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堀江 暁*; 富田 武郎*; 斉木 朝子*; 河野 秀俊; 高 ひかり*; 峯木 礼子*; 藤村 務*; 西山 千春*; 葛山 智久*; 西山 真*
Nature Chemical Biology, 5(9), p.673 - 679, 2009/09
被引用回数:41 パーセンタイル:67.64(Biochemistry & Molecular Biology)Although the latter part of the lysine biosynthesis, conversion of -aminoadipate (AAA) to lysine, of is similar to that of the arginine biosynthesis, enzymes homologous to ArgA and ArgJ are absent in the Thermus pathway. Since ArgA and ArgJ are known to modify the amino group of glutamate to avoid intramolecular cyclization of intermediates, their absence suggests an alternative N-modification system in the pathway. We reconstituted the conversion of AAA to lysine, and revealed that the amino group of AAA is modified with the -carboxyl group of C-terminal Glu54 of a small protein, LysW, and the side-chain of AAA is converted to the lysyl side-chain in LysW-attached forms. Lysine is subsequently liberated from LysW/lysine-fusion. Recognition of the acidic globular domain of LysW by biosynthetic enzymes indicates that LysW acts as a carrier protein or protein scaffold of the biosynthetic enzymes in this system. This study reveals the previously unknown function of a small protein in the primary metabolism.
吉田 彩子*; 富田 武郎*; 河野 秀俊; 伏信 進矢*; 葛山 智久*; 西山 真*
FEBS Journal, 276(11), p.3124 - 3136, 2009/06
被引用回数:14 パーセンタイル:29.62(Biochemistry & Molecular Biology)Crystal structures of the regulatory subunit (TtAK) of Thr-sensitive aspartate kinase (AK; EC 2.7.2.4) from Thermus thermophilus were determined at 2.15 ; in the Thr-bound form(TtAK-Thr) and at 2.98 ; in the Thr-free form (TtAK-free). Although both forms are crystallized as dimers, the contact surface area of the dimer interface in TtAK-free (3,200 ) is smaller than that of TtAK-Thr (3,890 ). Sedimentation equilibrium analyzed by ultracentrifuge revealed that TtAK is present in equilibrium between a monomer and dimmer and Thr binding shifts the equilibrium to dimer formation. In the absence of Thr, outward shift of b strands near the Thr-binding site (site 1) and concomitant loss of the electron density of the loop region between 3 and 4 near the Thr-binding site are observed. Based on the crystal structures, the regulatory mechanism by Thr is discussed. TtAK has higher thermostability than the regulatory subunit of AK (CgAK) with a difference in denaturation temperature () of 40C. Comparison of the crystal structures of TtAK and CgAK showed that the well-packed hydrophobic core and highproline content in loops contribute to the high thermostability of TtAK.