Kor. J. Microbiol. Biotechnol. Vol. 35, No. 3, 196 202(2007) z ww z Homocysteine wz, Cystathionine β-synthase yw p Ÿy*ÁxûÁ 1 Á 1 œw w ywœ, 1 w m y l Biochemical Characteristics for the Cofactor Free Mutant of Yeast Homocysteine Catalyzing Enzyme, Cystathionine β-synthase. Jhee, Kwang-Hwan*, Hyun-Nam Cho, Seun-Ah Yang 1, and In-Seun Lee 1. Dept. of Applied Chemistry, Kumoh National Institute of Technology, Gumi 730-701, Korea, 1 The Center for Traditional Microorganism Resources, Keimyung University, Daegu 704-701, Korea Mutations in the cystathionine β-synthase (CBS) gene cause homocystinuria, the most frequent inherited disorder in sulfur metabolism. CBS is the unique enzyme using both heme and pyridoxal 5-phosphate (PLP) for activity. Among the reported 140 mutations, one of the most common disease-causing alterations in human CBS is G307S mutation. To investigate the pathogenic mechanism of G307S by spectroscopic methods, we engineered the full length and the truncated G247S mutation of yeast CBS that is corresponding mutation to human G307S. Yeast CBS does not contain heme and thus gives a merit to study the spectroscopic properties. The UV-visible spectra of the purified full length and the truncated G247S yeast CBSs showed the total absence of PLP in the protein. The absence of PLP in G247S mutation was also confirmed by the PLP-cyanide adduct formation experiment, which was conducted by the incubation of the purified enzyme with KCN. The adducts were detected using a circular dichroism (CD) and a spectrofluorimeter. Radio isotope activity assay of full length and truncated G247S proteins also gave no activity. Our yeast G247S mutation data suggested that G307S might make the distortion of the active site so that cofactor PLP and substrate can not fit inside the active site. Our yeast CBS study addressed the reason why the G307S mutation in human CBS makes the enzyme inactive that consequently leads to severe clinical phenotype. Key words: Homocysteine, Cystathionine β-synthase, Mutant, PLP 1964 homocystinuriaš w (NIH) w w š[16]. cystathionine β-synthase(cbs) ü homocysteine w ƒ,, p. 30 w wù j ñ wù, homocysteine y y y» w x w w, yw ƒ t š [17, 20]. š, CBS y homocysteine xƒ ƒw xš [9, 13]. w homocysteine Alzheimer disease x w *Corresponding author Tel: 82-54-478-7825, Fax: 82-54-478-7710 E-mail: khjhee@kumoh.ac.kr» w» w[18]. L-Homocysteine y ww L-methionine L-cysteine ü v. ww L-homocysteine L- methionine py (re-methylation) jù L- serine ww L-cystathionine transsulfuration k homocysteine w jš. û homocysteine re-methylation wù homocysteine ƒ CBS w w (Fig. 1). CBS ywƒ l [8]. homocysteine w w w, CBS w y š. x 140ƒƒ CBS ƒ š, ƒ w clinical phenotype š[10, 12]. š G307S ƒ wš [5]. G307S w z (Saccharomyces cerevisiae)» z w ww
Fig. 1. Reaction scheme catalyzed by cystathionine β-synthase. Fig. 2. Homology between human CBS and yeast CBS. Numbers indicated the start amino acids and final amino acids that were compared. Shadow box indicated the identical amino acids. Symbol as + represents the similar amino acid for isoleucine and valine. Glycine residues for G307 of human and G247 of yeast are indicated and represented as bigger fonts. PLP-FREE MUTATION OF YEAST CYSTATHIONINE β-synthase 197 š w[11, 19]. CBS z CBS 1 BLASTP 2.2.17 w alignment 62% š p ww y G307( ) G247(z) 93% p (Fig. 2), CBS G307 ww z CBS G247 serine eyw z G247S z w CBS x zyw ³wš w. wr CBS z heme pyridoxal 5 phosphate (PLP, k B 6 ) wwš, z CBS z PLP ƒš heme Ÿw f t PLP Ÿw p qw CBS z PLP» w, x, x z f ƒ Ÿ w ww[6]. wr CBS 551 N heme w(1-55)ƒ š 119 lysine»ƒ z PLP wwš. š 441 proteolysisƒ ù C ƒ w [7]. š (truncated) z z 2-3 w y. 1-441¾ N (Nterminal domain) (catalytic domain) tx wš 442-551¾ C (C-terminal domain) (inhibitory domain) txwš. w S-adenosyl methionine(sam) C ww CBS y š. CBS heme PLP z wš hinge site ƒš w SAM w y x allosteric z[15]. z ƒƒ 3 x w C-terminal, heme, PLP, SAM w w w z w» w. z CBS 507 CBS 44 CBSƒ ƒš heme wƒ w» [14], z CBS 53 lysine»ƒ PLP wwš. š 354» proteolysisƒ w[7]. SAM w y [7]. z CBS z CBS heme w C SAM wƒ w. CBS w z CBS w z, 1, z PLP w š w»p w f œ w z CBS CBS wš w heme w Ÿw ¼w z[8]. CBS G307» ww z G247 serine ey w full-length z CBS (F-CBS) 354» C w truncated z CBS (T-CBS) w Ÿw y š z rš w. š š CBS 3 w clinical phenotype G307 3 e w zyw l (seriousness) š w. CBS w f wwš qw ª CBS y mw homocystinuria e. x L-cystathionine, L-serine, PLP, L-homocysteine thiolactone, SAM Sigma w. L-[U- 14 C]Serine (160 mci/ mmol) NEN Life Science w. Gigapite hydoroxylapatite, Seikagaku w. L-Homocysteine L-homocysteine thiolactone š w w[3]. L-Homocyteine
198 JHEE et al. w», 5,5 -dithiobis(2-nitrobenzoic acid) 412 nm Ÿ 13,600 M -1 cm w -1 w[4]. w Buffer B 50 mm Na/bicine 1 mm EDTA 1 mm DTT ww ph 7.8 bufferš Buffer K buffer 1 mm EDTA 1 mm DTT ww ph 7.5 buffer. z G247S l x x z CBS š» w[6, 7]. x full length CBS (F- CBS) pf-sec, truncated CBS(T-CBS) pf- SEC w. G247S PCR w ww quick change site-directed mutagenesis kit pfu DNA polymerase (Stratagene) w. w primer. Forward primer, 5 -GTTGAGGGTATT- TCTTATGATTTTGTTCC-3 ; Reverse primer, 5 -GGAACA- AAATCATAAGAAATACCCTCAAC-3. G247S-F pf-sec template wš G247S-T pt-sec template w. plasmid ww E.coli ƒw, pf-sec ATCC No. 87791, pt- SEC ATCC No. 87792. x CBS(x x) x x š w [6, 7]. G247S x š w w wš G247S-F G247S-T z š w[6, 7]. zyd zy d ƒ w. C L-serine k universalw 14 k» w z k x L- cystathionine TLC w k Phosphor- Imager w w[6]. 1 unit 37 C w o g 1 mmol L-cystathionine xw y w.» L-serine L-homocysteine 5 mm w. yy(h 2 S) d w. CBS β-mercaptoethanol L-cysteine S-hydoroxyethyl-L-cysteine yyƒ x, yy lead acetate g lead sulfide (PbS) e xw, UV-visible ŸŸ 390 nm Ÿ dw[2, 7]. β- mercaptoethanol L-cysteine ƒƒ 30 mm w, lead acetate 0.4 mm w. lead sulfide (PbS) x Native-PAGE gel lead acetate k gel dw [2]. z PLP w d z PLP w Adams w[1]. r CBSz 5 mm buffer (ph 7.5) nw z, 11% trichloroacetic acid 50 o C 15 k z, 1.8 mm KCN 50 o C 25 k. x pyridoxal-p-cn excitation q 325 nm emission q420 nm dw. PLP w w w. Y=Y max [X]/K d + [X]», Y zy X PLP ùkü. PLP 0.1 N NaOH 388 nm PLP Ÿ e = 6.55 mm -1 cm w -1 w. Ÿ UV-visible rp Hewlett Packard 8453-diode array rp w. d j y 25 C o w w Peltier l w. CD r p Jasco J-175 w. š z x CBS G247S z x full length CBS (WT-F) truncated CBS (WT-T) š IPTG induction g wš 50% SDS-PAGE w w(data not shown). z G247S-F G247S-T, x šxwš x SDS-PAGE vj w(fig. 3). CBS e, column chromatography e ƒ SDS-PAGE mw ywš zy dw. zy d 1 w w x vwš TLC ww z, TLCq 12 radiography v w rw d 2, yy x lead acetate w dw w. ù G247S-F G247S-T 1 2 y SDS-PAGE w w. w x CBS š G247S-F G247S-T x w ƒ j ww. š G247S-F w j» 56 kda š, G247S-T 39 kda j
P Fig. 3. SDS-PAGE analysis of the purified yeast G247S proteins. Lanes; M, molecular mass standards (molecular mass in kda in parentheses) myosin (220), phosphorylase b (97), bovine serum albumin (72), ovalbumin (46), carbonic anhydrase (29), β-lactoglobulin (20), and lysozyme (15). 1, purified truncated form of G247S (G247S-T), indicating 39 kda; 2, purified full length form of G247S (G247S-F), indicating 56 kda. Yeast CBS mutants, G247S-F and G247S-T were purified from host cell XL-1 blue harboring mutated pf-sec and pt-sec. Each enzyme was purified using DEAE-Sephacel, Gigapite, and Butyl-Sepharose column chromatography. About 10 mg of protein was applied per lane. E.coli 기를 보였다(Fig. 3). 그리고 SDS-PAGE 상에서의 단백질이 정확히 CBS 단백질임을 확인하기 위하여 Fig. 3의 샘플의 N 말단순서를 Perkin-Elmer사의 protein sequencer를 이용하여 확인한 바 56 kda과 39 kda의 양 샘플 모두 M-T-K-S-E를 나타내었는데 이는 CBS의 N 말단의 다섯 아미노산의 순서 와 일치하므로 정제한 단백질이 CBS임이 확인되었다. 효모 G247S의 spectrum data 순수 정제된 야생형 CBS (WT-F)와 G247S (G247S-F)의 UV-visible 흡광도를 조사하였다. 야생형의 경우에는 보고된 바와 같이 280 nm와 412 nm에서 피크를 나타내었다. 280 nm의 피크는 단백질을 나타내고 412 nm의 피크는 CBS의 활성부위에 있는 lysine 잔기와 결합하고 있는 조효소 PLP 의 존재를 나타내고 있다. 그리고 그 비율이 280 nm : 412 nm = 1 : 0.152를 나타내었다(Fig. 4). 이는 보고된 수치 1 : 0.16과 매우 근사치를 나타내고 있으며 Fig. 3의 SDS-PAGE 의 결과와 더불어 CBS0.1%가 매우 순수함을 나타내고 있다. 280 nm에서의 WT-F의 A 280 값이 0.94이므로[7], Fig. 3의 단백 질의 농도는 정확히 1 mg/ml이 된다. 그리고 WT-T의 경우 는 WT-F의 흡수스펙트럼과 동일하였다(data not shown). 반 면 G247S-F의 경우에는 280 nm의 피크는 0.86으로 야생형 의 apo-enzyme의 경우와 정확히 일치하였다[7]. 즉 G247S 는 PLP를 함유하고 있지 않으며 야생형 CBS와는 다르게 412 nm에서 피크가 전혀 보이지 않았는데 이는 G247S-F가 조효소인 PLP를 전혀 함유하고 있지 않다는 증거이다. 이는 LP-FREE MUTATION OF YEAST CYSTATHIONINE β-synthase 199 Fig. 4. Absorption spectra of yeast CBS. Data exhibit the full length form of wild-type (WT-F, bold line) and full length form of G247S (G247S-F, dashed line). Each protein concentration was approximately 1 mg/ml in buffer B. All spectra were taken at 25oC. 왜 G247S가 전혀 활성을 보이지 않았는지에 대한 이유이기 도 하였다. L-homocysteine과 L-serine과의 반응에서 Lcystathionine 을 형성하는 반응이거나, β-mercaptoethanol과 L-cysteine과의 반응으로 S-hydoroxyethyl-L-cysteine과 황화 수소가 형성되는 반응이든 모두 PLP가 없으면 불가능하기 때문에 G247S는 촉매할 수 있는 반응이 없게 되는 것이다. G247S-T의 CBS의 경우에도 흡수스펙트럼을 측정하였으나 G247S-F의 CBS와 동일하였다(data not shown). 그리고 효 소에 기질인 L-serine을 넣어도 흡수spectrum에 영향이 없었 다. 이는 G247S-F가 기질과 결합하지 않음을 나타내고 있 다. 야생형의 경우에는 aminoacrylate 중간체의 형성으로 인 하여 460 nm 부근으로 흡수극대치가 이동한다[6]. 효모의 WT-F 와 WT-T, G247S-F와 G247S-T의 CD spectrum을 측정하였다. CD spectrum으로써 조효소, PLP의 함량을 알 수 있다[6]. 야생형의 CBS는 WT-F와 WT-T 모 두 412 nm 부근에서 (+)의 CD를 나타내지만 G247S-F와 G247S-T는 412 nm에서 CD를 보이지 않고 있다(Fig. 5). 이는 UV-visible 흡수스펙트럼과 같은 결과로써 G247S 변 이는 G247S-F와 G247S-T 모두 PLP를 함유하고 있지 않다 는 결과이다. 그리고 효소에 기질인 L-serine을 넣어도 CD spectrum에 아무 영향이 없었는데 이는 G247S 변이 단백질 이 기질과 전혀 반응을 하지 않음을 나타내고 있다. 야생형 의 경우에는 중간체의 형성으로 인하여 460 nm 부근에서 (-)의 CD를 보인다[6]. 그리고 야생형의 apo 단백질 역시 412 nm에서 CD를 보이지 않는다[6]. PLP 함량과 PLP와의 친화력 측정 G247S의 UV-visible과 CD spectrum의 결과를 보면
200 JHEE et al. Fig. 5. Circular Dichroism spectra of yeast CBS. Spectra exhibit the full length form of wild-type (WT-F), truncated form of wildtype (WT-T), full length form of mutant G247S (G247S-F), and truncated form of mutant G247S (G247S-T). Each protein concentration was 1 mg/ml in buffer K. All CD spectra were taken at 25 o C. G247S-F G247S-T PLPƒ x w. ù rp š PLP w dw. G247S trichloroacetic acid w g ü w PLP k z KCN j PLP-CN w ƒ š yw excitation q 325 nm emission q 420 nm xÿrp ùkü ü w PLP w. PLP Ÿ w w. WT-F WT-T ƒƒ 0.98 0.96 w PLPƒ 0.98 0.96 ƒš PLPƒ 1: 1 w. ù G247S-F G247-T ƒƒ 0.01 0.02 PLPƒ. wr zy d2 w WT-F WF-T apo z PLP ƒj y dw PLP w (association constant), Ka dw WT-F 0.8 µm, WF-T 0.7 µm ùkü. ù G247S G247S-F G247S-T PLP (2 mm, x 2,500) zy 1 z y 2 y. G247S PLP ww. CBS z y CBS zy ƒ dw. L- Homocysteine kƒ C L-serine» 14 w z k z TLC mw C 14 L-cystathionine C û» 14 L- Fig. 6. CBS activity assay. The reaction mixture, which contained 200 mm Tris-HCl, ph 8.6, 20 mm PLP, 0.25 mg/ml bovine serum albumin, 5 mm L-[U- 14 C]serine (800 cpm/nmol), and CBS (0.02-0.1 mg) in 18 ml, was preincubated for 5 min at 37 o C. The reaction was initiated by adding 2 ml of 5 mm L- homocysteine and was terminated after 10 min by adding 5 ml of 50% trichloroacetic acid. Reaction mixture was centrifuged for 3 min, and 5 ml of supernatant was applied to a cellulose thin layer chromatography plate (Kodak). The product, L-[ 14 C] cystathionine, was separated from L-[ 14 C] serine by ascending thin layer chromatography in 2-propanol/formic acid/h 2 O (80/6/20, v/v). Radioactivity of the product was determined by PhosphorImager (Molecular Dynamics) and the data was analyzed by AlphaImager HP software. The lanes are: C, L-[ 14 C] serine without CBS; 1, reaction with wild-type (WT-F); 2, reaction with truncated form of wild-type (WT-T); 3, reaction with full length form of mutant G247S (G247S-F); and 4, reaction with truncated form of mutant G247S (G247S-T). serine w z PhosphorImager L-cystathionine w(fig. 6). w L-homocysteine ƒ w PhosphorImager ùkù. spotalphaimager HPvp w x L-cystathionine z w control G247S-F, G247S-T y x š, WT-F WT-T ƒƒ 420 unit 780 unit» š w[7]. CBSyd β-mercaptoethanol L-cysteine S-hydoroxyethyl-L-cysteine x yy (H 2 S) dw. y G247S-F G247S-T x y. w Native-PAGE gel Pbacetate k z y dw G247S-F G247S-T x y (data not shown). w w yd, š PLPƒ w (2 mm¾ w) y x. G247S w G307S ƒ homocystinuria y, PLP n x y ƒ y ƒ
Fig. 7. Active site and G307 position in 3D structure of human CBS. Rasmol software was used and the human CBS 3-D coordination was from PDB code 1JBQ, human CBS[14]. Heme, PLP and active site lysine119 was represented. The distance between G307 residue and PLP was 7.2Å. w w. š G307S z y x (unpublished data). w G307S z complementation x x z CBS ew w[19]. zy w ww G307S z G247S w yw š G247S w Ÿw ƒ ƒw š PLP ƒ y. CBS G307 3 w x CBS 3 Rasmol vp mw y PLP lysine119 G307 e r. PLP lysine119 ε Schiff s base mw wwš N ew heme group PLP 11Å. G307 y PLP 7.2Å ù G307Sƒ PLP w x R group glycine w j R PLP-FREE MUTATION OF YEAST CYSTATHIONINE β-synthase 201 group ƒ serine ƒ ù PLPƒ y ö š ƒ. w» w w e š ƒ, w w bulky group» w m ww š. š ƒ» w j w G307 loop ew 3 y w w y. ƒ ƒw w CBS G307S 3 ww w š z CBS 3 x z x CBS G347S 3 z 3 y w w ƒ x z CBS» w w. x y x w homocysteine ü yj z cystathionine β-synthase w ww. cystathionine β-synthase ƒ» zy ƒ w homocysteine j homocystinuria» G307 serine ey ƒ w. wr cystathionine β-synthase heme prosthetic group ƒš rp ƒ ù,» š cystathionine β-synthase ƒ z cystathionine β-synthase heme swwš rp w w G307 ww G247 serine ey, w yw p r. z G247S C truncated form w full length form ƒ w xw.» L-homocysteine L-serine w C 14 w y dw y x β-mercaptoethanol L-cysteine» w yy w. w UV-visible spectrum CD spectrum PLP p 410 nm x w. w PLP KCN incubation x PLP w. š cystathionine β-synthase 3 w, G307 z PLP ew bulkyw R group serine ey z PLP w w y w ƒ. G247S š PLP incubationw y z PLP
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