Crystal Structure and Product Analysis of an Archaeal myo-Inositol Kinase Reveal Substrate Recognition Mode and 3-OH Phosphorylation

Abstract

The TK2285 protein from Thermococcus kodakarensis was recently characterized as an enzyme catalyzing the phosphorylation of myo-inositol. Only two myo-inositol kinases have been identified so far, the TK2285 protein and Lpa3 from Zea mays, both of which belong to the ribokinase family. In either case, which of the six hydroxyl groups of myo-inositol is phosphorylated is still unknown. In addition, little is known about the myo-inositol binding mechanism of these enzymes. In this work, we determined two crystal structures: those of the TK2285 protein complexed with the substrates (ATP analogue and myo-inositol) or the reaction products formed by the enzyme. Analysis of the ternary substrates-complex structure and site-directed mutagenesis showed that five residues were involved in the interaction with myo-inositol. Structural comparison with other ribokinase family enzymes indicated that two of the five residues, Q136 and R140, are characteristic of myo-inositol kinase. The crystal structure of the ternary products-complex, which was prepared by incubating the TK2285 protein with myo-inositol and ATP, holds 1d-myo-inositol 3-phosphate (Ins(3)P) in the active site. NMR and HPLC analyses with a chiral column also indicated that the TK2285 reaction product was Ins(3)P. The results obtained here showed that the TK2285 protein specifically catalyzes the phosphorylation of the 3-OH of myo-inositol. We thus designated TK2285 as myo-inositol 3-kinase (MI3K). The precise identification of the reaction product should provide a sound basis to further explore inositol metabolism in Archaea.

Identification of a pyrophosphate-dependent kinase and its donor selectivity determinants

“…On the other hand, the values of K m and k cat toward myo -inositol are 12 ± 2 mM and 10.8 ± 0.3 s −1 , respectively. The K m value is 40 times higher than that of MI3K_TK (0.30 ± 0.03 mM) 19 . This raises the possibility that the genuine phosphate acceptor of TM0415 is a compound other than myo -inositol: for example, compounds related with the myo -inositol metabolic pathway that is composed of enzymes encoded by the TM0411–TM0416 operon in T. maritima , such as 2-keto- myo -inositol, diketo-inositol, 5-keto- l -gluconate, and d -tagaturonate (Supplementary Fig.…”

“…4a ), whereas D12, G26, Q136, R140, and D219 in MI3K_TK interact with all six hydroxyl groups (Fig. 4b ) 19 . As shown in Fig.…”

“…A PPi-dependent kinase belonging to the ribokinase family was identified based on structural similarity to a myo -inositol 3-kinase from the hyperthermophilic archaeon Thermococcus kodakarensis (MI3K_TK), which is an ATP-dependent member of the ribokinase family 19 , 20 . A Dali search 21 with the substrate-complex structure of MI3K_TK (Protein Data Bank (PDB) ID 4XF7) showed that the structure is the most similar to the unliganded structure of TM0415 from the hyperthermophilic bacterium Thermotoga maritima (PDB ID 1VK4; root-mean-square (RMS) distance 2.2 Å for 254 Cα atoms out of 283 Cα atoms of TM0415; Fig.…”

“…4 m yo -inositol-binding mode. a , b The myo -inositol-binding sites in the PCP-complex of TM0415 (cyan) and the substrate complex of MI3K_TK (green, PDB ID 4XF7 19 ). Dotted lines represent the interactions between myo -inositol and the residues.…”

Perdeuterated and 13C-enriched myo-inositol for DNP assisted monitoring of enzymatic phosphorylation by inositol-3-kinase

“…26, 27 In some archaea and plants enzymatic phosphorylation at the 3 position of soluble myo-inositol also occurs. 28, 29 Interestingly, small peptides have been found to catalyse similar stereo-specific phosphorylation. 30 Here, we use the TK2285 gene product from Thermococcus kodakarensis (myo-inositol- 3-kinase, EC 2.7.1.64) as an easily accessible system to illustrate the spectral changes that occur on phosphorylation of inositol containing substrates, and the advantages of DNP-enhanced NMR.…”