䠇䌝䠋㻃 䝃䝷䝕 䜳䠅䠂䠂䠂䝛 䝱䜼 䜫䜳 䝌㻃 ୯ ᶭ㛭ᠺᯕ ሒ 㻃 ࢡ よᯊ ࢡ 㸝 ㅨ 㸞 ୯ ᶭ㛭 ኬ㜨ኬᏕኬᏕ㝌 Ꮥ ಲ ක ᠺ 㸦㸣ᖲᠺ 1㸬ᖳ 10 ᮆ ࡄ ࢡ ධమ ᴏゝ ᑊࡌ ᴏ 㐅ᤎ Ἓ ᴣさ ࡗ Ṟ ሾ ᮮ ㅨ ࢰ ࢠ ࡗ 㸡ᵋ ᶭ よᯊ ࡖࡒࠊ ይ Ⳟ ࢰ ࢠ ࡗ ኬ㔖 ᬏよᯊ ຝ ࡂ ࡒ 㸡୯ ᶭ㛭 ᪃ス ࢡ ධమ Ὡ ࡊࡗࡗ㸡 ᣈⅤ ᖲ ࡊ ᵋ ᶭ よᯊ 㐅 ࠊ ᵾ よᯊᩐ 㐡ᠺࡊ ࡀ 㸡ࡈ ᚋ ࡒᠺᯕ セ ࡒ ᴏ ࡗࡄ ງ ງ ࡖ ࠊᵋ ᶭ よᯊ ᠺᯕ ࡊ 㸡ḗ ᣪࡅ ࡆ ࡀ ࠊ A. Ꮥ㛭 ࢰ ࢠ A-1. Ț ࢡ ࢰ ࢨࢪ ᠺ㓕 ᅒ A-1 㸯 Ț-ࢡ ࢰ ࢨࢪ ᠺ㓕 (GCS) 㸡ࢡ ࢰࢲ ᠺࡌ 㝷 ᚂ ṹ㝭 ཬᚺ㸝ࢡ ࢰ 㓗㸠ࢨࢪ 㸞 り፳ࡊ㸡మහ ࢡ ࢰࢲ ಕࡖ ࠊࡊ ࡊ㸡ᐞ ཋ 㸡ࢡ ࢰࢲ 㓗 ࢪ ࢪ㜭ᚒࢨࢪ ᝇ ࡌ ࡆ ࡖ ኣ ᛮ ᚋࡊ ࠊኬ Ⳟ ᮮ GCS ᵋ 㐼 ࢡ 々 మ ᬏᵋ Ử ࡊࡒ ᯕ㸡ᚉᮮ 㒼า ῼ ࡖࡒࢨࢪ ᵕᘟ ཬᚺᶭᵋ ᥆ ྊ ࡖࡒࠊGCS Ὡᛮ ຝᯕ ᢒโࡌ スゝ ࡀ ࠉ ຝ ᢘᐞ ཋ ᢘ 㛜 ࡗ ᙽࡈ 㸡 ぞ㜴ᐐ モ㥺 㛜ጙࡊ ࠊ A-2. 3- ࢨ㓏㓗 ࢣ ࢭ㸯 ᑺ 㸡 㓗 ㅨ ஸ㐅 ࡋ ࢢ మ ࢨ ࢨࢪ ࡊ ណㆉ㝸ᐐ ᣅࡂࡒ 㸡 ୯ࢢ మ ᗐ ࢰ ࡌ ᚪさ ࠊ3- ࢨ㓏㓗 ࢣ ࢭ 㸡 3- ࢨ㓏㓗 ࢬ 㓉㓗㸝 ࡍ ࢢ మ㸞 Ề ཬᚺ り፳ࡌ 㓕 㸡 ᑺ チ ࡊ ࡈ ࠊᮇ㓕 ᬏᵋ 㓕 NADH ࡊࡒ 㓕 ࡗ Ử ࡊࡒࠊ A-3. ᆵࢬ ࢬ ࢭ (ᅒ A-3)㸯 D-ࢬ 㸡୯ᯙ NMDA ࢬ ࢰ ࡌ ࢥ ࢪ 㸡 㧏ḗᶭ ࡖ 㔔さ ᙲ ᯕࡒࡊ ࠊࢬ ࢬ ࢭ (SerR) L-ࢬ D-ࢬ ᠺࡌ 㓕 㸡 ᅂ㸡ฦ 㓕ẍ SerR ᬏᵋ Ử ࡊࡒࠊࡐ Ὡᛮ㒂న 㸡り፳ᇱ ࢨ ࢰ B6 㛣 D- ᯣᶣࡊࡒಞ㣥ᵋ ᣚࡖ ࡒࠊ ࡒ㸡ATP open-closed ᵋ ን 㐅ࡌ ࡆ SerR Ὡ ᛮ ࡌ ࡆ ࡖࡒࠊࡆ ሒ 㸡SerR 㜴ᐐ ୯ ንᛮ ᝀ ᑊࡌ ࡊ 㛜 ࡌ 㐠 㛜ࡄࡒࠊ A-4. Ⳟ ᢘ ฝ ࢪ ࢰ (ᅒ A-4)㸯 㝌හវ ཋᅄⳞ ᢘ ᅏ㞬 Ⳟ 㸡 ᢘ ฝ ࢪ ࢰ ࡌ ࡆ ࡖ ኣ ᛮ ࡌࠊࡆ ฝ 㸡 አ ᆵ OprM㸡 አ හ 㛣 న ࡌ MexA㸡 හ ᆵ మ MexB 3 ࡗ ࡖ ᵋᠺࡈ ࠊ ᅂ㸡OprM MexA ᬏᵋ ࡊࡒࠊOprM 㔖మ አ ࡌ ș 㒂ฦ 㛣㝵 ฝࡌ Ș 㒂ฦ 㸡 ὕ ᙟᠺࡊ ࡒࠊMexA 3 ࡗ 㸡OprM MexB ࡌ ฦᏄࢠ ᵋ ࡖࡒࠊࡆ ᵋ ฝ 㜴ᐐ 㛜 ࡀ 㸡ᑛ㔖 ష ᑛ ຝᯕ ᙽ ࡀ ࡓࡄ ࡂ㸡ᚉᮮ ᢘ ྊ 㸡ኣࡂ Ⅴ ᙽࡈ ࠊ Ț ࢡ ࢰ ࢨࢪ ᠺ㓕 ᆵࢬ ࢬ ࢭ OprM + MexA 々 మ (A-4) (A-1) (A-3) (MexB ᵋ ࡎࡒ) A-5. ࡐ 㸯 ࢦ ࢯ ᆵ ฦᒪ㙈 㓗 ᇱ 㓕 ࢲ 㸝 㸞 々 మ㸡 ࢨ Ⳟ ࢰẐ Ia ᠺฦ㸡ࢬ ࢪⳞࢪ ࢥ ࢭ㸝ȘẐ 㸞 㸡ᑺ㓗㓗 㓕 ࢭ㸝チ 㸞 㸡 ࢪ ࢯ ࢰ ࢠ Dsg1 ࡗ 㸡ᵋ ᶭ よᯊ ࡖࡒࠊ
B. B-1. L- ( B-1) L- B-2. NAD ( B-2) (GluDH) GluDH GluDH GluDH NAD NADP GluDH B-3. 1 O- 85-100 O- B-4. 2- -5- -trna L- (B-1) NAD (B-2) C. C-1. N- -L- ( C-1) N- - NADPH N- -L- NADPH NADPH N- -L- L- C-2. 2- ( C-2) (S)-2- (S2-CPA) 2- (2-CAA) NADPH 2-CAA S2-CPA
C-3. (R-CN) (R-CHN-OH) C-4. N- N- -L- (C-1) 2- (C-2) D. D-1. D-2. L- L- 10% D-3. L- 17 4 17 10 14 4 17 3 (1) PDB 1 45 118 (2) PDB 1 (3) PDB 1 2 0 1 42 135 17 4 17 10 14 4 17 3 46 225 (1) 17 4 17 10 9 0 14 4 17 3 48 4
(2) (3) (4) 17 4 10 17 14 4 17 3 26 DNA 1-B-1 DERA N- -L- 1-C-1 heterofructosaccharide 1-B-2 DERA 1-B-4 L- 1-B-1 N- N- -L- N- -L- 1-C-1 AIST 1-B-3 4-(2)-(3) 1) 15 14 10 - - -5- DNA 6-N- -3-2- 2) 16 15 16 - -1,6-4- - GDP- D- GMP 17 3) N- -, - 2- -5-3- D-
1) N N N N 2) Streptomyces 3) 4) (1) (2) (3) (3) 5) ab initio ACORN, SHARP, warp 1.9 Å ab initio 1.6 Å directed evolution 100 17 X 16 1) 2) 3) 4) 10 3 16 17 1) 14, 15, 16 17
16 17 16 17 (17 ) 1) 2) 3) SPring-8 PF X SPring-8 4) 5) (http://www.bio.sci.osaka-u.ac.jp/~rmasui/p3k/p3kmeta_toppage.html) 10 14 15 16 17 307 342.6 259 258 1166.6
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