醣苷水解酵素家族29與64的反應機制與重要催化殘基之鑑定

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National Chiao Tung University

Department of Applied Chemistry

Ph.D Thesis

29

64 Catalytic mechanistic and identification of essential residues of

Glycohydrolase in family GH-29 and GH-64

(Sheng-Wen Liu)

(Dr. Yaw-Kuen Li)

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NMR KK

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glycobiology [glycoconjugates (glycoproteins) (glycosphingolipids) (proteoglycans) ] (lipopolysaccharides) (biosynthesis) (Glycoside hydrolases) (Glycosidic bond) (Organelle) (Substrate) (D- L-form )

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(retention configuration) (inverting configuration) A, B, H, Lewis L-(Apoptosis) Fucosidosis -1,3/6--L- ( -L-Fucosidase, EC 3.2.1.51) -1,3-(Laminaripentaose-producing -1,3-glucanase, EC 3.2.1.39) (Transition state) 3D

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29

64

29

-L--L- (Human -L-fucosidase h-Fuc) 29 (EC 3.2.1.51) -L-pET22b(+) BL21(DE3) pH 6.0 LBA IPTG (SP Q) G-75 95

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h-Fuc h-Fuc pH 5.0 Km kcat 0.105 mM 48.6 s-1

h-Fuc (Km = 0.43 mM kcat = 16.3 sec-1) h-Fuc (kcat/Km)

12 pH 4.5 pH 6.5 pH 3.0 7.5 1_H-NMR _h-Fuc _pNPF / h-Fuc h-Fuc fucosyl-enzyme h-Fuc SN1

Br nsted relationship Br nsted constant log kcat lg -0.13 log

kcat/Km lg -0.27 (fucosylation) Tm-Fuc h-Fuc -pNPF D225G E289G kcat/Km 20,000 450 pH pH-profile D225G E289G D225G E289G kcat 6 24

-fucosyl_azide -fucosyl_azide E289G

pNPF

-h-Fuc

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-L-64

DIC-108

-1,3-Streptomyces matensis DIC-108

-1,3-(Laminaripentaose-producing -1,3-glucanase, LPHase) 64 (EC 3.2.1.39) (single-displacement) -1,3 laminaripentaose 24 PCR LPHase pRSET_A BL21(DE3) IPTG (SP Q CM) 95 40 kDa (curdlan) LPHase 55 0_{C pH 7.5~8.5} _pH _3.5 _11.0 1_H-NMR _LPHase SN2 LPHase laminaripentaose p-NLPG 0C 50 mM (pH 7.1)

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LPHase Km kcat 1.6 mM 8.1 s-1 LPHase

pH-profile (bell-shaped curve) LPHase

pKa1 pKa2 6.0 10.1 GH-64

Asp143 Glu154 Asp170 Asp376 Asp377

p-NLPG

E154G D170G kcat/Km

1,700~2,000 E154G

D170G D170G

-laminaripentaosyl_azide LPHase

( ) Asp170 _Glu154 _LPHase

Arg115 _pH Tyr232 X-ray LPHase (SeMet-LPHase) (MAD) 1.62~2.3 Å LPHase 6 - 18 - 1 - - / (open groove) Asp170 _Glu154 SN2 -1,3 laminaripentaose 8 ± 0.5 Å

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Catalytic mechanistic and identification of essential residues

of Glycohydrolase in family GH-29 and GH-64

Student : Sheng-Wen Liu Advisor : Dr. Yaw-Kuen Li

Department of Applied Chemistry

National Chiao Tung University

Part I : Expression, mutagensis, mechanistic study and identification of

essential residues of human -L-Fucosidase in family GH-29

ABSTRACT

Fucosylated glycoconjugates play critical roles in various biological processes, but the limited availability of -L-Fucosidase hampers investigations at a molecular level. This thesis aims to clone and express human Fucosidase .

A gene from human encoding -L-fucosidase (Fuc) was cloned into pET22b(+) plasmid. Protein was successfully expressed in E. coli BL21 (DE3). After applying a series of ion-exchange and gel-filtration chromatography purification steps, recombinant h-Fuc with 95% homogeneity can be obtained. The molecular weight of the enzyme was analyzed by SDS-PAGE to be about 50 kDa. The optimal temperature of h-Fuc was 70 0_C

and the optimal pH was 4.5 and 6.5. The h-Fuc was stable at pH 3.0~6.0, while it was unstable at 75 0_{C or high. The catalytic mechanism of h-Fuc was confirmed a S}

N1-like with

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spectroscopy. Based on careful sequence alignment of GH-29 enzymes and extensive structure analysis of the close homologues of h-Fuc, nine residues of glutamate and aspartate in h-Fuc were selected for mutagenic study to determine the essential residues. Among the mutants, D225N, E289Q and E289G lost catalytic activity significantly; their kcat values are 1/5,700, 1/430 and 1/340, respectively, of that of the wild-type enzyme. Based on kcat values of aryl- -L-fucopyranosides catalyzed by wild-type -L-fucosidase, a

small Brønsted constant, lg = −0.13, was derived, indicating that the rate-limiting step of

the enzymatic reaction is fucosylation. The Brønsted plot for kcat/Km for the E289G mutant

is linear with lg = −0.93, but that for kcat is biphasic, with lg for poor substrates being

−0.88 and for activated substrates being −0.11. The small magnitude of lg for the activated

substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter lg value for the poor substrates indicates that the

rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that Asp225 functions as a nucleophile and Glu289 as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and

1_{H-NMR spectral analysis confirmed the formation of -fucosyl azide and -fucosyl azide}

in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu289, an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/MS analysis.

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Part II Artificial gene cloning protein mutagensis and crystal structure

study and identification of essential residues of LPHase from

Streptomyces matensis DIC-108 in family GH-64

ABSTRACT

An artificial gene of laminaripentaose-producing -1,3-glucanase (LPHase) from

Streptomyces matensis DIC-108 was reconstructed by PCR and overexpressed in E. coli.

Although the polypeptide sequence of LPHase exhibits significant similarity with the catalytic domains of -1,3-glucanase of Arthrobacter sp. YCWD3 and Cellulosimicrobium

cellulans, it uniquely catalyzes the hydrolysis of -1,3-glucans to release

laminaripentasaccharide as the predominant product. The recombinant wild-type enzyme and mutants were purified to >90% homogeneity by ionic-exchange chromatography. The optimal temperature of the LPHase was 55 oC and the optimal pH was 8.5. The LPHase was stable at pH 5.0~9.0, while it was unstable at 65 0C or higher. The catalysis of LPHase is confirmed to follow a one-step single displacement mechanism by 1H-NMR spectroscopy. p-Nitrophenyl- -1,3-laminaripentaopyranoside (p-NPLPG) was synthesized to facilitate the kinetic analysis. Michaelis constant (Km) and catalytic activity were

determined with p-NPLPG and were found to be 1.6 mM and 8.1 s-1, respective. To determine the amino acid residues essential for the catalysis of LPHase, more than 10 residues including five highly conserved residues, D143, E154, D170, D376 and D377, were mutated. Among the mutants, E154 and D170 mutants, such as E154Q, E154G, D174N, and D174G, significantly lost their catalytic activity. Further investigation with chemical rescue on E154G and D174G confirmed that E154 and D170 function as the general acid and the general base in the LPHase catalysis, respectively. This study

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provided the first hand information on identification of the essential residues GH-64 through kinetic examination. Further studies on structural and enzymatic characterizations were also conducted to understand the detail catalytic mechanism of LPHase. The crystal structure of LPHase was solved to 1.62 Å resolution using multiple-wavelength anomalous dispersion methods. The LPHase structure reveals a novel crescent-like fold; it consists of a barrel domain and a mixed (α/β) domain, including six α-helix, eighteen β-sheet, and one η structure, forming a wide-open groove between the two domains. The liganded crystal structure was also solved to 1.80 Å, showing limited conformational changes. Within the wide groove, molecular modeling using a laminarihexaose as a substrate suggests roles for Glu154 and Asp170 as acid and base catalysts, respectively, whereas the side chains in active sitedemarcate subsite +5. Site-directed mutagenesis of Glu154 and Asp170 confirms that both carboxylates are essential for catalysis. Together, our results suggest that LPHase uses a direct displacement mechanism involving Glu154 and Asp170 to cleave a β-1,3-glucan into specific -pentasaccharide oligomers.

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...i

...ii

...iv

...viii

...xii

...xxiii

...xxv

29 -L-..………...…………...

2 1-1

-L- ( -L-fucose) ...2 1-2 -L- …………...5 1-2-1 -L- ……...…5 1-2-2 -L- …………...7 1-2-3 -L- ………...………....8 1-3 -L- ( -L-Fucosidase) ……...10 1-4 -L- ………...………...…..15 1-5 ………....………...…..23

………...………....…

.27

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2-2 ………..………...28 2-3 (competent cell) …………..………...28 2-4 (transformation)……….………..28 2-5 -L- ...29 2-5-1 ...29 2-5-2 -L- pET22b(+) (ligation)………...………...31 2-5-3 (sequencing)……….…...31 2-5-4 (site-directed mutagenesis)………...………..32 2-5-5 ………..………..………....33 2-5-6 ………..……….………...34 2-6 ………..………..…...35 2-7 -L-(expression)...35 2-7-1 DNA ………...………….………...35 2-7-2 -L- ……....35 2-7-3 -L- ………...……..36 2-8 -L- ..………...36 2-8-1 ..………..………...………...36 2-8-2 ……..……….………...36 2-8-3 ..……….………...37 2-8-4 HiTrap SP ………...…...38 2-8-5 HiTrap Q ………..………...38 2-8-6 G-75 ………...…....39

2-9 6-His h-Fuc D225G/N E289G/Q …….……….………...40

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2-11 ……….43 2-11-1 (LC-MASS) ……….….43 2-11-2 ………....43 2-11-3 ………...43 2-12 -L- ……….44 2-12-1 …...44 2-12-2 -L-(p-nitrophenyl- -L-fucopyranoside pNPF)...45 2-12-3 -L- ………...…...48 2-12-4 2- -L-(2-chloro-4-nitrophenyl- -L-fucopyranoside CNPF) ………..48

2-12-5 O-hydroxyl-1-(2,3-epoxypropyl)- -L-fucopyranoside (EPFP) ………...50 2-12-6 Fuconolactone ………..……….51 2-13 -L- ……….…..52 2-13-1 ………...52 2-13-2 (Thermostability)………...52 2-13-3 ………...53 2-13-4 (pH stability)………..54 2-13-5 ………...…....55 2-14 -L-...55 2-15 -L-………..56 2-16 -L- kcat Km ……...…....57 2-16-1

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-L-2-16-2

-L-kcat Km (pH-profile ) ……….…...58

2-16-3 -L-kcat Km (Br nsted plot ) ………...59

2-17 ………...…...60 2-18 -L- …...61 2-19 1H-NMR ………..62 2-20 E289G ………..63 2-21 CD (circular dichroism) …....64 2-22 ………...65

……….…...…

66 3-1 -L- …………..….66 3-2 -L- ………...68 3-3 -L- ………..…….……69 3-4 -L- ...………71 3-4-1 ……….……….71 3-4-2 ………..72 3-5 -L- …..73 3-5-1 -L- …….….74 3-5-2 -L- ………...……...75 3-5-3 -L- ………...…………...76 3-5-4 -L- ………...………...76 3-5-5 -L- ……...78

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3-6 - -L- (aryl- -L-fucopyranosides) ..79 3-6-1 (substrate specificity) ………...……...81 3-7 ………...82 3-8 -L- ………...85 3-8-1 -L-………...86 3-8-2 (Common intermidiate) ………88 3-9 -L- ………...90 3-10 -L-………...94 3-11 ……….103 3-11-1 ……….103 3-11-2 ……….106 3-12 -L- ……….110 3-13 -L- Km …111 3-14 -L- …….112 3-15 -L- ……...117 3-16 Glu289 ……..…120 3-17 Glu289 ………...123 3-18 E289G ……….125

3-19 E289G Br nsted plot ……….129

3-20 E289G ……….132

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64 DIC-108

-1,3-..………..

138 5-1 - ...138 5-2 -1,3- ………...………..…..142 5-3 -1,3- ( -1,3-Glucanase) …………..…146 5-4 -1,3- ………...………....147 5-5 (Curdlan) ………...………..152 5-6 -1,3- ………...………....153 5-7 ………...………...158 5-8 Streptomyces matensis -1,3-(LPHase) ………...……….……….161 5-9 ……….………...…164

……….………...…….

165 6-1 ………..…………..…………...….165 6-2 -1,3- ………...…...166 6-2-1 ……..…..………....166 6-2-2 (oligonucleotide template) ...………...166 6-2-3 ……….………...169 6-2-4 -1,3- …………..…170 6-3 (competent cell) ………..………....171

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6-5 (sequencing) ……….………...172 6-6 -1,3- ..173 6-6-1 -1,3- pRSET_A (ligation)………...………....173 6-6-2 (site-directed mutagenesis)……….174 6-6-3 ...………..…...…………..…..175 6-6-4 ………..………..….…...177 6-7 ………..…...………...178 6-8 -1,3-(overaxpression) ………...….………...178 6-8-1 DNA ………...………...178 6-8-2 ………....178 6-8-3 ………...179 6-9 -1,3- ………..180 6-9-1 ………..……….………...180 6-9-2 ……..………...180 6-9-3 HiTrap SP ………...…...181 6-9-4 HiTrap Q ………...181 6-9-5 CM SepharoseTM ………...182 6-9-6 SDS-PAGE (homology) ………...182 6-10 ………....183 6-10-1 (LC-MASS) ………..183 6-10-2 ………....183 6-10-3 …...183 6-11 -1,3-………...184

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6-11-2 ………....185 6-12 Laminaripentaose ( -D-1,3- ) …...185 6-13 -1,3- 186 6-13-1 ………....186 6-13-2 - -D-1,3-(p-nitrophenyl- -D-1,3-pentaglucopyranoside) ...187 6-13-3 O-hydroxyl-1-(2,3-epoxypropyl)- -1,3-D-pentaglucopyranoside (EPLPG) ………...191 6-13-4 1-O-(2-difluoromethyl-4-nitrophenyl)- -1,3-D-pentagluco-pyranoside (2DFMNLPG) …...193 6-14 -1,3- …...195 6-14-1 ……….…...195 6-14-2 (Thermostability) ………...195 6-14-3 ………...196 6-14-4 (pH stability)………..197 6-14-5 ………...198 6-15 -1,3- kcat Km ……..199 6-15-1 -1,3-kcat Km (pH-profile ) ………....200 6-15-2 ………....200 6-16 ………...201 6-17 -1,3-D170G azide ………...…202 6-18 -1,3-D170E ………...…....203

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D170C …...203 6-20 CD(circular dichroism) …..204 6-21 -1,3- …………..205 6-21-1 (wild type) ………...205 6-21-2 (Selenomethionine) ………....205 6-21-3 ………....206 6-21-4 ………....206 6-21-5 ………....206 6-21-6 (Tertiary structure) ………..207

…

………...…208 7-1 -1,3-……….208 7-1-1 -1,3- ……...208 7-1-2 -1,3- ...210 7-1-3 -1,3- ………..212 7-1-4 -1,3- ………...213 7-1-5 -1,3- …..………216 7-1-5-1 ………...………..216 7-1-5-2 ……….…217 7-1-6 -1,3- 217 7-1-6-1 LPHase …………...218 7-1-6-2 LPHase ………..219 7-1-6-3 -1,3- …..220 7-1-6-4 -1,3- …………...220

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7-1-6-6 -1,3-substrate specificity ………..……….223 7-1-7 -1,3- ……...225 7-1-8 -1,3- (Laminaripentaose) ………..229 7-1-9 - -D-1,3- ……….229 7-1-10 LPHase …………...231 7-1-11 -1,3- ...235 7-1-12 -1,3- ………...238 7-1-13 LPHase …………...240 7-1-14 ……….………243 7-1-15 LPHase ………...246 7-1-16 E154D D170E ……….252 7-2 -1,3- ………...259 7-2-1 ………...……...…………...259 7-2-2 -1,3- …………..……….265 7-2-3 -1,3- ...268 7-2-4 -1,3-………..…...…...……….277 7-2-5 …...280

..………

……….………..………287

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I

…

..………..………313 II

…

..……….314 III

…

..………315 IV

…

..………317 V

…

..……….………318 VI

…

..………319 VII

…

..………..326 VIII

…

..……….…328 IX

…

..………329 X

…

..……….330 XI

…

..………332 XII

…

..………..333 XIII

…

..……….…335 XIV

…

..……….338 XV

…

..………..…339 XVI

…

..……….……340 XVII

…

..………..……….……341

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1-1 -L- ……….5

1-2 -L- ………...8

1-3 -L- ..15

3-1 h-Fuc ……..………...69

3-2 Vector NTI h-Fuc ………..71

3-3 h-Fuc ……….78 3-4 h-Fuc Km kcat ……….81 3-5 -L- Km kcat …83 3-6 NMR …...…89 3-7 h-Fuc (Ki) ……….105 3-8 h-Fuc pNPF Michaelis-Menten ………...110 3-9 h-Fuc D225G 1 M pH 6.8 ………...114 3-10 h-Fuc E289Q ……….124 3-11 E289G Km kcat ………...129 3-12 E289G 500 mM azide Km kcat………..……...129 5-1 -1,3-………...154 7-1-1 LPHase ………..213

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7-1-4 LPHase

………...224

7-1-5 LPHase …...225

7-1-6 LPHase p-NLPG

Michaelis-Menten ………....239 7-1-7 wild type E154D D170E D170C LPHase

p-NLPG Km kcat ………...253 7-2-1 7-2-12 PDB ………..276 7-2-2 3-2-15 LPHase ……….281

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1-1 L-fucose ……….…….2 1-2 Typical mammalian complex N-glycans………..………...3

1-3 ………...………13 1-4 GH-29 Thermotoga maritima -L- 3D ………...14 1-5 GH-95 Bifidobacterium bifidum -1,2-L- 3D ………...14 1-6 GH-29 T. maritime …..16 1-7 ……….17 1-8 h-Fuc Asp ……….18 1-9 ……….…..19 1-10 CD ……...20 1-11 h-Fuc (rescure) ………...22 1-12 -L- ………25 1-13 3D-CPHmodel ………..26 2-1 -L- :…………..45 2-2 CNPF :………...48 2-3 EPFP ……….50 2-4 Fuconolactone :………..51

3-1 h-Fuc Fuc(NCBI AAA52481)

………...67

3-2 pET22b(+) h-Fuc ……….67

3-3 h-Fuc BL21(DE3) …….68

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3-6 h-Fuc SDS-PAGE………...73

3-7 h-Fuc ……….………74

3-8 h-Fuc 25 oC ……….75

3-9 h-Fuc …..………...77

3-10 h-Fuc Br nsted plot………...84

3-11 -L- ………...85 3-12 pNPF C 1 ……….86 3-13 h-Fuc pNPF ………...87 3-14 h-Fuc ...88 3-15 h-Fuc pNPF/MeOH ……….90 3-16 (A)Michaelis-Menten equation (B) pNPF Km kcat………..92 3-17 h-Fuc ……….93

3-18 h-Fuc kcat kcat/Km……….93

3-19 -L- pH …….93 3-20 GH-29 T. maritima -L-……….95 3-21 3D ……….96 3-22 MuSiC-ME ………...98 3-23 T. maritime -L-……….101 3-24 Fuconolactone Ki ………105 3-25 h-Fuc EPFP 4 ………107 3-26 h-Fuc EPFP ………108

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3-28 D225N pNPF pH-profile..113 3-29 D225G

…………..……….115 3-30 -fucosyl azide 1H-NMR ………..116

3-31 h-Fuc ( ) E289Q ( ) E79G ( ) pH-profile

…. …………..………...121 3-32 E289G kcat/Km . …………..………...124 3-33 E289G aryl- -L-kcat ……….126 3-34 E289G CNPF (7.5 mM) 27 o_{C pD 6.0} 1_H-NMR _{………...128} 3-35 E289G …….128

3-36 h-Fuc ( ) E289G( ) Br nsted plot…………..130

3-37 E289G ( ) ( ) 500 mM Br nsted plot…130

3-38 E289G pNPF 8 ………...133 5-1 ……….138 5-2 -1,3- ………...141 5-3 -1,3- ( -1,3-glucan) …145 5-4 GH-5 16 17 -1,3- 3D ….150 5-5 (active site) …. …………...151 5-6 (Curdlan) ………...152 5-7 X -1,3- ………...153 5-8 GH- 55 -1,3- 3D ...154 5-9 Edge ………...158

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5-11 Eileen Recursive PCR ………...159 5-12 ………...160

5-13 -1,3- ……..….163

7-1-1 PCR ………...209 7-1-2 LPHase S. matensis DIC-108(NCBI BAA34349)

………...211 7-1-3 pRSET_A LPHase ………….211 7-1-4 LPHase BL21(DE3) ……….212 7-1-5 LPHase ………..…214 7-1-6 LPHase SDS-PAGE………..……….215 7-1-7 LPHase ………..……216 7-1-8 LPHase ………..……218 7-1-9 LPHase 25 o_C _{……….…219} 7-1-10 LPHase …………..…221 7-1-11 LC/ESI-MS LPHase 224 7-1-12 LC/MS HPLC LPHase ………..……….226 7-1-13 1-O-methyl- -1,3-laminarihexaose( -laminariheptaose)

LPHase LC/MS ………..………...228 7-1-14 1-O-methyl- -1,3-laminariheptaose . ………..………...228 7-1-15 ………...227 7-1-16 GH-64 -1,3- …....233 7-1-17 GH-64 (Bacteria) (Eukaryota)

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7-1-18 LPHase ………….236 7-1-19 LPHase E154Q D170N ………...237 7-1-20 p-NLPG pH-profile…241 7-1-21 LPHase ………...245 7-1-22 LPHase E154G D170G ………247 7-1-23 D170G ………...248 7-1-24 p-NLPG pH-profile………..……...249 7-1-25 D170G azide …..250 7-1-26 D170G 1_H-NMR _{………...252} 7-1-27 D170E 1H-NMR …...254 7-1-28 D170C ………...256 7-1-29 D170C ………...256 7-1-30 LPHase D170C ………..258 7-1-31 10 ……….258 7-2-1 ………..…..260

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7-2-4 LPHase (sweet tasting protein) Thaumatin

(superposition) ………...262

7-2-5 LPHase (A) (B) (topology) 263

7-2-6 LPHase (surface representation) ……..…....264

7-2-7 LPHase Cysteine ……….266

7-2-8 LPHase Ramachandran Plot (disallowed region) Arg308_{………...267} 7-2-9 LPHase 64 40 % ...272 7-2-10 LPHase LPHase

-

....273 7-2-11 LPHase ……….273 7-2-12 ………...…..275 7-2-13 (Asp143 Glu154 Asp170 Asp376 Asp377)

LPHase ……….278

7-2-14 Glu154 Asp170 Asp377

………...………....278 7-2-15 LPHase

………...….….281 7-2-16 LPHase Arg115 _SDS-PAGE _CD _…....284

7-1-17 LPHase R115Q pH-profile…….…...285 7-2-18

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-L-1-1

-L-

( -L-fucose)

L- ( 1-1 ) 6- -L-6-deoxy-L-galactose L- D- L--L- 145 L-N-linked O-linked -OH L-(1) (1) (2) (2) (3) (selectins) (2) (4) (2 , 3) ₍₅₎ (4) ₍₆₎ (2) (5 , 6)

Leukocyte adhesion deficiency type II, congenital disorder of glycosylation IIc (7)

O

H₃C OH

OH HO

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L- -1, 2 -1, 3 -1, 4 -1, 6 galactose N- N-acetyl glucosamine

L-- ( A, B, H, Lewis ) L-(2,8) 1-2 N-glycans L- (a) (9, 10) _(b) (c) (apoptosis) (d) (4)

23 kDa 40 kDa (UEA)

(LCA) 17 20

(AAL) LCA

AAL-(100±4.9) % (48.1±2.5)

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LCA-A E- L-(e) (Fucosidosis) (2,11,12) -L-(0.5 mol/g/hr) Lewis_X ( ( -1,2-fucosylation)

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-L-1-2

-L-1-2-1

-L-1-1 -L-Fucosidosis(13,14) -L-Immunoprecipitation -L- Antigenic sites (epitopes) (15) -L-(16) 1-1 -L- (28)

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IEF isoelectric focusing -L-isoform

-L-(sialyltransferase) CMP-N-acetylneuraminic acid

-L-(sialic acid)

-L--L- (17)

-L- (18)

(19) _Alhadeff

-L- goat anti-human liver -L-fucosidase -L-7-8 % (oligomannoside) N- (N-acetyl lactosamine) 80 Beem (20) Argad (21) Pronase digestion) -L- 9 6 N-Fukushima (22) _Aronson(23)

(38)

-L-(24)

-L-1-2-2

-L--L- -1,2 -1,3 -1,4 -1,6 N-(25, 26) -L-(27) -L- p-nitrophenyl- -L-fucopyranoside

pNPF 4-methylumbelliferyl- -L-fucopyranoside 4-MU-fucoside

(28) _1-2

pH 4.0-6.0 pH 6.0-7.0

pNPF Michaelis constant Km 0.1-1.3 mM Maximal velocity (Vmax) 11-27

(39)

1-2 -L- (28)

1-2-3

-L--L- ( -L-Fucosidase)

-L- 439

Fuc_1 Fuc_2-1 Fuc_2

1 (1 P34) (Fuc_1 Fuc_2)

Fucosidosis silent (29)_{Fuc_0}

-L-Fuc_1 Fuc_2 Fuc_2-1

Fuc_1 Fuc_2-1

Fuc_2 (Fuc_1, Fuc_2)

-L-Fuc_2-1)

(40)

-L-(Fucosidosis)(30)

-L-neurovisceral storage disease

-L-26 Fucosidosis (31, 32) ₄₂₂ Cytosin) (Thymine) -L- -L-Fucosidosis glycoasparagine(30) glycoasparginase glycoasparagine -1,6 -1,6 (33, 34) -L-cystic fibrosis -L-(35, 36) ₈₀ _Deugnier (37) -L-(38, 39)

-L-(tumor marker) AFP -fetoprotein

(38, 39)

-L-(40, 41)

(41)

-L-(40, 41)

1-3

-L-

( -L-Fucosidase)

-L- (glycohydrolase)

HOR (42)

-L- -L-Fucosidase -L-Fucoside fucohydrolase (exo) (43, 44) (45-49) IUB E.C.3.2.1.X (50) (broad substrate specificity) -L- (hydrolase) (EC 3.) C-O O- (O-glycoside hydrolases EC 3.2.1.-)

(42)

(50,51) L--L- -L-( -L-1,2 -L-1,3 -L-1,4 -L-1,6 ) -L-EC 3.2.1.51 -L- -L-1,2 EC 3.2.1.63 -L-1,3/1,4 EC 3.2.1.111 -L-1,6 EC 3.2.1.127 (fold) (52) (sequence comparision) (53) _2,000 115 (family) ( )(54,55) CAZy (http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html) 3,000 115

-L- ( -L-Fucosidase -L-Fucoside fucohydrolase) 29 CAZY GH-29

95 CAZY GH-95

-L-GH-29

(SN1-like) (56) GH-95 (SN2-like) (57)

(retention configuration) (inverting configuration)

(43)

(retaining glycoside hydrolases)

(glycosylation) (carboxylic group) (leaving group)

(glycosyl-enzyme intermediate)

(deglycosylation) ( )

(transglycosylation) 5.5 Å (B) (Inversion mechanism)

(inverting glycoside hydrolases)

-- ( - - ) (C1) (protonation) (aglycone) (single-displacement inverting mechanism) ( ) 6.5 ~ 10.5 Å 5.5 Å (42, 61, 62) -L-115 70

Protein Data Bank (PDB)

(44)

(clan) GH-A GH-B …… GH-N (GH glycosylhrdrolase) -L-GH-29 Thermotoga maritime -L- ( / )8 dimer dimer (63) _1-4 _GH-95 _{Bifidobacterium bifidum -1,2-} L-( / )6 (64) 1-5_A 1-3

(45)

1-4 GH-29 Thermotoga maritima -L- 3-D (63)

(46)

1-4

-L--L- (Enzymology)

GH-29 Thermotoga maritima Sulfolobus solfataricus

-1,3(4)-L-(56,65,66) _{(retaining glycosidases)}

(double-displacement mechanism)

(single-step mechanism) GH-95 Bifidobacterium

bifidum Bacillus halodurans -1,2-L- (57,64,67)

1-3

-L-GH-29

-L-(nucleophile) (general acid/base)

1-3 -L-Glycoside

hydrolase family Mechanism

Catalytic Nucleophile/Base Catalytic Proton Donor 3D structure status

GH-29 Retaining Asp Glu ( / )8

GH-95 Inverting Asp Glu ( / )6

(wide type)

(mutant enzyme) (68-72)

(47)

(1)

-L-GH-29 T. maritime -L- (Tm-Fuc) 2005 X-ray (63) _PDB _{1HL8 1HL9 1ODU} _GH-95 _{B. bifidum} -1,2-L-(Bb-Fuc) 2007 X-ray (64)

PDB 2EAB 2EAC 2EAD

Tm-Fuc N- -4 ( Asp224₎ _{6 (} _/ _Glu266₎ -( 1-6) Bb-Fuc N- -- ( Asp766 _/ Glu566 _{1-5_B)} 1-6 GH-29 T. maritime (63)

(48)

(74)

Asp Glu

kcat)

1,000

(2)

(active site) (75)

(active site affinity labelling and tandem mass spectrometric localization)

( )

(group-specific labels)

(mechanism-based inhibitors) (transitional complex analogue)

(glycone moiety) (aglycone moiety)

1-7

(49)

(1) (2) (inhibitor binding) ki Ki kreact. 1_H-NMR Withers T. maritime

-L-(Tm-Fuc) 1,2(cis)-difluoro- -L-fucopyranoside Tm-Fuc

(peptide mapping)

reactivation ligands (ki) (Ki) kreact.

(76) _Tm-Fuc

Asp224Gly Asp224

(56,63) _1-8

(50)

( ) (RP-HPLC) (peptide mapping) ESI-MS/MS (77) 1-9 1-9

(3)

(78) _{(thermal stability)}

(pH-dependence) (structure/reactivity studies) (secondary deuterium kinetic isotope effects)

(51)

(a)

(circular dichroism CD)

L--helix -sheet unordered

CD 190~250 nm ( 1-10)

1-10 CD

(b) ( log kcat pH ) (Glycoside

hydrolase) pH-profile (bell-shaped curve) pKa (apparent pKa)

(pKa2)

1Å ( )

(52)

(c) (secondary deuterium kinetic isotope effect)

Br nsted plot (log kcat )

kcat Km 103~104

(4)

(general acid/base) GH-29

Withers Antonio Trincone T. maritima S. solfataricus

(56,65,66) ₍₁₎

(2) pre-steady-state steady-state

(Km kcat) (71) (3) pH 3.0~8.0 (pH dependence)

(4)

kcat/Km fucosylation step

103~105 (5) (79) _{(defucosylation step)}

(azide) (formate) ( defucosylation step) kcat

30-300 -fucosyl_azide 1H-NMR 1-11 -fucosyl_azide -fucosyl_azide (6) (80) 1/450

(53)

1-11 h-Fuc (rescure)

(81)

(66)

(54)

-L-1-5

(carbodiimide) (84, 85) (84, 85) 29 1987 White -L- (86) -L-(65,66,82,83)

(carboxylate group -COO-₎

oxocarbenium ion (87,88) -L- - ( -L-fucosyl_enzyme intermediate) 1-12 -L-(Fuc_1 461 ) (Fuc_2 467 ) 68 % homology -L-(Fuc_1 -L-Fucosidase_1 EC 3.2.1.51) 29 (CAZY GH-29)

(55)

GH-29 -L- 2005 T.

maritima X-

-L-(87) _Tm-Fuc

Tm-Fuc h-Fuc

34 % identity 38 % homology Insight II package program (MSI,

San Diego, CA, USA) (Swiss-Model server

http://swissmodel.expasy.org/) h-Fuc 3D-CPH model ( 1-13

) h-Fuc Tm-Fuc

N ( / )8( TIM-barrel-like) C

-sandwich like

h-Fuc Asp Glu

Br nsted plot -L-(1) DNA -L- ( -L-fucosidase) (2) 1_H-NMR (3) -L-(nucleophilic attack) (4) -L-(general acid/base)

(56)

(57)

1-13 -L- 3D-CPH model

-L- 3D

(58)

2-1

1.

(Tryptone yeast extract) Merck Difco Merck Aldrich TCI Sigma

Kit (T_A cloning Kit PCR extract Gel extract Plasmid extract Kit T4 ligase Kit) GeneMark Viogene NEB

human -L-fucosidase pCMV Stratagene 2

(EYELA NDO-450ND)

(RISEN refrigerated circulators

FIRSTEK SCIENTIFIC B602D, S300R, S302R)

UV (Agilent 8453) (KUBOTA 7700)

ESI-Q-TOF (Micromass)

EYELA rotary vaccum evaporator N-N series Misonix, ultransonic processor

(GeneAmp PCR system 2400, 9700 PC808 Program temp control system) 3. FPLC system (Pharmacia Biotech FPLC3467)

HiTrap Desalting column (Pharmacia, 5 mL) HiTrap SP column HiTrap Q column G-75 column HisTrapTM HP Pharmacia

(ISCO Pharmacia Biotech FRAC-100)

(59)

2-2

-L- (h-Fuc) pCMV

Stratagene

pCMV cDNA

2-3

(competent cell)

TSS (LB / 10% PEG_8000 / 5% DMSO / 20 mM MgSO4) 5X KCM

(0.5 M KCl / 0.15 M CaCl2/ 0.25 mM MgCl2) 121 °C 20 1 ml (Escherichia coli) 100 ml LB 37 °C 150 rpm OD600 0.4 ( 2~3 ) 20 4 °C 350 g ( 1,800 rpm) 8 ( ) ( ) 20 ml 4 °C 350 g 8 1 ml TSS -80 °C 15 100 l -80 °C

2-4

(transformation)

10 l DNA 20 l 5X KCM 50 l heat shock 100 l JM-109 20 42 °C 60 20 50 l LB 37 °C 200 rpm 1 100 l 50 l

(60)

LB-Amp LB-Amp 37 °C 150 rpm 16 3~5 ml (cycle sequencing) 40 l

2-5

-L-2-5-1

PCR pCMV -L-(h-Fuc) 1. h-Fuc pCMV

2. Nde I Hind III HAFUh

5’-CTGCAGCACATATGGTGCGTCGGGCCCAGCCTC-3’ HAFUt 5’-AAGCTTGGATCCTCACTTCACTCCTGTCAGCT-3’

250 l

(1) human -L-fucosidase (h-Fuc) pCMV 3.5 l (2) 10X reaction buffer 5 l (3) HAFUh (125 ng/ l) 1.5 l HAFUt (125 ng/ l) 1.5 l (3) 10 mM dNTP mix 1 l (4) ddH2O 50 l (5) 1 l vent DNA (2.5 U/ l) 3. PCR

(61)

1 1 95 °C 2 min 2 20 95 °C 30 sec 55 °C 35 sec 69 °C 1 min/kb (vent) 3 1 69 °C 1 min 1 4 °C 30 min PCR 5 l DNA PCR

(62)

2-5-2

-L-

pET22b(+)

(ligation)

1. pET22b(+) ( I) Nde I Hind III

DNA DNA 5,300bp

2. PCR h-Fuc Nde I Hind III

DNA 1,400bp h-Fuc

3. 1. 2. 1:5 1 l T4 DNA

(ligase) 1 l (ligase buffer) ddH2O

10 l 16 °C 24

4. JM-109 2-4

5. Nde I Hind III DNA DNA

DNA (

DNA pET22b(+)_h-Fuc)

2-5-3

(Sequencing)

pET22b(+)_h-Fuc JM-109

(Ampicillin 100 g/ml) (Agar plate) pET22b(+)

Ampicillin (single colony) (

(63)

2-5-4

(site-directed mutagenesis)

Strategen Quick Change

1 ( ) 2 pfu 3 Dpn DNA 4 DNA JM-109

(64)

2-5-5

28~35 primer 5

E70G(+) 5’ –GCC TGG GGC AGC

GGC TGG TTC TGG TGG CAC - 3’

E70G(-) 5’ – GTG CCA CCA GAA CCA

GCC GCT GCC CCA GGC - 3’

E79G(+) 5' –GCA CTG GCA GGG

CGG GGG GCG GCC GCA - 3'

E79Q(-) 5' –TGC GGC CGC CCC

TGG CCC TGC CAG TGC - 3'

E135G(+) 5’ - ACG ACA AAG CAT CAC

GGC GGC TTC ACA AAC - 3’

E135G(-) 5’ - GTT TGT GAA GCC

GCC GTG ATG CTT TGT CGT - 3’

D158G(+) 5’ - GGG CCT CAT CGG

GGC TTG GTT CGT GAA - 3’

D158G(-) 5’ - TTC ACC AAC CAA

GCC CCG ATG AGG CCC - 3’

D225G(+) 5’ - CTG ATA TGG TCT

GGC GGG GAG TGG GAA - 3’

D225N(-) 5’ - TTC CCA CTC CCC

GTT AGA CCA TAT CAG - 3’

D258G(+) 5’ - GTG GTA GTA AAT

GGC CGA TGG GGT CAG – 3’

D258N(-) 5’ - CTG ACC CCA TCG

GTT ATT TAC TAC CAC – 3’

E275G(+) 5' - GGA TAC TAT AAC TGT

GGA GAT AAA TTC AAG - 3'

E275Q(-) 5' - CTT GAA TTT ATC

CTG ACA GTT ATA GTA TCC - 3'

D276G(+) 5’ - TAC TAT AAC TGT GAA

GGC AAA TTC AAG - 3’

D276G(-) 5’ - CTT GA A TTT

GCC TTC ACA GTT ATA GTA - 3’

E289G(+) 5’ - GAT CAC AAG TGG

GGG ATG TGC ACC - 3’

(65)

2-5-6

1. Sample reaction (2) 10X reaction buffer 5 l (3) dsDNA (50 ng/ l) (pET22b(+)_h-Fuc ) 3.5 l (4) (125 ng/ l) 1 l (125 ng/ l) 1 l (5) 10 mM dNTP mix 1 l (6) ddH2O 50 l 2. 1 l pfu DNA (2.5 U/ l) 3. PCR 1 1 95 °C 2 min 2 20 95 °C 30 sec 57.5 °C 30 sec 69 °C 2 min/kb (pfu) 3 1 69 °C 1 min 1 4 °C 30 min 4. PCR 5 l DNA 1 l Dpn I 37 °C 8~10 5. JM-109 2-4

(66)

2-6

25 % -80 °C

2-7

-L-(expression)

2-7-1

DNA

BL21(DE3)

pET22b(+)_h-Fuc E70G E79G/Q E135G D158G D258G E275G D276G D225G/N E289G/Q -80 o_C 30 42 °C 60-65 1-2 100 L LB 37oC 1 LB-Amp 37 °C 12 ( 2-4)

2-7-2

-L-LB-Amp 5 ml LBA 8 1 ml 50 mL LBA OD600 0.4~0.5 2 mM IPTG

(67)

-L-2-7-3

-L-5 mL 200 l 20 mM (pH 5.7) (sonication) 10 l (1 mM pNPF) 210 l (50 mM pH 7.0) 30 l UV 400 nm

2-8

-L-2-8-1

pET22b(+)_h-Fuc BL21(DE3) -L-BL21(DE3)

-L-2-8-2

-80 °C pET22b(+)_h-Fuc BL21(DE3) 5 ml LB 37 °C 120 rpm 8 5 ml 1 L LBA 2 ml 1 M IPTG ( 2 mM) 37 °C 100 rpm 30 3 ml 14,000 rpm pH 5.7 0.2 ml

(68)

(45W 75 % pulse) 14,000 rpm 3 pNPF 37 °C UV 400 nm pNPF 4 °C 7,000 rpm 10 10 ml (10 mM pH 5.7) 10 mM PMSF 10 l/ml (45W 75% pulse) 4 °C 17,000 rpm 20

2-8-3

1. (Ammouium sulfate) 20 % 2. 17,000 rpm 4 °C 15 3. 20 % 85 % 4. 3. 5. 17,000 rpm 4 °C 15 20 % ~ 85 % 6. 5. (20 mM pH 5.5) 1.5 ml

HiTrap (Desalting column)

2 ml HiTrap

(69)

2-8-4 HiTrap SP

1. 5 ml 6. 20 %~85 % (20 mM pH 5.5) HiTrap SP 2. 0 1 M 1.5 ml/min 300 ml 3. 2.5 ml 4. pNPF 37 °C pNPF

2-8-5 HiTrap Q

1. HiTrap SP 10,000 2. 1.5 ml (10 mM pH 6.2) HiTrap 2 ml HiTrap 3. 5 ml 2. (20 mM pH 6.2) HiTrap Q 4. 0 1 M 1.2 ml/min 300 ml 5. 2.0 ml 6. pNPF 37 °C pNPF

(70)

2-8-6

G-75

1. HiTrap Q 10,000 2. 1.5 ml (10 mM pH 5.7) HiTrap 2 ml HiTrap 3. 1 ml 2. (50 mM pH 5.7) G-75 4. 50 mM ( 10 mM NaCl pH 5.7) 0.7 ml/min 5. 1.5 ml 6. pNPF 37 °C pNPF

(71)

2-9

6-His

h-Fuc

D225G/N E289G/Q

2-5-6 h-Fuc D225G/N E289G/Q

pET22b(+) 6-His h-Fuc

6-His

1. plasmid extract kit D225G/N E289G/Q h-Fuc

pET22b(+)_h-Fuc

2. Nde I Hind III HAFUh

5’-CTGCAGCACATATGGTGCGTCGGGCCCAGCCTC-3’ HAFUt1 5’-GCGGCCGCAAGCTTCTTCACTCCTGTCAGCT-3’ ( h-Fuc

) 2-5-1 h-Fuc

PCR

3. 2 PCR h-Fuc pET22b(+)

( ) Nde I Hind III DNA

4. 2-5-2 3.

D225G/N E289G/Q h-Fuc pET22b(+)

5.

6. BL21(DE3) 6-His D225G/N

E289G/Q h-Fuc 2-8-3

(72)

2-10 SDS-PAGE

(homology)

Laemmli 1970 SDS-PAGE

1.

1. (Stacking gel) 7 % (Separation gel) 17.5 % 10 cm ×7.4 cm ×0.1mm

Running gel Stacking gel 50% Acrylamide / Bisacrylamide (48.28 g Acrylamide, 1.72 g Bisacrylamide) 1.64 mL ( ) 0.35 mL ( ) 2M Tris-H3PO4 (pH = 8.9) 1.23 mL 10%SDS 65.63 l 24.75 l APS in IPA (10%) 32.81 l 12.75 l TEMED 10 l 3.5 l H2O 3.59 mL 1.78 mL 1M Tris-H3PO4 (pH = 6.5) 312.75 l

2. Sample loadind buffer

(1) 1 M Tris-HCl (pH 6.8) 0.6 mL (2) 50% glycerol 5 mL (3) 2-mercaptoethanol 0.5 mL (4) 0.1% bromophenol blue 1 mL (5) 10% SDS 2 mL (6) H2O 0.9 mL Total volume 10 mL

(73)

3. 30 l 5 l Sample loading buffer 95 0C 2

4. Tris-glycine rumming buffer (4x) (1) Glycine 56 g

(2) Tris-Base 12 g (3) SDS 4 g

Total volume 1L

800 mL running buffer (1x) Sample loading buffer 150 Tris-glycine running buffer 1.5

2. Coomassie Brilliant Blue G-250

(1) Stain solution 200 mL acetic acid, 500 mL Isopropanol, 0.6 g Coomassie blue, 1.3 L Mini-Q H2O

(2) Destain solution 400 mL acetic acid, 400 mL Isopropanol, 3.2 L Mini-Q H2O

(3) ( 10 cm ×7.4 cm ×1 mm ) Stain solution 30 ~ 1

(4) Stain solution Destain solution 30 ~ 1

(74)

2-11

2-11-1

(LC-MASS)

1. 3.5~4 mg/ml -L- Zip-Tip Millipore 0.1 mL/s H20:CH3CN:1% HCOOH = 80:10:10 IPA:CH3CN:HCOOH = 10:10:4 2. 3.5~4 mg/ml E289G 9 mM FPNF pNPF 8 1. E289G

2-11-2

Bicinchoninic Acid (BCA) Assay

Bicinic acid : 4% (w/v) Copper (II) sulfate 50:10

Standard Working Reagent (SWR) (bovine serum albumin, BSA) SWR 1:20 (BSA:SWR) 37 °C 30

10 562 nm

2-11-3

10 l 20 l

SWR BCA 562 nm

(75)

2-12

-L-2-12-1

1. (NMR) Bruker DRX-300

D2O D2O 4.72 CDCl3 CDCl3

7.24 ppm (coupling constant) Hz

s (single) d (doublet) t (triplet) q (quarte) m (multiplet) b (broad peak)

2. (TLC) Merck Silica gel 60 F254 (aluminium sheet

TLC) ICN SiliTech 32-63 60Å (230~400 mesh)

3. TLC Ninhydrin solution(0.3 ninhydrin in 100 n-butanol add 3 acetic acid) Anisaldehyde solution(9.2

Anisaldehyde 3.75 Acetic acid 338 EtOH(95%) 12.5 H2SO4 )

4. Sigma-Aldrich Acros (TCI)

5. Activated CH Sepharose 4B Amersham Bioscience

6. TEDIA Merck

7. (developing solvent)

8. EYELA ROTARY VACUUM EVAPORATOR N-N series

(76)

2-12-2

-L-(p-nitrophenyl- -L-fucopyranoside pNPF)

Acceptor

-L

-R= 3-nitrophenyl 4-nitrophenyl 2,4-dinitrophenyl phenyl 2-methyl-4-nitrophenyl 3,4-dinitrophenyl 4-cyanophenyl 2-fluro-4-nitrophenyl

2-1 -L- :

(a) Ac2O Pyridine rt 8 h

(b) H4N2-HOAc DMF rt 30 min

(c) CCl3CN Cs2CO3 CH2Cl2 rt 8 h

(d) ROH (acceptor) TMSOTf 4Å MS CH2Cl2 -20 °C 30 min

(e) NaOMe MeOH rt 30 min

3 4

(77)

2,3,4-Tri-O-acetyl- -fucopyranosyl acetate

OAc

Acetylation

1. 1 (10 mL) 15 2. 15 ml overnight 3. TLC (dichloromethane DCM) 1N HCl (MgSO4) 4. Hexane/EA=9/1 Hexane/EA=7/3 0.88 (1) 88 %

1-(trichloroacetimidate)-2,3,4-Tri-O-acetyl- -fucopyranoside

1. acetylation (1) 600 183 Hydrazine acetate 3 DMF 2. 30 (ethyl acetate) (MgSO4)

(78)

3. 2. CCl3CN CCl3CN 1:1 4. 8 (ethyl acetate) (MgSO4) (2) 93 : = 7:1

1-(4-nitrophenyl)-2,3,4-Tri-O-acetyl- -fucopyranoside

1. (2) 364 140 4-nitrophenol Acceptor 500 4Å 5 3 2. 3 150 TMSOTf -20 °C 30

3. ethylacetate / hexane = 1/6 silica-gel

(3) ethylacetate hexane 56 %

1-(4-nitrophenyl)-2,3,4-Tri-O-hydroxyl- -fucopyranoside

1. (3) 1 30 32

NaOMe 2

(79)

3. IR-120

(4) 82 %

2-12-3

-L-(3) Acceptor 3-nitrophenyl

4-nitrophenyl 2,4-dinitrophenyl phenyl 2-methyl-4-nitrophenyl 3,4-dinitrophenyl 4-cyanophenyl 2-fluoro-4-nitrophenyl -L

-2-12-4 2-

-L-(2-chloro-4-nitrophenyl- -fucopyranoside CNPF)

2-2 CNPF :

(a) H2 Pd(OH)2/C MeOH_EtOAc 77% for 2

(b) TBDMSCl imidazol DMF 78% for 3

(c) NIS TMSOTf CH2Cl2 73% for 4

(80)

1. 5 L-Fucose (89) 1 4.3 2. 0.415 (0.71 mmol) 1 6 ml MeOH/EtOAc (2:1) Pd(OH)2/C (40 mg) 3. (H2) 80 mL/min 10 4. TLC 5. petroleum ether/EtOAc (3:1) 2 ( 0.306 ) 79 % 6. 1.04 (5 mmol) 2 5 mL N,Ndimethylformamide( DMF) imidazole (2.04 30 mmol) Tert-butyldimethylsilyl chloride (TBDMSCl) (2.26

15 mmol) 0 °C 30 40 °C 12 7. TLC (MgSO4) 8. petroleum ether/EtOAc (20:1) 3( 0.8 ) 78 %

9. 2.0 (3.73 mmol) 3 0.712 (4.1 mmol) 2-chloro-4-nitrophenol

60 °C 10 ml NIS (0.923

4.01 mmol) TMSOTf (30 l 0.19 mmol)

-42 °C 30

10. TLC Et3N

11. petroleum ether/EtOAc 30:1 20:1 4 ( 1.25 ) 61 %

(81)

HF (70 %) 0 oC 20

13. TLC

14. EtOAc 5( 0.335 )

68 %

2-12-5

O-hydroxyl-1-(2,3-epoxypropyl)- -L-fucopyranoside (EPFP)

2-3 O OH OH HO CH3 OH Ac2O/Et3N O OAc AcO AcO CH3 OAc Allyltrimethylsilane BF3OEt2 O OAc AcO AcO CH3 K2CO3 MeOH O OH OH HO CH3 + isomer TBDMSOTf

diisopropyl ethyl amine O OTBDMS TBDMSO TBDMSO CH3 2,6-di-t-butyl-4-methyl phenol m-CPBA O OTBDMS TBDMSO TBDMSO CH3 O O OH OH HO CH3 O TBAF/THF

(82)

2-12-6

Fuconolactone

Fuconolactone -L-oxocarbenium ion Fuconolactone O OBnOBn OBn OH O OBnOBn OBn O O OHOH OH O a b 2-4 Fuconolactone : (a) PCC, CH2Cl2, 4Å MS, rt, 1 h (b) H2, 5 Pt C , EtOH, rt, 12 h 1. (1) 40 ( (90)) 3 170 PCC 250 4Å 1

2. ethylacetate / hexane = 1/4 silica-gel (2)

3. 80 (2) 10 5 Pt(C)

12

4. celite Pt(C)

methanol / dichloro methane = 1/6 silica-gel Fuconolactone.

(83)

2-13

-L-2-13-1

pH -L-1. 370 l 50 mM (pH 7.0) 0.25 mM pNPF 4 °C 2. 30 l h-Fuc 400 nm (initial velocity) 3. 1 2. 16 °C 25 °C 37 °C 45 °C 55 °C 65 °C 75 °C 85 °C 95 °C 400 nm 4.

2-13-2

(Thermostability)

pH -L-1. 30 l h-Fuc 25 °C 370 l 0.25 mM

(84)

(initial velocity) 1 2. 500 l h-Fuc 25 °C 1 , 20 , 40 , 60 , 80 , 100 , 130 , 160 1. 400 nm 3. 2. 1. 25 °C 4. 2. 3. 37 °C 45 °C 55 °C 60 °C 65 °C 75 °C 5.

2-13-3

-L-pH

Glycine/HCl (50 mM pH 2 2.5 3) Citric (50 mM pH 3.5) NaOAc (50 mM pH 4 4.5 5 5.5) MES (50 mM pH 5.5 6) Na2HPO4 (50 mM pH 6.5 7 7.5) Hepes

(50 mM pH 7.5 8) Tris (50 mM pH 8 8.5) Glycine/NaOH (50 mM pH 9 9.5 10) Caps (50 mM pH 10 10.5)

1. pH 25 °C

2. 30 l h-Fuc 0.25 mM pNPF 348 nm

(85)

3. pH pH

2-13-4

(pH stability)

-L-pH

Glycine/HCl (50 mM pH 2 3) NaOAc (50 mM pH 4 5) MES (50 mM pH 6) Na2HPO4 (50 mM pH 7) Tris (50 mM pH 8) Glycine/NaOH (50 mM pH 9)

1. 30 l h-Fuc 25 °C 370 l 0.25 mM pNPF 50 mM 400 nm (initial velocity) 1 2. 500 l (50 mM pH 5.0) 25 °C 3. h-Fuc 25 oC 1 , 20 , 40 , 60 , 80 , 100 , 130 , 160 1. 400 nm 4. 3. 1. 25 °C pH 5.0 5. 2. 4. pH 6. pH pH pH

(86)

2-13-5

-L-Cu2+ _Cd2+ _Ni2+ Ba2+ _Zn2+ _Co2+ _Mn2+ _Ca2+ _Mg2+ _Fe2+ _Pb2+ _Hg2+ _EDTA _DTT 2-mercaptoethanol 1. 30 l h-Fuc 0.25 mM pNPF 25 °C pH 7.0 50 mM 400 nm (initial velocity) 1 2. 1. 30 l h-Fuc 0.25 mM pNPF 25 °C pH 7.0 5 mM 50 mM 400 nm 3. 2. 1. 5 mM h-Fuc

2-14

-L--L- pNPF

(87)

1. D2O (20 mM pD 7.0) pNPF H 2. -L- D2O H2O H 3. H2O pNPF NMR 4. 0.5 ml 1. pNPF NMR 5. 0 ~ 200 6. (chemical shift) 5.12 4.48

-L-2-15

-L-CNPF pNPF pCPF 2 mg 700 l (20 mM pH 5.5) 200 L 100 l h-Fuc ( 2.36 g / L) 1 ml 6 M (~20%) 200 l 37 °C 36 0.5 mL D2O 2~3 500 mL D2O NMR tube 0.5 ml NMR 50 °C

(88)

2-16

-L-

k

cat

K

m

Km

Michaelis-Menten equation (double reciprocal plot) (19)

E + S

ES

E + P

K

_m

k

cat

steady-state

V = Vmax [S] / Km + [S] Vmax = kcat x [E]t

[E]t kcat 1 / V = Km / Vmax 1 / S + 1 / Vmax Vmax Km Km / Vmax 1 / Vmax - 1 / Km Km h-Fuc ( 0.95 g/ l ) 50 mM pH 6.8 aryl substrates ( ) UV

(89)

2-16-1

-L-p-nitrophenyl- -L-fucopyranoside (pNPF)

p-nitrophenyl- -D-arabinopyranoside (pNPAP) p-nitrophenyl- -L-arabinofuranoside (pNPAF) p-nitrophenyl- -D-Galactopyranoside (pNP-Gal) p-nitrophenyl- -D-Glucopyranoside (pNP-Glc)

p-nitrophenyl- -N-acetylglucosamine (pNP-GlcNAc)

1. 0.93 g/ l h-Fuc 0.25 mM~2 mM

pNPF 37 °C pH 6.8 50 mM /100 mM

400 nm (initial velocity)

2. kcat Km

3. pNPF pNPAP pNPAF pNP-Gal pNP-Glc

pNP-GlcNAc 1. 400 nm kcat

Km

2-16-2

-L-k

cat

K

m

(pH-profile

)

1. pH Glycine/HCl (50 mM pH 2 2.5 3) Citric (50 mM pH 3.5) NaOAc (50 mM pH 4 4.5 5 5.5) MES (50 mM pH 5.5 6) Na2HPO4

(90)

25 °C 2. 0.93 g/ l h-Fuc 0.5 mM~2 mM pNPF 37 °C UV 348 nm (initial velocity) 3. Lineweaver-Burk method kcat Km pH pH 4. h-Fuc 1. 3. kcat Km pH

2-16-3

-L-k

cat

K

m

(Br nsted plot

)

1.

pNPF 50 mM (pH 6.8) (0.05 mM ~2 mM)

0.93 g/ l h-Fuc 37 °C UV 400

nm

2. pNPF Michaelis-Menten equation Km Vmax

pNPF Km Vmax 3 1.~2. pNPF 2,4-dinitrophenyl-fucopyranoside (DNPF 0.02 mM-0.25 mM) 2-chloro-4-nitrophenyl-fucopyranoside (CNPF 0.02 mM- 0.25 mM) 2-fluoro-4-nitrophenyl-fucopyranoside (FNPF 0.02 mM- 0.5 mM) 2-methyl-4-nitrophenyl-fucopyranoside (MeNPF 0.2 mM- 1.5 mM) 3-nitrophenyl-fucopyranoside (mPNPF 0.5 mM- 1.2 mM) 4-cyanophenyl-fucopyranoside (pCPF 0.1 mM- 0.5 mM)

(91)

Phenyl-fucopyranoside (PF 0.1 mM- 0.5 mM) UV

( II) (initial velocity V0)

Lineweaver-Burk method (double reciprocal plot) Km kcat

4. h-Fuc 1. 3. kcat Km pKa

2-17

O-hydroxyl-1-(2,3-epoxypropyl)- -L-fucopyranoside (EPFP) (nucleophile general acid/base) 1. EPFP (3.5 7 15 22.5 35 mM) h-Fuc 20 mM (pH 5.7) 0.25 mM pNPF 50 mM (pH 7.0) UV 400 nm ( EPFP ) Enzfit kobs ( s-1) 2. 5 EPFP 5 kobs

3. 5 kobs EPFP (y = ax +b) EPFP

h-Fuc (ki) (Ki)

4. 1.~ 4.

(92)

1. 0.25 mM pNPF (50 mM pH 6.8 37 oC)

2. (0.25~2 mM) ( -Fuconolactone)

3.

( ) _Ki

4. (L-Fucose 1-O-Methyl- -L-Fucose

)

Ki

2-18

-L-1. 50~3000 mM pH 6.8 50 mM 2 [S]»Km E289G/Q 10 l ( 0.93~1.14 g/ l) CNPF FNPF pNPF ( 0.5~2.5 mM) 37 °C 400 nm ( kcat ) 3 pNPF 4 kcat 24 0.5 M E289Q pNPF 2-16-2

(93)

2-19

1

H-NMR

D225G

1. 1.5 M sodium azide ( ) 50 mM (pH 6.5) 80 mM FNPF 2. D225G ( 1.69 g/ l) 1. 3. TLC FNPF FNPF 4. 3. (5 mL) 15 5. 10 ml overnight 6. TLC (dichloromethane DCM) (MgSO4) 7. Hexane/EA=9/1 Hexane/EA=7/3 8. 7. D- NMR 1H-NMR 9. (chemical shift) D225G

(94)

E289G

1. D2O 1.5 M sodium azide ( ) 5 mM (pD 6.8) 7.5 mM CNPF 2. E289G ( 1.4 g/ l) 1. 3. CNPF NMR 4. 0.5 ml 1. CNPF NMR 5. 0.1 ml 2. 0 ~ 300 6. (chemical shift) E289G

2-20

E289G

kcat 24 0.5 M aryl-fucopyranosides 1 (CNPF FNPF pNPF MeNPF mPNPF pCPF PF) 500 mM 50 mM (pH 6.8) (0.5 mM~2.5 mM) E289G 37 °C 400 nm

(95)

2 Lineweaver-Burk method Km Vmax ( kcat )

3 log kcat log kcat/Km (kcat= Vmax/[E]t) pKa Br nsted

plot

2-21 CD (circular dichroism)

(AL) (AR) A= AL AR (CD ) 1. Jasco J-815 h-Fuc ( 200~250 nm) 25~30 M (1 mM pH 7.0) 16 2. ellipticity ( ) ellipiticity ([ ]MRW).

**[ ]MRW = /(10Crl) Cr = (n1000cg)/Mr**

ellipticity in mdeg Cr l n peptide bond cg (g/mL) Mr 2 -1

(96)

2-22

GH-29 Thermotoga maritima (Tm-Fuc

Q9WYE2 PDB 1ODU) Swiss-Model server

(http://swissmodel.expasy.org/) -L- 3D-CPHmodel CAZy (http://www.cazy.org/) GH-29

-L- InterPro

(http://www.ebi.ac.uk/interpro/) ( / )8 (

TIM-barrel domain IPR017853) Halocynthia

roretzi (Q8TA71 Hr-Fuc) Schistosoma japonicum (Q86FH9 Sj-Fuc) Pongo abelii

(Q5RFI5 Pa-Fuc) Drosophila melanogaster (Q9VTJ4 Dm-Fuc) Caenorhabditis elegans (P49713 Ce-Fuc) Dictyostelium discoideum (P10901 Dd-Fuc) h-Fuc

70 %

-L-Tm-Fuc 7-356 h-Fuc 31-365 Hr-Fuc 55-392 Sj-Fuc 33-369 Pa-Fuc 31-366 Dm-Fuc 33-368 Ce-Fuc 17-350 Dd-Fuc 20-361 PSIPRE

(http://zeus.cs.vu.nl/programs/pralinewww/)

Combinatorial Extension (CE)

(97)

3-1

-L-Fuc_1 Fuc_2-1 Fuc_2

-L- (Fuc_1)

439

pGEX-2T Glutathione

S-transferase(GST) (fusion protein)

(inclusion bodies)(91) -L-( III) 90 % pRSET_A pET DNA BL21(DE3) pET22b(+) -L-JM-109 -L-pCMV NdeI HindIII PCR -L-(MRSRPAGPALLLLLLFLGAAES) N ATG( Met) ( 3-1 ) PCR NdeI HindIII 1,400bp T4 pET22b(+) BL21(DE3) 3-2

(98)

(99)

3-2

-L-PCR BL21(DE3) h-Fuc pET22b(+) lacZ IPTG -L-pH 4.0~6.0 pH 5 mL LBA OD600 0.6~0.8 1 L pH 4.0~8.0 LBA ( 2 mM IPTG) OD600 ( 3-3_A ) pNPF UV 400 nm ( 3-3_B) pH 6.0 24~30 pH 6.0

(100)

3-3

-L-BL21(DE3) HiTrap SP pH 5.5 HiTrap SP ( 3-4_A) pH 5.5 -L- 500~700 mM -L-HiTrap Q pH 6.2 250~350 mM ( 3-4_B) -L-G-75 0.70 ml/min pH 5.7 ( 3-4_C) -L-SDS-PAGE ( 3-5_A) 95 % ( 3-1 ) 3-1 h-Fuc

Step Protein Total (mg) Total Activity (units) Specific Activity (units/mg) Purity Fold Yield (%) Crude enzyme extract* 102 651 6.4 1 100 HiTrap SP ( pH 5.5 ) 34.8 342 9.8 1.5 34 HiTrap Q ( pH 6.2 ) 6.7 266 39.7 6.2 20 Superdex G-75 (pH 5.7) 0.34 72 211.7 33.1 5.1

(101)

(A)

(B)

OD/280 nm (- -) NaCl, % (—) h-Fuc activity (- -)

(C)

3-4 h-Fuc

(102)

(A) (B)

3-5 h-Fuc SDS-PAGE

(A) lane M protein marker 14.5-97 kDa

lane A SP h-Fuc

lane B Q h-Fuc

lane C G-75 h-Fuc

(B) lane M protein marker 14.5-97 kDa

lane A E79G h-Fuc

lane B D225G h-Fuc ( 6-His )

(103)

3-4

-L-3-4-1

-L- SDS-PAGE 50 KDa Q-TOF LC/MS 50895.0 Da ( 3-6) Vector NTI ( 3-2) 51028.5 Da 133 Da N Met (M.W.= 131 Da) 3-6 h-Fuc

Analysis Entire Protein

Length 439 aa

Molecular Weight 51028.5 m.w.

1 microgram = 19.655 pMoles

Molar Extinction coefficient 178430

1 A[280] corr. to 0.29 mg/ml

A[280] of 1 mg/ml 3.51 AU

Isoelectric Point 6.03

(104)

3-4-2

Bradford Assay Lowry Assay UV BCA Assay

Bicinchoninic Acid (BCA) Assay

BCA OD562 (Y = aX + b) ( IV ) OD562 UV 280 nm 1 OD280 g / L OD280 BSA ( ) 1 g / L h-Fuc (10 L) BCA OD562 0.71 1.63 g / L OD280 1.42 OD 1 OD280 = 1.16 g / L

3-5

pH - profile pH pH

(105)

3-5-1

-L-h-Fuc

Glycine/HCl (50 mM pH 2 2.5 3) Citric (50 mM pH 3.5) NaOAc (50 mM pH 4 4.5 5 5.5) MES (50 mM pH 5.5 6) Na2HPO4 (50 mM pH 6.5 7 7.5)

Hepes (50 mM pH 7.5 8) Tris (50 mM pH 8 8.5) Glycine/NaOH (50mM pH 9 9.5 10) Caps (50mM pH 10 10.5) 0.25 mM pNPF 37 oC UV 400 nm h-Fuc pH pH ( 3-7) pH 3.5~5.5 pH pH pH 8.0

10

8

6

4

2 0.0008

0.0006

0.0004

0.0002

0 pH

E

nz

ym

e

ac

tiv

ity

(O

D

4

00 n

m

)

Glycine-HCl NaOAc MES Na2HPO4 Hepes Tris Glycine-NaOH Caps 3-7 h-Fuc

(106)

3-5-2

3-9 h-Fuc 25 o_C

pH (50 mM pH 7.0) 0.25 mM

pNPF 37 oC UV 400 nm pH

(residual activity ratio) 25 o_C _{pH 5.7}

3-8 25 oC 4 pH 3.0

pH 6.0 95 % pH 2.5 pH 7.0

70 %~85 % pH 8.0

pH 3.0 6.0

(107)

3-5-3

h-Fuc 4 16 25 37 45 55 65 75 85 95 o_C _{50 mM} _{(pH 7.0)} _{0.25 mM} _pNPF UV 400 nm ( 3-9_A ) h-Fuc 75 oC 75 oC 65 oC ~70 o_C _h-Fuc _pNPF ₁

3-5-4

3-9_B h-Fuc 25 oC ~ 75 oC 0.25 mM pNPF 37 o_{C 50 mM} _{(pH 7.0)} _UV _{400 nm} 4 oC h-Fuc pNPF h-Fuc h-Fuc 4 25 oC ~ 55 oC 60 oC 60 oC

(108)

(A)

(B)

3-9 h-Fuc

(A) (B)

(109)

3-5-5

-L-(active site) metalloenzyme metalloenzyme 37 0C h-Fuc 0.25 mM pNPF 5 mM 3-3 h-Fuc Hg2+ h-Fuc 90 % h-Fuc Hg2+ DTT 2-mercaptoethanol h-Fuc Cys h-Fuc h-Fuc Cys (92) 3-3 h-Fuc None Cu2+ Cd2+ Ni2+ Ba2+ Zn2+ 100 % 103 % 107 % 102 % 96 % 108 % Hg2+ Co2+ Mn2+ Ca2+ Mg2+ Fe2+ 0.7 % 109 % 105 % 97 % 92 % 106 % Pb2+ EDTA DTT 2-mercaptoethanol 104 % 94 % 3 % 1 % -L- 5 mM pNPF 37 0C UV 400 nm

(110)

3-6

- -L-

(aryl- -L-fucopyranosides)

-

-L-pKa (coupling) pKa

- (1,2-cis) Acetobromoglycoside (94,95) phenols - -L-2-1 2-2 1-(trichloroacetimidate)-2,3,4-Tri-O-acetyl- -fucopyranoside ( 2-1 2) -20 0C (kinetic control) (phenols) -- --L-- 65~75% - -L-(92) - -L- 1H-NMR (300MHz) V VI

1. TAF : 8.22-8.19 (m, 2H, ArH), 7.17-7.13 (m, 2H, ArH), 5.86 (d, 1 H, J = 4.0Hz, H-1),

5.58 (dd, 1 H, J = 6.0,12.0Hz, H-3), 5.37 (ddd, 2 H, J = 6.0,6.0,12.0Hz, H-2, H-4), 4.29 (q, 1 H, J = 7.0, 14.0 Hz, H-5), 2.19 (s, 3 H, AcO), 2.05 (s, 3 H, AcO), 2.01 (s, 3 H, AcO), 1.21 (d, 3 H, J = 7.0Hz, H-6) C14H20O9

2. 2,4-Dinitrophenyl- -L-fucopyranoside (DNPF) : 1.32 (d, 3H, J = 6.0 Hz, H-6),

3.71-3.8 (m, 2H, H-4, H-5), 3.84 (dd, 1H, J = 3.6, 9.3 Hz, H-3), 4.14 (dd, 1H, J = 8.1, 9.5 Hz, H-2), 5.60 (d, 1H, J = 5.1 Hz, H-1), 7.50 (d, 1H, J = 9.0Hz, ArH), 8.45 (dd, 1H, J = 2.7, 9.3Hz, ArH), 8.69 (d, 1H, J = 2.7Hz, ArH) C12H14N2O9

(111)

3. 2-Fluoro-4-nitrophenyl- -L-fucopyranoside (FNPF) : 1.09 (d, 3 H, J = 6.5 Hz, H-6), 3.81 (m, 2 H, H-4, H-5), 3.94 (dd, 1 H, J = 3.5, 9.6 Hz, H-3), 4.08 (dd, 1 H, J = 7.3, 9.6 Hz, H-2), 5.77 (d, 1 H, J = 3.16 Hz, H-1), 7.37 (d, 1 H, J = 9.2 Hz, ArH), 8.06 (dd, 1 H, J = 2.6, 9.2 Hz, ArH), 8.85 (d, 1 H, J = 2.6 Hz, ArH) C12H14NO7F 4. 2-Chloro-4-nitrophenyl- -L-fucopyranoside (CNPF) : 1.13 (d, 3 H, J = 6.6 Hz, H-6), 3.74-3.83 (m, 2 H, H-4, H-5), 3.95 (dd, 1 H, J = 3.6, 9.6 Hz, H-3), 4.33 (dd, 1 H, J = 7.6, 9.6 Hz, H-2), 5.71 (d, 1 H, J = 3.78 Hz, H-1), 7.31 (d, 1 H, J = 9.2 Hz, ArH), 8.08 (dd, 1 H, J = 2.6, 9.2 Hz, ArH), 8.85 (d, 1 H, J = 2.6 Hz, ArH) C12H14NO7Cl 5. p-nitrophenyl- -L-fucopyranoside (pNPF) : 1.22 (d, J = 6.0 Hz, 3H, H-6), 3.72-3.8 (m, 2H, H-4, H-5), 3.94 (dd, J = 3.0, 9.0 Hz, 1H, H-3), 4.30 (dd, 1 H, J = 7.2, 9.1 Hz, H-2), 5.87 (d, 1 H, J = 4.0 Hz, H-1), 7.33 (d, 2 H, J = 8.7 Hz, ArH), 8.32 (d, 2 H, J = 9.3 Hz, ArH) C12H15NO7

6. 2-Methyl-4-nitrophenyl- -L-fucopyranoside (MeNPF) : 1.20 (d, 3 H, J = 6.4 Hz,

H-6), 2.37 (s, 3 H, CH3), 3.72-3.78 (m, 2 H, H-4, H-5), 3.96 (dd, 1 H, J = 3.6, 9.6 Hz, H-3), 4.02 (dd, 1 H, J = 7.6, 9.6 Hz, H-2), 5.70 (d, 1 H, J = 4.88 Hz, H-1), 7.28 (d, 1H, J = 9.8 Hz, ArH), 8.08 (dd, 1 H, J = 2.6, 9.6 Hz, ArH), 8.11 (d, 1 H, J = 2.6 Hz, ArH)

C13H17NO7 7. m-nitrophenyl- -L-fucopyranoside (mNPF) : 1.25 (d, 3H, J = 7.0Hz, H-6), 3.66-3.72 (m, 2H, H-4, H-5), 3.8 (dd, J = 3.5, 9.6 Hz, 1H, H-3), 4.16 (dd, 1H, J = 6.9, 9.3 Hz, H-2), 5.73 (d, J = 3.6Hz, 1H, H-1), 7.41-7.37 (m, 1H, ArH), 7.50-7.45 (m, 1H, ArH), 7.94-7.90 (m, 2H, ArH) C12H15NO7 8. p-cyanophenyl- -L-fucopyranoside (pCPF) : 1.21 (d, 3 H, J = 6.8 Hz, H-6), 3.7-3.76 (m, 2 H, H-4, H-5), 3.87 (dd, J = 3.0, 9.0 Hz, 1H, H-3), 4.16 (dd, 1 H, J = 7.2, 9.1 Hz, H-2), 5.83 (d, 1 H, J = 3.6 Hz, H-1), 7.12 (d, 2 H, J = 8.7 Hz, ArH), 7.65 (d, 2 H, J = 8.9 Hz, ArH) C13H15NO5 9. phenyl- -L-fucopyranoside (PF) : 1.25 (d, 3 H, J = 7.0 Hz, H-6), 3.74-3.82 (m, 2 H, H-4, H-5), 3.97 (dd, 1 H, J = 3.6, 9.0 Hz, H-3), 4.3 (dd, 1 H, J = 7.2, 9.5 Hz, H-2), 5.74 (d, 1 H, J = 3.6 Hz, H-1), 7.07-6.81 (m, 3 H, ArH), 7.33-7.21 (t, 2 H, ArH)

(112)

3-6-1

(substrate specificity)

(exo-type)

-L--L- Km kcat DNPF CNPF FNPF

pNPF MeNPF mNPF pCPF PF pNPAP pNPAF pNP-Gal pNP-Glc pNP-GlcNAc Km kcat 3-4 3-5

3-4 h-Fuc a Km kcat

Phenyl substrate Km (mM) kcat (s-1) kcat/Km (s-1.mM-1)

pNP- -L-Fuc p-NPAPc p-NPAFc pNP-Galc pNP-Glcc pNP-GlcNAcc 0.284 ± 0.002 0.73 ± 0.02 NDb ND ND ND 17.07± 0.02 1.9 ± 0.1 ND ND ND ND 60.11 2.6 a. 37 °C, 400 nm, pH 6.8 50 mM /0.1 M 0.25 ~ 2 mM b. ND

c. p-Nitrophenyl- -D-arabinopyranoside (p-NPAP) p-Nitrophenyl- -L-arabinofuranoside (p-NPAF) p-Nitrophenyl- -D-galactopyranoside (pNP-Gal) p-Nitrophenyl- -D-glucopyranoside (pNP-Glc) p-Nitrophenyl- -N-acetylglucosamine (pNP-GlcNAc) (from sigma)

3-4

-L-pNPF kcat = 17.07 s-1 Km = 0.284 mM

pNPAF pNP-Gal pNP-Glc pNP-GlcNAc

pNPAP pNPF 1/23 kcat = 1.9 s-1 Km = 0.73 mM pNPAP pNPF arabinose C5 -CH3 -H pNPAP kcat Km C5 (93) 3-5

(113)

CNPF FNPF MeNPF mNPF pCPF PF Km kcat 2 4 aryl- -L--L- 3-7

3-7

-L-Km Km (enzyme-substrate complex ES complex)

(apparent dissociation constant) Km

kcat turnover number

ES complex kcat

-L-(k2 k3)

k2 k3

NPF nitrophenyl fucoside NP nitrophenol F Fucose

kcat (s-1) (k2 k3)

kcat/Km (s-1.mM-1)

(114)

-L-(97) (56, 65) -L-SN1 -L -DNPF 3,4--DNPF CNPF FNPF pNPF MeNPF mNPF pCPF PF h-Fuc Km kcat Km kcat

pKa Br nsted plot

-L-pKa 2,4-dinitrophenyl- -L-fucopyranoside (pKa=3.96) 3,4-dinitrophenyl- -L-fucopyranoside (pKa=5.36) h-Fuc

-L- CNPF FNPF pNPF MeNPF mNPF pCPF PF pKa 5.42 9.99 37 0C 50 mM (pH 6.8) h-Fuc CNPF FNPF pNPF MeNPF mNPF pCPF PF Km 0.27 mM 0.28 mM 0.28 mM 0.48 mM 0.34 mM 0.71 mM 1.18 mM Km kcat pKa

3-5 3-10_A logkcat pKa Br nsted plot

logkcat/Km pKa Br nsted plot 3-10_B

3-5 -L- Km kcat

substrate pKa kcat (s-1) Km(mM) kcat/Km (s-1.mM-1) log kcat log (kcat/Km)

CNPF 5.42 18.77 0.27 68.77 1.27 1.84 FNPF 6.20 18.22 0.28 65.29 1.26 1.81 pNPF 7.18 17.07 0.28 60.11 1.23 1.78 MeNPF 7.42 13.64 0.48 28.42 1.13 1.45 mNPF 8.39 8.97 0.34 26.53 0.95 1.42 pCPF 8.49 8.95 0.71 12.58 0.95 1.1 PF 9.99 5.5 1.18 4.66 0.74 0.67

(115)

fucosylation

pKa kcat kcat/Km

3-10 log kcat pKa

Br nsted plot ( lg -0.13)

( )

log kcat /Km pKa ( lg =

-0.27) h-Fuc ( E.S complex) Km pKa 3-5 (C1) C-O (rate-limiting step) fucosylation step (A) (B)

(116)

3-8

-L--L- ( -L-fucosidase Fuc)

GH-29 GH-95 GH-29 GH-95

-1-3,4 -1-2

-L-(catalytic of the anomeric configuration)

GH-95 (inversion mechanism) GH-29

(retention mechanism) T. maritime S.

solfataricus

(essential amino acid)

(-AH) (leaving group)

(nucleophilic attack) fucosylation step defucosylation step

(Transition-State) carbocation fucosyl-enzyme intermediate 3-11

(117)

3-8-1

-L-25 °C

h-Fuc pNPF 3-12 3-13 pNPF

1 H (doublet) (chemical shift)

=5.87 ppm J1;2 = 4.0 Hz 3-12 -form 1 = 4.48 ppm J1;2 = 3.9 Hz -form 1 = 5.12 ppm J1;2 = 8.1 Hz 3-13 = 4.48 ppm 5.12 ppm 15 = 4.48 ppm 30 = 4.48 ppm = 5.12 ppm = 95 5 60 88 12 -form -form mutarotation -form 150 -form -form 72:28

-form h-Fuc (double displacement

mechanism) 3-11

- (fucosyl-enzyme

(118)

-3-13 h-Fuc pNPF

(a) pNPF h-Fuc

(b) h-Fuc pNPF 15

(c) h-Fuc pNPF 30

(119)

3-8-2

(Common intermidiate)

- phenol -3-14 3-14 h-Fuc pH-profile -L- (retenting) (two-step) (CNPF pNPF pCPF) (6.0 M) h-fuc D2O