Photon Factory Activity Report 2017 #35 (2018)

BL-1A, BL-5A, AR-NE3A/2017G696

Structure study on a new pyrophosphate-dependent kinase
Masahiro Fujihashi*, Ryuhei Nagata, and Kunio Miki
Department of Chemistry, Graduate School of Science, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan

1 Introduction
Most kinases use ATP as a phosphate donor, while a few
kinases use pyrophosphate (PPi) instead. Three types ofPPi-dependent kinase have been reported so far. However,mechanisms of PPi-specific recognition in these enzymes
remain unclear.
Here, we identified a new type of PPi-dependent kinase,
TM0415, and determined its crystal structures. The
structures revealed important residues for the PPi-specificrecognition, and these residues enabled us to findadditional PPi-specific kinases from the genome database.
2 Experiment
Identification of TM0415 was performed using the Daliserver [1]. An ATP-dependent myo-inositol 3-kinase from
Thermococcus kodakarensis (MI3K_TK, PDB ID 4XF7)[2] was used as a query structure. Donor specificity ofTM0415 was investigated by LC-MS.
Methylenediphosphonic acid (PCP) was used as a PPi
analog in crystallization. 2 or 20 mM ammonium sulfate
was added for preparing a PCP complex or a sulfate ioncomplex, respectively. Diffraction data sets were collected
at beamlines of BL-1A, 5A, and NE3A. The phases weredetermined by molecular replacement with the atomic
coordinates of an unliganded structure of TM0415 (PDBID 1VK4).
The additional PPi-specific kinases were discovered
using a BLAST search. PPi-dependent activity ofcandidate PPi-dependent kinases was analyzed by LC-MSand a malachite green assay.
3 Results and Discussion
The Dali server showed that the substrate-complex
structure of MI3K_TK is the most similar to theunliganded structure of TM0415 from Thermotoga
maritima. TM0415 has been annotated as a carbohydrate
kinase and thought to be involved in myo-inositol
metabolism. However, this enzyme exhibited no ATPdependent kinase activity toward various carbohydrates,
including myo-inositol [3,4]. Structural comparison
between TM0415 and MI3K_TK showed that F221, R232,
and M266 of TM0415 occupy a potential ATP-bindingpocket (Fig. 1A). We investigated the TM0415 activity
using ATP, ADP, or PPi as the phosphate donor. This
investigation revealed that TM0415 phosphorylates myoinositol using PPi but neither ATP nor ADP.
The PCP-complex structure of TM0415 was determinedto elucidate the PPi-binding mode. This structure revealed
residues involved in PPi binding (Fig. 1B). R229 is locatednear PCP, but its side chain was disordered in this structure.
In a sulfate-ion-complex structure, which possesses asulfate ion in the PPi-binding site instead of PCP, R229
interacts with the sulfate ion (Fig. 1C). This suggests thatR229 recognizes PPi in the reaction because the sulfate ion
is well superposed on one of the phosphoryl groups of PCP.
Indeed, a R229A mutation resulted in a drastic decrease in
the activity. Three of the PPi-binding residues (K171, R229,and R232) are not conserved in homologous ATP/ADPdependent kinases, including MI3K_TK. This suggeststhat these three residues contribute to the PPi-specificrecognition. Notably, R232 is also expected to prevent ATPbinding as described above. We considered the fiveresidues (K171, F221, R229, R232, and M266) as key
residues for the PPi-specific recognition.
We sought additional PPi-dependent kinases from thegenome database based on conservation of the five key
residues. Fifty candidates of PPi-dependent kinases werefound. PPi-dependent activity of four candidates wasinvestigated. Two of them exhibited PPi-specific kinase
activity toward myo-inositol, suggesting that the five key
residues can be collectively used as a signature to discoverunidentified PPi-dependent kinases [5].

Fig. 1: Phosphate donor binding site of TM0415 (green).
(A) Superposition of the substrate complex of MI3K_TK(cyan) on the unliganded TM0415. (B,C) The PCP- orsulfate-ion-complex structure.
References[1] L. Holm, et al. Nucleic Acids Res. 38, W545–9 (2010).
[2] R. Nagata, et al. Biochemistry 54, 3494–503 (2015).
[3] I. A. Rodionova, et al. J. Bacteriol. 194, 5552–63 (2012).
[4] I. A. Rodionova, et al. Environ. Microbiol. 15, 2254–66(2013).
[5] R. Nagata, et al. Nat. Commun. 9, 1765 (2018)* mfuji@kuchem.kyoto-u.ac.jp