ドージンニュース_134号.indd

Size: px

Start display at page:

Download "ドージンニュース_134号.indd"

いおりたもん
5 years ago
Views:

1 Generation of Artifi cial Enzymes in Directed Evolution. Advances in methods for conformational prediction, structural analysis and site-directed mutagenesis of proteins and peptides have contributed to the understanding of their structure and function. However, with the exception of a few successes, the generation of practical functional molecules solely by rational design remains a difficult challenge. The aim of our study is to investigate molecular design relying on evolutionary processes, called as directed evolution, to generate a novel class of biofunctional molecules. This evolutionary approach consists of three steps; 1) constructions of protein/peptide libraries based on structural information, 2) expressions of the libraries on phage particles, and 3) selections with investigator-imposed selective pressures. In this work, we study on generation of artificial 20 DNA DNA DNA DNA biocatalysts by immune system that uses the evolutionary processes to give receptor-like molecules. As a natural enzyme binds the transition state of the chemical reaction to lower the activation energy, immunization with a putative transition-state analog (TSA), with the expectation that the induced antigencombining site could be both geometrically and electronically complementary to the transition-state, provides catalytic antibodies. We have succeeded to generate antibodies catalyzing highly stereo and regio-specific hydrolyses with immunization of phosphonate transition-state analogs. Furthermore, to evolve catalytic antibodies toward higher catalytic activity, phage-displayed antibody (Fab) libraries were constructed to screen antibodies with optimized differential affinity for the transition state relative to the ground state. Recently, we have demonstrated a new strategy for generating catalytic antibodies, namely, by the generation of antigen-combining site that function as an apoprotein for binding functionalized components, called holoabzyme. Replacement of the functionalized components enables a single antibody to catalyze multiple reactions, -elimination, decarboxylation and aldol reactions. Catalytic antibodies have great potential for generating novel catalysts as well as for providing opportunities to examine the evolutionary dynamics of enzymes. 1) 2) 3) 4) 5) 6) 7) 2 1 H V H, D, J H Lκ Vκ, Jκ V H 100 D 1

2 1 15 J H 5 Vκ Jκ 5 B B 10 8 B 1 B 2 3 B 3 1 1, (S ) (P) (S) (S ) (DG uncat ) E) (S ) (DG TS ) (DG cat ) (S) DGS (DGcat) (K TS ) (K m ) K m /K TS = k cat /k uncat 2

3 3 A A B (1) (2) 4 11 (2) (3) KLH 12 (2) 6 6D , 11 ph (3) C10 K TSA = μm k cat = min -1 6D9 1 (min -1 ) (min -1 M -1 ) 3

4 5 k cat = min -1 K TSA = μm 5A (k cat /k uncat ) K m /K TSA 6D9 8D11 4B5 9C10, 3G6 5 k cat /k uncat K m /K TSA 5B 6D9 X 13) 6D9 6 2 Tyr (H58) Trp (H100i) 6 6D9 X Fab L :,H ;, His(L27d) His(L27d) Ala 14) 6D9 L CDR-1 His(L27d) 15, 16) B , 19,

4.2 6D9 19, 20 2 1 3 2 4 4 3 X 6D9 L CDR1 His L27d 6 13,14)

5 4.2 6D9 19, X 6D9 L CDR1 His L27d 6 13,14) His (6D9) (8Hg) His (L27d) Ser(L27e) Tyr ( 6) His (L27d) His (L27d) Tyr L27e 2 Tyr L27e Ser Ser (AGT) Tyr (TAT, TAC) D9L CDR1 2 L CDR1 5

6 8 5.1 (5) (6) (7) 22) (6) 86% 5.2 (10) (8) - (9) 4) (11) (12) 5) L- D

7 9 ( 9) 7) (13) 4 4 (14) Michaelis- Menten 4 k cat = min -1, K m = 6.6 μm, k cat / k uncat = 2700 (15) ) Holoabzyme 6.1 B B 11 (16) (16) 2 p- N- 2 (17) (18) (19) (20) - (23) (24) - (19) ( 11) 6.2 (16) KLHBSA KLH- (KLH-1) Balb/c (16) (16) (17) (18) 22 2 (25E2, 27C1) (18) (19) (20) (18) 198 (25E2) 109 (27C1) (19) (25E2) (27C1) (20) (25E2) (27C1) (16) 7

8 NHAc AcHN X AcHN 2N P H 18:X=H 19:X=NH 2 20:X=SH 24 AcHN b- 2N 17 18,19,20 X 21:X=0,22:X=NH 2N H 2N F N 25 2N H 19 2N H 2N 19 2N H (25E2: K d = 27 nm; 27C1: K d = 8.7 nm) ( - ) (16) 6.3 (24) - 25E2 - (23) (25) - 27C1 - K m 24 = 122 μm, K m 23 = 864 μm, k cat = 0.89 min -1 k cat /K m 23/k uncat = C-C I (19) I I I - (19) I - (28) 11 27C1 K m 19 = 6.9 mm, 8

9 K m 28 = 2.1 mm, k cat = 1.29 min -1 k cat /K m 28/ k uncat = E2, 27C REFERENCES 1) Schultz P. G., Lerner R. A., Science, 1995,269, ),,1999, 35,695. 3) Teraishi K., Saito M., Fujii I., Nakarura H., Tetrahedron Lett.,1992, 33, ) Fujii I., Lerner R. A., Janda K. D., J. Am. Chem. Soc.,1991,113, ) Tanaka F., Kinishita K., Tanimura R., Fujii I., J. Am. Chem. Soc.,1996,118, ) Tanaka F., da M., Fujii I., Teterahedron Lett., 1998, 39, ) Iwabuchi Y., Miyashita H., Tanimura R., Kinoshita K., Kikuchi M.,Fujii I.,J. Am. Chem. Soc., 1994,116,771. 8) Iwabuchi Y., Kurihara S., da M., Fujii I., Teterahedron Lett.,1999, 40, ) Kurihara S., Tsumuraya T., Suzuki K., Kuroda M., Liu L., Takaoka Y., and Fujii I., Chem. Eur. J., 2000,6, ) Kakinuma H., Fujii I., Nishi Y., J. Immu. Method,2002, 269, ) Miyashita H., Karaki Y., Kikuchi M., Fujii I., Proc. Natl. Acac. Sci. USA, 1993, 90, ) Fujii I., Tanaka F., Miyashita H., Tanimura R., and Kinoshita K., J. Am. Chem. Soc., 1995,117, ) Kristensen1., Vassylyev D. G., Tanaka F., Morikawa K., Fujii I., J. Mol. Biol., 1998, 281, ) Miyashita H., Hara T., Tanimura R., Fujii I., J. Mol. Biol., 1997, 267, ) da M., Ito N., Tsumuraya T., Suzuki K., Sakakura M., and Fujii I., J. Mol. Bi ol., 2007, 369, ) Tsumuraya T. and Fujii I., Bull. CHem. Soc. Jpn., 2008, 81, ) Hoogenboom H. R., Trends Biotechnol., 1997,15, ) Fujii I., Fukuyama S., Iwabuchi Y., Tanimura R., Nat. Biotechnol., 1998,16, ) Takahashi N., Kakinuma H., Liu L., Nishi Y., Fujii I., Nat. Biotechnol., 2001, 19, ) Takahashi-Ando N., Kakinuma H., Fujii I., Nishi Y., Journal of Immunological Methods, 2004, 294, 1. 21),,,2009, ) Ikeda S., Weinhouse M., Janda K. D., Lerner R. A., Danishefsky S. J., J. Am. Chem. Soc., 1991,113, ) Ishikawa F., Tsumuraya T., Fujii I., J. Am. Chem. Soc., 2009,131, (Ikuo Fujii) Tel: Fax: fujii@b.s.osakafu-u.ac.jp HP: () ( )

DojinNews_Vol.134_#699B44.eps

DojinNews_Vol.134_#699B44.eps Review 1 5 10 Topics on Chemistry Bicyclic peptides 12 14 C16-PEG-SAMs Amino-EG 6 -hexadecanethiol, hydrochloride A505 10 mg 45,000 Carboxy-EG 6 -hexadecanethiol C463 10 mg 32,000 Hydroxy-EG 6 -hexadecanethiol