP-PCP and myo-inositol as ligands and using
polyethylene glycol 3350 and ammonium iodide asprecipitants.
Crystals of the products-complex wereprepared using ATP and myo-inositol as ligands. The
mixture of TK2285 and ligands were incubated at 85 ºC
to allow the enzyme to produce ADP and thephosphorylated myo-inositol product, and crystallizedusing polyethylene glycol 3350 and ammonium iodide as
precipitants.
The crystals were flash-frozen and thediffraction datasets were collected at the beamline NE3A.
Both types of crystals were belonged to the space group
P212121 and the cell constants were a = ~77 Å, b = ~81 Å,and c = 81~82 Å. The resolutions were 1.93 Å and 2.08
Å for substrates-complex and products-complex,respectively. The phases were determined by Fourier
synthesis using 3W4S structure (unliganded TK2285 [1])as the model. Both of the structures were refined to R
and Rfree factors of 20%~21% and ~25%, respectively [2].
3 Results and Discussion
Overall structures were shown in Figure 1. The liddomain of both complexes were closed comparing to theunliganded structure. The substrates-complex showedthat the 3-OH group is nearest to the -phosphate ofAMP-PMP (Figure 2A). The structure suggests that the3-OH group is phosphorylated by TK2285. The 3-OH
phosphorylation was confirmed by the products-complex(Figure 2B). 70% of the 3-OH of the myo-inositol wasphosphorylated in the crystal structure. The other 30% is
non-phosphorylated myo-inositol.
HPLC with chiralcolumn and NMR analyses of the TK2285 product also
identified that the enzyme produces myo-inositol-3-phosphate. Thus, we named TK2285 myo-inositol 3kinase (MI3K) [2].

Fig. 1: Superposition of overall structures of TK2285.
The structures of the substrates-complex, the productscomplex and the unliganded structure are shown inorange, green and gray, respectively. The red ellipse andthe arrow show the lid domain and its motion,respectively.

Fig. 2: Fo-Fc omit map contoured at 3 superposed on theligand binding sites of the substrates-complex (A) and theproducts-complex (B). Water molecules and Mg2+ ion are
shown as red and black spheres, respectively. Gray
broken lines show interactions regarding Mg2+ ion andmyo-inositol. A black broken line is drawn between the 3OH group of myo-inositol and the -phosphate of AMPPCP. Red characters represent the atom numbering.

References[1] T. Sato, M. Fujihashi, Y. Miyamoto, K. Kuwata, E.
Kusaka, H. Fujita, K. Miki, and H. Atomi, J. Biol.
Chem. 288, 20856 (2013)[2] R. Nagata, M. Fujihashi, T. Sato, H. Atomi, and K.
Miki, Biochemistry 54, 3494 (2015).
* mfuji@kuchem.kyoto-u.ac.jp