Shrivastava Et Al 2010 Benzyl Alcohol- and Benzaldehyde Dehydrogenase from P. Putida CSV86

Shrivastava et al 2010 Benzyl alcohol- and Benzaldehyde dehydrogenase from P. putida CSV86

Revised

ArchMicr-2011-0010

Supplementary material/Online resource

Archives of Microbiology

Purification and characterization of benzyl alcohol- and benzaldehyde- dehydrogenase from Pseudomonas putida CSV86

Rahul Shrivastava*, Aditya Basu*, and Prashant S. Phale†

Department of Biosciences and Bioengineering

Indian Institute of Technology-Bombay,

Powai, Mumbai 400076, India

*Both authors contributed equally.

†Corresponding author:

Professor PS Phale,

Email:

Ph: +91-22-2576 7836

Fax: +91-22-2572 3480

Section I. Purification of benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH). Both BADH and BZDH were purified simultaneously from P. putida CSV86. All purification steps were carried out at 4°C or on ice.

Preparation of cell-free extract.Cells grown on benzyl alcohol (0.1 %) were harvested by continuous flow centrifugation (CEPA, Switzerland) at 37000 × g and washed twice with Buffer A [glycine-KOH (50 mM, pH 8.5), glycerol (5 %), dithiothreitol (2 mM)]. The cells (~23 g, 7.5 l culture) were suspended in the same buffer in the ratio of 1:4 (w/v). The cell suspension (~95 ml) was sonicated with an ultrasonic processor (GE130, USA), 25 cycles of 15 pulses (each pulse of 2 second and 15 watts) and 2 min interval between each cycle. The cell homogenate obtained was centrifuged twice at 37000 ×g for 30 min. The clear amber colored supernatant was referred to as the cell-free extract (~100 ml) and used for the purification of both enzymes.

Ammonium sulfate fractionation. The cell-free extract was brought to 0-20 % followed by 20-55 % ammonium sulfate saturation by addition of solid ammonium sulfate, incubated for 30 min with constant stirring and centrifuged at 37000 × g for 30 min. Both BADH and BZDH were co-precipitated in 20-55% fraction. The protein pellet was dissolved in Buffer A and dialyzed against Buffer B [Buffer A + benzyl alcohol (5 mM)] for 5 h.

DEAE-Sephacel ion exchange chromatography. The dialyzed ammonium sulfate (20-55 %) fraction was loaded onto DEAE-Sephacel column (120 × 17 mm, vol. 25 ml) pre-equilibrated with Buffer C [Buffer B + isopropanol (10 %)], washed with Buffer C (200 ml) containing KCl (75 mM) and eluted with a linear gradient of 75 - 200 mM KCl in Buffer C (200 ml). Fractions (2.5 ml, flow rate-30 ml.h-1) were collected using a fraction collector (Redifrac 920, GE healthcare). Both enzymes were co-eluted in the range of 110-180 mM. The fractions having BADH and BZDH activities greater than 1 unit were pooled and dialyzed for 5 h in Buffer D [potassium phosphate (pH 6.5, 50 mM), glycerol (5 %), DTT (2 mM), benzyl alcohol (5 mM)].

Blue-agarose column chromatography.The dialyzed enzymes were then loaded onto Blue-agarose column (120 × 12 mm, vol. 13 ml) pre-equilibrated with Buffer E [Buffer D + isopropanol (10 %)]. The column was washed with Buffer E (100 ml). BZDH was bound to the matrix while BADH remained in the unbound fraction. Bound BZDH was eluted with a linear gradient of NAD+ (0 to 1 mM) in Buffer E (200 ml). Fractions (2.5 ml, flow rate-30 ml.h-1) were collected and those having more than 1 unit of BZDH activity were pooled and concentrated by ultra-filtration (membrane mol. wt. cut-off 30 kDa, Amicon).

Phenyl-Sepharose chromatography.The unbound fractions containing more than 1 unit of BADH activity from Blue-agarose chromatography were pooled, loaded onto Phenyl-Sepharose column (120 × 12 mm, vol. 13 ml) equilibrated with Buffer E and washed with Buffer E (150 ml). The bound enzyme was eluted with a linear gradient of 0 to 75 % ethylene glycol in Buffer E (200 ml). Fractions (2.5 ml, flow rate-30 ml.h-1) were collected and those having activity more than 1 unit were pooled and dialyzed (membrane cut off 12 kDa) against Buffer B for 5 h.

Q-Sepharose column chromatography.The dialyzed BADH was loaded onto Q-Sepharose column (120 × 12 mm, vol. 13 ml) equilibrated with Buffer C. The column was washed with Buffer C (200 ml) and the bound enzyme was eluted with a linear gradient of 0 - 500 mM KCl in Buffer C (200 ml). Fractions (2.5 ml, flow rate-30 ml.h-1) were collected and those having more than 1 unit of BADH activity were pooled and concentrated by ultra-filtration.

Sephacryl S-300-HR gel filtration chromatography.Concentrated BADH and BZDH were loaded separately onto Sephacryl S-300-HR gel filtration column (750  12 mm, vol 90.5 ml, void vol. 34 ml) pre-equilibrated and developed with Buffer D. Fractions (1 ml, flow rate-6 ml.h-1) were collected. Peak fractions of BADH and BZDH were pooled, concentrated and dialyzed against Buffer D containing glycerol (40 %) and stored at -20oC.

Section II. Molar absorption coefficient (ε) for various aromatic alcohols and aldehydes and calculated correction factors.To determine the molar absorption coefficient, the stock solution of various compounds was prepared in dimethylsulphoxide (DMSO). The presence of DMSO (≤ 1%, v/v) in the reaction mixture did not affect the activity of BADH and BZDH and it does not absorb at 340 nm. The molar absorption coefficients () of substrate and product were determined under standard assay condition. Absorption coefficients of more than 1 % of the value of NADH at 340 nm were taken into consideration and used to calculate correction factor (Online resource,Table S.1) using equation, Correction factor = (NADH) / [(NADH)340 – (substrate) + (product)].

Section III. HPLC analysis.To identify and confirm the reaction product, the bulk enzyme reactionwith various aromatic substrates and purified enzymes were performed and analyzed by an HPLC (Agilent, HP 1100, USA)using octadecyl silica reverse-phase column (250 × 4.6 mm; particle size 5 , Zorbax), maintained at 40°C. The column was developed (flow rate- 1 ml.min-1) using solvent A (0.1 % phosphate in water) for 1.5 min followed by the gradient of 0 - 50 % of solvent B (0.1 % phosphate in acetonitrile) for 1.5 min followed by 50 - 70 % of solvent B for 20 min. The metabolites were detected by a diode array detector at five different wavelengths (230–280 nm with 12 nm increments). Identification of the reaction product was done by HPCHEM ™ software by comparing the absorption spectra and retention time to those of authentic compounds (Online resource,Table S.2).

Shrivastava et al 2010 Benzyl alcohol- and Benzaldehyde dehydrogenase from P. putida CSV86

Table S.1. Molar absorption coefficient () for various aromatic alcohols and aldehydes and calculated correction factors.

Compound / BADH forward* / BADH reverse / BZDH
 (M-1.cm-1) / Correction Factor /  (M-1.cm-1) / Correction Factor /  (M-1.cm-1) / Correction Factor
Substrate / Product / Substrate / Product / Substrate / Product
balc† / bald / bald / balc / bald / bacid
2-Methoxy- / 0 / 300 / 0.954 / 2900 / 0 / 1.873 / 2000 / 0 / 1.474
3-Methoxy- / 0 / 100 / 0.984 / 700 / 0 / 1.127 / 600 / 0 / 1.107
4-Methoxy- / 0 / 0 / 1.000 / 0 / 0 / 1.000 / 0 / 0 / 1.000
2-Hydroxy- / 0 / 900 / 0.874 / 2200 / 0 / 1.547 / 1900 / 0 / 1.440
3-Hydroxy- / 0 / 700 / 0.899 / 800 / 0 / 1.148 / 1200 / 0 / 1.239
4-Hydroxy- / 0 / 19700 / 0.240 / 3700 / 0 / 2.468 / 20700 / 0 / -0.430
2,3,4-Chloro- / 0 / 0 / 1.000 / 0 / 0 / 1.000 / 0 / 0 / 1.000
2,3,4-Methyl- / 0 / 0 / 1.000 / 0 / 0 / 1.000 / 0 / 0 / 1.000
2-Nitro- / 600 / 800 / 0.969 / 600 / 700 / 0.984 / 600 / 700 / 0.984
3-Nitro- / 500 / 400 / 1.016 / 200 / 500 / 0.954 / 200 / 300 / 0.984
4-Nitro- / 600 / 1600 / 0.861 / 500 / 600 / 0.984 / 1200 / 600 / 1.107
1-Hmn-‡ / 0 / 2800 / 0.690 / 4500 / 0 / 3.616 / 4100 / 0 / 2.934
2-Hmn- / 0 / 900 / 0.874 / 1800 / 0 / 1.407 / 1600 / 0 / 1.346

*BADH forward reaction (pH 9.0) includes substituted aromatic alcohol as substrate and aldehyde as product, while BADH reverse reaction (pH 7.0) includes substituted aromatic aldehyde as substrate and alcohol as product. On the other hand, BZDH reaction (pH 9.6) includes substituted aromatic aldehyde as substrateand aromatic acid as product. Molar absorption coefficients were determined at 340 nm under the standard assay conditions (See Materials and Methods).

†Abbreviations used are balc, benzyl alcohol; bald, benzaldehyde; bacid, benzoic acid; 1-Hmn, 1-hydroxymethylnaphthalene; 2-Hmn, 2-hydroxymethylnaphthalene.

‡In case of 1-Hmn and 2-Hmn, their aldehydes and acids are known as naphthaldehydes (1-nald or 2-nald) and naphthoic acids (1-nacid or 2-nacid) respectively.

Table S.2 HPLC analysis of the products obtained after bulk enzyme reactions of benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH) from Pseudomonas putida CSV86.

BADH / BZDH
Rt† / Forward‡ / Rt / Reverse / Forward / Rt
Balc* / 6.8 / 36.5 (100) / Bald / 8.5 / 147 (100) / Bald / 419 (100) / Bacid / 7.0
2-Methoxybalc / 7.0 / 23.9 (65) / 2-Methoxybald / 8.9 / 86.7 (59) / 2-Methoxybald / 11.9 (3) / 2-Methoxybacid / 6.7
3-Metthoxybalc / 6.9 / 41.8 (114) / 3-Methoxybald / 9.0 / 131 (89) / 3-Methoxybald / 741 (177) / 3-Methoxybacid / 7.2
4-Methoxybalc / 6.7 / 150 (411) / 4-Methoxybald / 8.4 / 109 (74) / 4-Methoxybald / 787 (188) / 4-Methoxybacid / 7.0
2-Hydroxybalc / 6.2 / 152 (417) / 2-Hydroxybald / 8.6 / 121 (82) / 2-Hydroxybald / 204 (49) / 2-Hydroxybacid / 7.4
3-Hydroxybalc / 6.0 / 53.4 (146) / 3-Hydroxybald / 6.7 / 97.9 (66) / 3-Hydroxybald / 1443 (345) / 3-Hydroxybacid / 6.1
4-Hydroxybalc / 5.8 / 183 (501) / 4-Hydroxybald / 6.1 / 513 (348) / 4-Hydroxybald / 749 (179) / 4-Hydroxybacid / 6.5
2-Chlorobalc / 7.8 / 13.3 (36) / 2-Chlorobald / 10.8 / 175 (118) / 2-Chlorobald / 58.5 (14) / 2-Chlorobacid / 7.4
3-Chlorobalc / 8.0 / 24 (66) / 3-Chlorobald / 10.7 / 154 (105) / 3-Chlorobald / 829 (198) / 3-Chlorobacid / 8.2
4-Chlorobalc / 7.9 / 46.4 (127) / 4-Chlorobald / 10.5 / 134 (91) / 4-Chlorobald / 1532 (366) / 4-Chlorobacid / 8.3
2-Nitrobalc / 7.2 / NA§ / 2-Nitrobald / 8.5 / 114 (77) / 2-Nitrobald / NA / 2-Nitrobacid / ND
3-Nitrobalc / 7.1 / 9.6 (26) / 3-Nitrobald / 8.4 / 98 (67) / 3-Nitrobald / 920 (220) / 3-Nitrobacid / 7.2
4-Nitrobalc / 7.0 / 10.5 (29) / 4-Nitrobald / 8.5 / 191 (129) / 4-Nitrobald / 492 (118) / 4-Nitrobacid / 7.3
1-Hmn / 8.7 / 19.8 (54) / 1-Nald / 12.6 / 156 (106) / 1-Nald / 15.9 (4) / 1-Nacid / 8.9
2-Hmn / 8.7 / 62.6 (172) / 2-Nald / 12.2 / 129 (87) / 2-Nald / 590 (141) / 2-Nacid / 9.0

*For abbreviations see Supplementary Table S.1. †Rt, is retention time in min. ‡The numbers in the columns denote specific activity (µmol.min-1.mg-1) and the value in parenthesis is the percentage activity for those particular substrates taking activity on benzyl alcohol and benzaldehyde as 100 %, for BADH and BZDH, respectively.

§NA or ND; no activity detected or the product was not detected by this method.

Shrivastava et al 2010 Benzyl alcohol- and Benzaldehyde dehydrogenase from P. putida CSV86

Figure S.1.

....|....| ....|....| ....|....| ....|....| ....|....|

10 20 30 40 50

1 ------MHTL MDQAVWQD-- --KLMCDGWR ASEAR---AA

2 ------MTDLLGGET WSGKIFDGNW VTASG-GSIK

3 ------MTRLLSEPV WKSKIFDGEW VRGSA-DDRI

4 ------MATQLK STENDYGIKS EYGHYIGGEW IAGDSGKTID

5 ------M QESPVQQSAK QVSLLINGDA TSARGGQTFQ

6 ------MGAA DATAFFN------FSGLK QAALGGGARD

7 ------MNAP HAMRFLDDAQ WDGLLFQGHW VPPLRRQTVD

8 ------MRETKEQPI WYGKVFSSNW VEARG-GVAN

9 ------MAVFASDSFG QLKVEKIMTA QWNHYINGEY VSPESEEYIH

10 ------MSIFTKEL WDKKLFNGSW QS-AQ-DTYS

11 MVLNNRKNEI IEKARTGRYE VYMTLLDASI WNKKLFNGGW FE-SG-QPYG

12 ------MSAS DGTPLLHRAI EAECVFNGDW IPSSS-PLLP

13 ------MN KTSKFLPEEL WSNCVFDGTW INISP-DVSQ

14 ------ML CVDTLLPNEL WNERLFTGTW VPGEL-PATA

Clustal Co .

....|....| ....|....| ....|....| ....|....| ....|....|

60 70 80 90 100

1 VTDKATGEQI GSIGVADAES VVRAAALARA AQPAWHAMPY EDRAAIFRKA

2 VTEPATGAVL GEIGAADASD IDRAARRAAA AQPDWAARTG HARADVLRKA

3 VVEPATGKQL TTVGMATPDD VAAAATKATA AQKNWAARPY TERAAVLRRA

4 LLNPSTGKVL TKIQAGNAKD IERAIAAAKA AFPKWSQSLP GERQEILIEI

5 RHNPLNGSVA SVAPAASVQD AVAAVHAASA AFPSWSNTGP GQRRALLMRA

6 VIEPATGQVL CTVGIADAAD VAAATATAAA AQKAWVAVPP REKAEIFRRA

7 VVEPATGRKL TRVGLADAQD VDAAVQAAAA AQSAWVALPP RERAKVFHRA

8 VVDPSNGDIL GITGVANGED VDAAVNAAKR AQKEWAAIPF SERAAIVRKA

9 EFIPTTALPG DSIARGSAAD VDKAVRAAAA AQPAWNARKP IERGRILLAI

10 VIEVATGQVL GEIGYATAAD VVSAAQQAKA AQQQWWALNY QERQAVFERA

11 VVEVATGEQL GQTGSASPTD VAQAAKEAQT AQRQWWALDY LERQAVFEKA

12 VIEPATGELL MNTAMADAAD IAVACREAAL AQPAWAALGP REKAEIFLLA

13 VIEPATGASL GCIGMANAAS VSAAARVANE VQADWFLTAY EERAAIMRKA

14 VTSPGNGVAL GALANAGAKQ VEQSAREAAT AQRSWYALPY DERAKVLRKA

Clustal Co .. . . : * . * : :.

....|....| ....|....| ....|....| ....|....| ....|....|

110 120 130 140 150

1 ARLVEEHWEE LATWIVRESG -AIRPKADVE LKAAAGILYE AAGMLTEPQG

2 AMTLDANRAE VERWLIREGG -AVPGKAAFE VDLVLSELWE ASALPTQPWG

3 GQLFEQYADE IGEWVIREAG -SIPAKAGLE THTAAEECYN AAALAAHPVG

4 ARRLKARQSH YATLETLNNG KPMRESMFFD MTQTIGQFEL FAGAAYGLHG

5 ADALEAKTQA FVDIMVLEIG -SSAGWAQFN VRLAAGALRE AAALTTQIGG

6 AALFQQHGDE LALYAARETG -GILAKGQHE IREAALICNL AASIPLQSQG

7 AMLFEQHFDE LALAIARETG -AALFKGEHE VREAITLCHA AAGMPLAAQG

8 AEKLKEREYE FADWNVRECG -AIRPKGLWE AGIAYEQMHQ AAGLASLPNG

9 ARLVRANAAA FCAKEAEETG KPLKMAA-FE IEACAQYFEY YGGLATAIQG

10 AALLTENQAE VIEWLVKESG -SLQLKAGFE VSIAIQVLKH CIASPTNEQG

11 VQIATEHQDE LVNWLIRESG -SLALKAQFE VKVSIQMLKN CIAFPQLDQG

12 ADHAVCAFDE LALYVARESG -GSLHKGQHE VNEAIVLLRQ AAGMLSQAHG

13 AEIAELHFGD IVEWLVRESG -STQLKAAFE TRVTIKALQE SAMMPGKSQG

14 AAIAEQYQAE IANWIVRESG -STQAKAGFE ASISIKALHE ACALPSRSQG

Clustal Co : * : *

....|....| ....|....| ....|....| ....|....| ....|....|

160 170 180 190 200

1 L--LLPSNPG --RMSFARRL PHGVVGVIAP FNFPLILAIR AVAPALATGN

2 H--LLPTSQE -GRVSIGRRV PLGVVGVISP WNFPLVLAMR AVAPALALGN

3 E--VLRSEQP --RLSFSRRV PVGTVGVIAP FNFPIILSIR AVAPALALGN

4 Q----TLDY- PDAIGIVHRE PLGVCAQIIP WNVPMLMMAC KIAPALASGN

5 E--VIPSDVP -GNLALAVRQ PAGVVLGIAP WIAPVILAVR AIATPLACGN

6 H--VLPSVPG --RTSIARRV PHGIVGVISP FNFPMILTIR SVAPALAAGN

7 Q--VLASQAG --RLSYARRA PFGVVGVISP FNFPLFLTLR SVAPALAAGN

8 T--LFPSAVP -GRMNLCQRV PVGVVGVIAP WNFPLFLAMR SVAPALALGN

9 E----TINLG PSYHAYTTRE PFGVVGVILP WNSPLNQAGR AIAPALVSGN

10 T--LLPTQNG --KLSIAKRL PLGVVGVISP FNFPLYLALR AVAPALAFGN

11 N--ILPSRNG --KLSLAKRL PLGVVGVISP FNFPLYLALR AVGPALAFGN

12 HGLMLPSAAG --RLSYARRV AHGVVGVISP FNFPLVLSMR SVAPALAAGN

13 D--VLPNAPG SGRLSLARRR PLGVVGIISP FNFPLYLAMR SVAPALAVGN

14 E--VLPSTAG --RLSIARRR PLGVIGVISP FNFPLYLAMR AVAPALATGN

Clustal Co * . * * * : *: :...*. **

....|....| ....|....| ....|....| ....|....| ....|....|

210 220 230 240 250

1 AVVLKPDLRT AVSGGVLIAR IFEEAGLPPG VLHMLP---- GGADAGGALC

2 AVVLKPDVQT AISGGVVIAQ LFAAAGLPEG VLHVLA---- GDAEPGQALS

3 SVILKPDPRT AICGGVVLAR IFEEAGLPAG VFSMLP---- GGADAGEAIV

4 TVVLKPAETV CLSVIEF--F VEMADLLPPG VINVVTG--- YGADVGEALV

5 TVVFKGSEQC PATHG-LVMH TLQEAGFPRG VLNFITNAPA DAATVVEAMV

6 AVVVKPDSRT PVTGGLMIAR IFAQAGLPKD LLQVLP---- GDAEAGEALV

7 AVVVKPDART PVSGGYLIAR VFAEAGLPAG LLHVLP---- GGAEAGEALV

8 AVILKPDLQT AVTGGALIAE IFSDAGMPDG VLHVLP---- GGADVGESMV

9 TVVVKPSEFT SVTMLQFAEL VVKEAGLPPG VLNVVTG--- TGKETGEPLV

10 AVVLKPDERT AVCSGYVIAR IFELAGLPKG LLHVLP---- GGVEVGEALT

11 AVVLKPDERT AVCSGYAIAR IFELAGLPKG LLHVLP---- GGADTGEALT

12 AVVLKPDPQT PISGGFLIAR LFEEAGLPKG LLHVLP---- GAADAGEALC

13 SVVLKPDPRT AVSGGLVIGR LFELAGLPKG VLHVLP---- GGGDAGAALV

14 AVVLKPDPRT AVCGGHVIAR IFELAGLPAG VLHVLP---- GGAEAGAALT

Clustal Co :*:.* :* . :: .:. .:

....|....| ....|....| ....|....| ....|....| ....|....|

260 270 280 290 300

1 TDPNIAMVAF TGSTRVGRTV GELCGRHLKK VSLELGGKNS LIVLDDADLD

2 EHPAVSLISF TGSTSVGRQV GATAGRTLKR VSLELGGNNA LIVLDDADLD

3 VHPSVRVIAF TGSTRAGRAV GELAGRYLKR AHLELGGNNA LIVMDDVDLE

4 TSPDVAKVAF TGSIATARRI IQYASANIIP QTLELGGKSA HIVCGDADID

5 AHPSVRRVNF TGSTQVGRRI ASACANHLKP VVLELGGKAP FLVLDDADLD

6 IDPLVPMIAF TGSPAVGRRI GELAGRHLKK VSLELGGANS LIILDDADLD

7 VHARVPMIAF TGSPGIGRRI GELAGRHLKK VSLELGGANA LIILDDADLD

8 ANSGINMISF TGSTQVGRLI GEKCGRMLKK VALELGGNNV HIVLPDADLE

9 KHPLIRKVAF TGSVRAGREI GKLAADRIIP LSLELGGKSP NIVFEDADLD

10 LDQNIASIQF TGSTQVGRIV GANAAKTLKK VSLELGGKNS LIILDDADIE

11 LDPHIASIQF TGSTQVGRII GANAGKTLKK VSLELGGKNS LIILDDADLD

12 RDTNVQMITF TGSTAAGRKV AEAAGRNLKK VSLELGGKNP LVILEDADLD

13 ESPDVAMIQF TGSTAAGRKV GEAAGRNLKK VSLELGGKNS LIILDDANLD

14 ASPSVAMIQF TGSTAAGRKV GEAAGRHLKK VSLELGGKNS LIILDDADLD

Clustal Co : : * *** .* : .. : ***** :: *.:::

....|....| ....|....| ....|....| ....|....| ....|....|

310 320 330 340 350

1 LAAGCAAFGA WLHQGQICMA TGRILAHESI AEQLLAKLAE KADHLPVGDP

2 AASSAGAWGS FLHQGQVCMT AGRHIVLESV ADRYLEKLTA RAANLPVGDP

3 KAASVGAFGS FMHQGQICMT TGRQLVHESI AADYTALLAE HADKLPVGDP

4 AAVESATMST VLNKGEVCLA GSRLFLHQSI QDEFLAKFKT ALEGIRQGDP

5 AAACAAVFGA FANSGQICIS TERIIVDRKV ADTFVQKLAD RAASLPLGDP

6 VAASNAAWGA YFHQGQICMA SNRILVHESM AAELTRRLIA KATHLPMGNG

7 LAASNAAWGA WLHQGQICMA ANRILVHASL ADALTARLVE KAQHLPVGDG

8 GAVSCAAWGT FLHQGQVCMA AGRHLVHRDV AQQYAEKLAL RAKNLVVGDP

9 RAVAGSVFAF TVNTGQVCLA GTRCLVHESI FEKFSKKLAG AVEALQFSD-

10 LAAENIAWGA FLHSGQICMT SGKILIHQKI YQQVKQRVIE KVQNFVVGNP

11 LAAENIAWGA FLHSGQICMT SGKILIHEKI YDALKARVIE KVKRFVVGNP

12 LAASNAAFGA WLHQGQICMA TGLILVHESI VVDLTRKLAD KARALTVGNA

13 LAVANAIWGT FLHQGQICMA TGRILVQRSL YPAFLKALTA KVGKLSVGDP

14 LAIANATWGV YLHQGQICMS TGRVLVQRGI YDAFVARLVA KARSLTVGDP

Clustal Co * . : *::*:: : : . : .:

....|....| ....|....| ....|....| ....|....| ....|....|

360 370 380 390 400

1 ASGRVALGPI ISDGQLRQID AVVKDSVAAG ARLCAGG------TYDQLF

2 NTGQVALGPL INEHQVANVD RIVRATRDAG AEIRTGG------TFDGLF

3 FTGQVALGPI IDAKQRDHIH SLVNGSIAAG AKLAAGG------TYEDLF

4 LDMATQLGAQ ASKMQFDKVE SYLQLATEEG AEVLTGGTRA DAANLAGGNF

5 RESAVALGAV VDMQTVHRCN ALIDDALAQG GRLVAGG------KADSTL

6 ASGQVALGPL IDARQRDRVH ALVQDSVKAG AKLEAGG------VFDGLF

7 AGGQAALGPL IDQKQLQRVH AIVQDSVAAG ARLLAGG------AYEQLF

8 NSDQVHLGPL INEKQVVRVH ALVESAQRAG AQVLAGG------TYQDRY

9 -GESFGLGPL TTKAQFEQVH RYNELAIQEG AHCLVGG--- EAPSDKTGWY

10 CDKNVTIGPL INAKQAQRVE QLVSAAVNEG ATLEIGG------HADGVF

11 LEQNVTIGPL INEKQSKRVE ALVQAAVEHG ATLEIGG------KANGPF

12 ARGEAALGPL INKRQLQHVH QVVSDSLQAG AQLETGG------EYGGLF

13 ATTKVSLGPL INAHQRDQVA KIVREAQQAG AKLEVGG------RYEELF

14 ASGQVHLGPL INAAQRDNAL RIVDAARAAG ATLATGG------AADGLF

Clustal Co :*. . : * . **

....|....| ....|....| ....|....| ....|....| ....|....|

410 420 430 440 450

1 YRPTVLGGVK PGMRAFEEEI FGPVACVTTF RDEDEAVALA NATEYGLSAA

2 YRPTVLAGVT PAMPAFQEEI FGPVAPVVVV KDEAEAIAVA NDTEYGLVAA

3 YRPTVLADTP LTAPAFADEV FGPVAPVTPF ATVEQAVAIA NDSEYGLSLG

4 IKPTVFTNVK NTMRIAQEEI FRPVTTVISW NDQTHMMQQA NATTYGLAGG

5 MPATLIDHVT PSMRLYREEA FGPVKGIVRV NGVEEAVACA NDNEYGLAAA

6 YKPTVLTGVK AGMRVYDEET FGPVANIITY KTDAEAIALA NNNTGSLSSA

7 YRPTVLGGVR PGMRVFDEEV FGPVASIIPF RDDDEAVALA NDSQGGLAAG

8 YQATVIMDVK PEMEVFKSEI FGPVAPITVF DSIEEAIELA NCSEYGLAAS

9 VRPTVYTNVN NSMRIAREEI FGPVLVLIPF KDENEAVAIA NDSDYGLAAG

10 FQPTVLTDVT ANNSIFSEEI FGPVAVLIPF SSDEQAIELA NDGDYGLSAG

11 FEPSVLSQVQ ADNPIFSEEI FGPVAVLIPF ASDEQAIELA NMGDYGLSAG

12 YRPTVLSGVK PGMRAFEEEI FGPVAIVVSF STDEEAIELA NRSEYGLAAA

13 FQPTVLSDVK PDNPAFREEI FGPVAVVVPF NSDEEAIRLA NDTEYGLAAG

14 FQPTVLSDVQ RDNPAFHEEI FGPVAVVVPF DSDEQAVELA NDTDYGLSAA

Clustal Co .:: . .* * ** : . : * * .* .

....|....| ....|....| ....|....| ....|....| ....|....|

460 470 480 490 500

1 VISRSTGRAL ALGNRLNTGL LHINDQTVAD EPHVPFGGRA ASGNGGRVGG

2 IQTGSVERGE RLADQLRTGM VHINDQTLNN DAYAPFGGTG ASGNGTRFGT

3 ILTSDAMRGL ELAQQIPTGL VHINDQTVGD EAVIPFGGMA SSGNGGRVGG

4 VWTKDLARAH RIARQLETGT VWIN-RYYNL KANMPLGGYK QNGFGREFSH

5 VFGRDVGRAI AVARRIESGI CHVNGATVHD EPQMPFGGIK DSGMG-RFGG

6 VISRSVGRAM AVGQQLKSGM VHINDQTVND DCVNPFGGPG IAGNGTSVGG

7 VISASVGRAM ALGQRLRVGM LHINDQTVND DCINPFGGPG IAGNGSSMGG

8 IHTRALATGL DIAKRLNTGM VHINDQPINC EPHVPFGGMG ASGSGGRFGG

9 VWTTDLARAH RVSAQIEAGQ VYVN-EYPSG GVETPFGGFK QSGHGREKGI

10 IITSNVGRGM QLGAQLKVGL LHINDQTVND ETVNPFGGFG SSGNGTRIGG

11 IISSNVGRAM QLGAQLNVGL LHINDQTVND ETINPFGGFG ASGNATRIGG

12 VISPNVGRAT AIGDRLRCGM LHINDQTVAD ECINSFGGRG ASGNGCSAGS

13 IISSNIGRAM KVGDKLRTGM LHINDQTVND EVVNPFGGWG ASGNGTSVGG

14 ILSRDTGRAL RLGEQLRTGL LHINDQTVND EVINPFGGVG ASGNGSSIGG

Clustal Co : . :. :: * :* .:** * . .

....|....| ....|....| ....|.

510 520

1 P-ANWEEFTQ WQWVTVKDTP PAYPF-

2 Q-SSWDEFTQ WQWRTSRSVA QQFPF-

3 IGANLDAFTE TQWVTAQSAL PVYPF-

4 EVLNHYTQTK SVVVNLQEGR VGMFDQ

5 K-AGIDAFTD LRWLTVQTTP RDYSF-

6 P-ADFEEYTM WQWMTIKDTA PNYPF-

7 P-ADWDAYTQ WQWVTVKDSA PRYPF-

8 P-ASIEEFTQ SQWISMVEKP ANYPF-

9 EALHHYTQTK TTIIRI------

10 P-ANADEFTQ WQWITVQAQA PHYPF-

11 P-ANPDEFTQ WQWMTIQAEA PHYPF-

12 P-SDWEEYSQ WQWVTVKNQA PTYPF-

13 A-ANWEEFTQ WQWLTIKDEA TPYAL-

14 A-ANWEEFTQ WQWLTLKGQA PAYPL-

Clustal Co :

Figure S.1. Alignment of amino acid sequences of BZDH from various micro-organisms. Cluster score representation: *, . and :denote identical sequences, conserved substitution and semi-conserved substitution respectively.

Figure S.2.

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

10 20 30 40 50 60 70

1 ------TCAGAAG GGATAGGCCG GCGGCGTATC CTTGACGGTG

2 ------TCAGAAC GGGAACTGTT GGGCCACAGA GCGGCTGGTG

3 ------ATG-ACGC GTTTGCTGAG CGA-ACCTGT GTGGAAATCG

4 ------ATGGCTA CGCAGTTGAA AAGTACAGAA AACGACTACG GCATTAAGTC CGAGTACGGC

5 ------TCAGAAC GAGTAATCGC GCGGGGTCGT CTGCACGGTC

6 ------ATGGGTGC AGCAGATGCA ACA-GCATTT TTT------

7 ------TCAGAAC GGGTAGCGCG GCGCGCTGTC CTTGACCGTC

8 ------AT GCTT-TGCGT GGA--TACCT TACTGCC-CA ACG-AACTCT GGAA--CGAG

9 ------ATGCGGGA AACAAAAG-A GCA-GCCTAT CTGGTACGGG

10 ------ATGCGGGA AACAAAAG-A GCA-GCCTAT CTGGTACGGG

11 ------ATGCGGGA AACAAAAG-A GCA-GCCTAT CTGGTACGGG

12 ATGGCGGTAT TTGCCAGTGA CTCTTTTGGC CAGC-TGAAA GTGGAGAAAA TTATGACTGC CCAGTGGAAC

13 ------AT GTCAGTGTCT GAAACCACCC ACCTGTTACA GCG-GGCCCT TGAGTCCGAA

Clustal Co

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

80 90 100 110 120 130 140

1 ACCCACTGCC ACTGGGTGAA CTCTTCCCAA TTGGCCGGCC CGCCCACGCG GCCGCCGTTG CCCGAGGCCG

2 CGCCACTGCC ATTGGGTGAA TTCGTCCCAG CTGCTCTGCG TTCCGAATCG CGTGCCGTTG CCGGACGCTC

3 AAGATCTTCG ACGGCGAAT- -GGGTTCGCG GTAGCGCCGA CGATCGTATT GTCGTCGAGC CGGCCACCGG

4 CATTATATCG GCGGCGAGT- -GGATTGCCG GGGAC--AGC GGCAAGACCA TCGATCTACT CAATCCCTCG

5 AGCCAGCGCA GATCGGTAAA GGCGTCGATG CCGGCCTTGC CGCCGAACCG CC--CCATT- CCGCTGTCTT

6 AATTTTTCCG GCCTGAAGC- -AAGCTGCCT TGGGTGGTGG TGCGCGCGAT GTGATCGAGC CCGCCACGGG

7 ACCCACTGCC ACTGGGTGTA CGCATCCCAG TCGGCCGGGC CGCCCATGCT GCTGCCGTTG CCGGCGATGC

8 CGGCTGTTCA CCGGTACCT- -GGGTACCCG GTGAGCTGCC CGCCACCGCC GTTACCTCCC CTGGCAATGG

9 AAGGTGTTTA GTTCTAATT- -GGGTGGAGG CGCGGGGAGG TGTTGCCAAT GTTGTCGACC CGTCCAATGG

10 AAGGTGTTTA GTTCTAATT- -GGGTGGAGG CGCGGGGAGG TGTTGCCAAT GTTGTCGACC CGTCCAATGG

11 AAGGTGTTTA GTTCTAATT- -GGGTAGAGG CGCGGGGAGG TGTTGCCAAT GTTGTCGATC CGTCCAATGG

12 CACTACATTA ACGGGGAATA CGTATCACCC GAATCTGAAG AGTATATCCA CGAGTTCATC CCAACCACGG

13 TGCCTGTTCA ACGGCGATT- -GGATACCCG CCTCGGGGTC CGTGATGCCT GTGATCGAAC CTGCCACCGG

Clustal Co *

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

150 160 170 180 190 200 210

1 CCCGGCCGCC GA---ATGGA ACATGGGGTT --CGTCCGCC ACGGTCTGAT CGTTGATGTG GAGCAGGCCG

2 CGGTACCGCC GA---ACGGC GCATACGCGT --CGTTGTTC AGGGTCTGGT CGTTGATGTG GACCATGCCC

3 CAAGCAGTTG ACG--ACGGT C-GGCATGGC --AACACCCG ACGACGTGGC GGCCGCTGCA ACAAAAGCTA

4 ACCGGCAAGG TGCTGACCAA GATCCAGGCC GGCAATGCCA AGGACATTGA GCGTGCGATT GCCGCTGCCA

5 TGATGCCGCC GA---ACGGC ATCTGCGGTT --CGTCGTGG ACCGTGGCGC CGTTGACGTG GCAGATGCCC

6 CCAGGTCTTG TGC--ACCGT G-GGCATTGC --CGATGCGG CCGACGTGGC CGCCGCCACC GCCACTGCCG

7 CGGGGCCGCC GA---AGGGG TTGATGCAGT --CATCGTTG ACGGTCTGAT CGTTGATATG GAGCATGCCG

8 CGTGGCACTG GGT--GCCCT G-GCCAACGC --CGGTGCCA AACAGGTCGA GCAAAGCGCG CGCGAGGCCG

9 AGATATTCTT GGC--ATTAC G-GGCGTTGC --TAACGGCG AAGATGTCGA TGCTGCTGTG AACGCAGCTA

10 AGATATTCTT GGC--ATTAC G-GGCGTTGC --TAACGGCG AAGATGTCGA TGCTGCTGTG AACGCAGCTA

11 AGACATTCTT GGC--ATTAC G-GGTGTTGC --TAACGGCG AAGATGTCGA TGCTGCTGTG AACGCAGCTA

12 CTTTGCCGGG TG---ACTCA ATCGCAAGG- GGCTCGGCAG CTGACGTTGA TAAGGCTGTT CGTGCCGCGG

13 CG-AGCCCTT GAT--GCGGT GCGCCATGGC --CAACGCGG CCGACATCGC CGTCGCCAGT CGCAGCGCGG

Clustal Co **

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

220 230 240 250 260 270 280

1 GTATTGAGGC GATTGCCGAG CGCCAGCGCC CGTCCGGTCG AACGCGAGAT CACCGCGGCC GAGAGGCCGT

2 GTGCGGAGCT GATCGGCCAG CCGCTCCCCG CGTTCGACCG AACCCGTCTG AATAGCGGCG ACCAGGCCGT

3 CTGCAGCTCA GAAGAATTGG GCGGCAAG-G CCATACACTG AACGAGCCGC CGTTCT--AC GCCGCGCCGG

4 AGGCTGCTTT TCCCAAGTGG TCGCAAAG-C CTGCCCGGCG AGCGCCAGGA GATCCT--GA TCGAAATTGC

5 GATTCGATGC GGCGCGCCAC CGCGATGGCA CGCCCAACGT CGCGTCCGAA CACCGCCGCC GCCAATCCGT

6 CTGCCGCGCA AAAAGCGTGG GTGGCGGT-G CCTCCGCGTG AAAAGGCTGA AATTTT--TC GCCGTGCTGC

7 ACGCGCAGGC GCTGCCCCAG CGCCATGGCC CGCCCCACCG AGGCCGAGAT CACGCCGGCG GCCAGCCCGC

8 CCACGGCCCA GCGCAGTTGG TATGCCCT-G CCCTACGACG AACGCGCCAA AGTCCT--GC GCAAGGCCGC

9 AGAGAGCGCA AAAGGAATGG GCCGCAAT-A CCATTTAGTG AAAGAGCCGC CATTGT--CC GCAAGGCTGC

10 AGAGAGCGCA AAAGGAATGG GCCGCAAT-A CCATTTAGTG AAAGAGCCGC CATTGT--CC GCAAGGCTGC

11 AGAGAGCGCA AAAGGAATGG GCCGCAAT-A CCATTTAGTG AAAGAGCCGC CATTGT--CC GCAAGGCTGC

12 CAGCGGCTCA GCCTGCCTGG AATGCACG-C AAGCCAATTG AGCGGGGTCG TATCCT--TC TCGCCATAGC

13 CCCTGGCCCA GCCGGCATGG GCCGCACTCG GCCCACGG-G AACGCGCCGC CGTGTT--TC GCAAGGCGGC

Clustal Co *

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

290 300 310 320 330 340 350

1 ATTCGGTGGC GTTGGCCAGG GCCACGGCTT CGTCCTCGTC GCGGAAGGTG GTGACGCAGG CCAC--CGGC

2 ACTCGGTGTC GTTCGCGACG GCGATCGCTT CCGCCTCGTC CTTGACGACC ACGACGGGAG CGAC--CGGA

3 TCAACTGTTC GAGCAGTACG CCGACGAAAT -CGGCGAATG GGTAATCCGC GAGGCCGGCA GCAT--CCCG

4 GCGCC-GGCT GAAGGCACGC CAGTCGCACT ACGCGACGCT CGAAACGTTG AACAACGGCA AGCCGATGCG

5 ATTCGTTGTC GTTGGCGCAG GCCACGGCTT CTTCCACGCC GTTGACGCGC ACGATGCCCT TGAC--CGGG

6 TGCCCTGTTT CAGCAGCATG GTGATGAATT -GGCGCTGTA CGCCGCACGC GAGACGGGCG GTAT--TTTG

7 CCTGGCTGTC GTTGGCGAGC GCCACGGCCT CGTCGTCGTC CCGGAAGGGG ATGATGCTGG CGAC--CGGG

8 CGCTATCGCC GAGCAATACC AGGCCGAGAT -CGCCAACTG GATCGTTCGC GAAAGTGGCT CGAC--CCAG

9 CGAAAAACTA AAGGAGCGCG AACATGAGTT -CGCCGATTG GAACGTGCGG GAATGCGGCG CAAT--TCGT

10 CGAAAAACTA AAGGAGCGCG AACATGAGTT -CGCCGATTG GAACGTGCGG GAATGCGGCG CAAT--TCGT

11 CGAAAAACTA AAGGAGCGCG AGTATGAATT -CGCCGATTG GAACGTACGG GAATGCGGCG CAAT--TCGT

12 TCGTTTGGTT CGCGCCAACG CAGCGGCTTT CTGCGCCAAA -GAAGCGGAA GAAACTGGCA AGCC--TCTG

13 GGACCTGGCT CAGCAGTGCT TCGCCGAACT -GGCGCTGTA TGTCGCCCGC GAAACCGGGG GCGC--GCTG

Clustal Co *

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

360 370 380 390 400 410 420

1 CCGAAGATCT CCTCCTCGAA CGCACGCATG CCCGGCTTCA CCCCGCCCAG CACGGTGGGC CGGTAGAACA

2 CCGAAGATCT CTTCCTGGAA TGCCGGCATC GCCGGGGTGA CGCCCGCGAG AACGGTCGGC CGGTAGAAGA