Biochemical characterization of the novel rice kinesin K23 and its kinetic study using fluorescence resonance energy transfer between an intrinsic tryptophan residue and a fluorescent ATP analogue

• Published in: Journal of biochemistry (Tokyo) Vol. 149; no. 5; pp. 539 - 550
• Main Authors: Umezu, Nozomi, Hanzawa, Nobue, Yamada, Masafumi D, Kondo, Kazunori, Mitsui, Toshiaki, Maruta, Shinsaku
• Format: Journal Article
• Language: English
• Published: England 01.05.2011
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Animals; Fluorescence Resonance Energy Transfer; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Isoenzymes - genetics; Isoenzymes - metabolism; Kinesin - chemistry; Kinesin - classification; Kinesin - genetics; Kinesin - metabolism; Molecular Sequence Data; ortho-Aminobenzoates - chemistry; ortho-Aminobenzoates - metabolism; Oryza - metabolism; Phylogeny; Plant Proteins - genetics; Plant Proteins - metabolism; Tryptophan - chemistry; Tryptophan - metabolism
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvr012

We previously demonstrated that the rice kinesin K16, which belongs to the kinesin-7 subfamily, has unique enzymatic properties and atomic structure within key functional regions. In this study, we focused on a novel rice plant kinesin, K23, which also belongs to the kinesin-7 subfamily. The biochemical characterization of the K23 motor domain (K23MD) was studied and compared with the rice kinesin K16 and other related kinesins. K23 exhibits ∼45-fold (1.3 Pi mol(-1) site mol(-1) s(-1)) lower microtubule-dependent ATPase activity than conventional kinesins, whereas its affinity for microtubules is comparable with conventional kinesins. MgADP-free K23 is unstable compared with the unusually stable MgADP-free K16MD. The enzymatic properties of K23MD are somewhat different from those of K16. We used a fluorescent ATP analogue 2'(3')-O-(N'-methylanthraniloyl)-ATP (mant-ATP) for the kinetic characterization of K23. The fluorescence of mant-ATP was not significantly altered during its hydrolysis by K23. However, significant fluorescence resonance energy transfer (FRET) between mant-ATP and W21 in the motor domain was observed. The kinetic study using FRET revealed that K23 has unique kinetic characteristics when compared with other kinesins.