cyp199a4/hapux/hapur cyp199a2/pux/pur

CYP199A4 Supplementary data

Table S1. Sequence similarities and alignment between the Class I CYP systems from R.

palustris HaA2 and R. palustris CGA009.

CYP199A4/HaPux/HaPuR CYP199A2/Pux/PuR

Identities Positives Gaps

CYP199A4 356/412 (86%) 383/412 (92%) 2/412 (0%)

HaPux 74/106 (69%) 91/106 (85%) 1/106 (0%)

HaPuR 357/405 (88%) 374/405 (92%) 0/405 (0%)

Table S2. Spectral properties of CYP199A2, HaPux and HaPuR from R. palustris HaA2.

Absorbance (nm) ε (mM–1 cm–1)

CYP199A4 279 48.6

357 27.9

419 103.1

538 9.4

569 10.7

HaPux 280 18.7

316 14.1

416 11.2

457 9.1

HaPuR 172 65.3

383 9.6

455 10.0

482 8.2

Supplementary Material (ESI) for Molecular BioSystemsThis journal is (c) The Royal Society of Chemistry, 2009

S. G. Bell, A. B. H. Tan, E. O.D. Johnson and L.-L. Wong

Figure S1. Sequence alignment of HaPuR, PuR and PdR from R. palustris HaA2, R.

palustris CGA009 and P. putida respectively.

10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| PuR --MNDTVLIAGAGHAGFQVAVSLRQAKYPGRIALINDEKHLPYQRPPLSKAYLKSGGDPN HaPuR --MNDTVLIAGAGHAGFQVAVSLRQAKYTGRIALINDEKHLPYQRPPLSKAYLKSGGDPN PdR MNANDNVVIVGTGLAGVEVAFGLRASGWEGNIRLVGDATVIPHHLPPLSKAYLAGKATAE Clustal Consensus **.*:*.*:* **.:**..** : : *.* *:.* . :*:: ******** . . .: 70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| PuR SLMFRPEKFFQDQAIELISD-RMVSIDREGRKLLLASGTAIEYGHLVLATGARNRMLDVP HaPuR SLMFRPEKFFQDQTIELIDG-RAVAIDRDAKTLLLASGDKIEYGHLVLATGARNRQLDVP PdR SLYLRTPDAYAAQNIQLLGGTQVTAINRDRQQVILSDGRALDYDRLVLATGGRPRPLPVA Clustal Consensus ** :*. . : * *:*:.. : .:*:*: : ::*:.* ::*.:******.* * * *. 130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| PuR N---ASLPDVLYLRTLDESEVLRQRMPDKKHVVVIGAGFIGLEFAATARAKGLEVDVVEL HaPuR N---ATLDDVLYLRTLDESEVVRQRMPEKKHVVVIGAGFIGLEFAATARGKGMEVDVVEL PdR SGAVGKANNFRYLRTLEDAECIRRQLIADNRLVVIGGGYIGLEVAATAIKANMHVTLLDT Clustal Consensus . .. :. *****:::* :*::: .:::****.*:****.**** .:.* ::: 190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| PuR APRVMARVVTPEISSYFHDRHSGAGIRMHYGVRAT--EIAAEGDRVTGVVLSDGNTLPCD HaPuR APRVMARAVTPEISSYFHDRHTAAGIRIHYGVRAT--EIEGEDGRVTGVALSDGRTLPCD PdR AARVLERVTAPPVSAFYEHLHREAGVDIRTGTQVCGFEMSTDQQKVTAVLCEDGTRLPAD Clustal Consensus *.**: *..:* :*:::.. * **: :: *.:. *: : :**.* .** **.* 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| PuR LVVVGVGVIPNVEIAAAAGLPTAAGIIVDQQLLTSDPHISAIGDCALFESVRFGETMRVE HaPuR LVVVGVGVIPNVELASAAGLPTAAGIIVNEQLLTEDPNISAIGDCALFNSVRFGEVMRVE PdR LVIAGIGLIPNCELASAAGLQVDNGIVINEHMQTSDPLIMAVGDCARFHSQLYDRWVRIE Clustal Consensus **:.*:*:*** *:*:**** . **:::::: *.** * *:**** *.* :.. :*:* 310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| PuR SVQNATDQARCVAARLTGDAKPYDGYPWFWSDQGDDKLQIVGLTAGFDQVVIRGSVAERS HaPuR SVQNATDQARCVAARLTGSPATYDGYPWFWSDQGDDKLQIAGLTAGFDQVVLRGSVAERS PdR SVPNALEQARKIAAILCGKVPRDEAAPWFWSDQYEIGLKMVGLSEGYDRIIVRGSLAQPD Clustal Consensus ** ** :*** :** * *. :. ******* : *::.**: *:*::::***:*: . 370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| PuR FSAFCYKAGKLIGIESVNRAADHVFGRKILPLDKSVTPEQAADLSFDLKKAAA------- HaPuR FSAFCYKDGQLIGVESVNRAADHVFGRKILPLGKSVTPEQAADLSFDLKKAMA------- PdR FSVFYLQGDRVLAVDTVNRPVEFNQSKQIITDRLPVEPNLLGDESVPLKEIIAAAKAELS Clustal Consensus **.* : .:::.:::***..:. .::*:. .* *: .* *. **: * .. PuR -- HaPuR -- PdR SA Clustal Consensus



Figure S2. Sequence alignment of HaPux, Pux and Pdx from R. palustris HaA2, R.

palustris CGA009 and P. putida respectively.

10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| Pux MPSITFILPDGERRTTEAAVGDTAMYAALSLGLDGVVAECGGNAVCATCHVYVEHG-LEK HaPux MPSITFIHPDGRSEIVDAAIGDSAMFAALNHGIDSIVAECGGNAVCATCHVYVDDLWLAK Pdx MSKVVYVSHDGTRRELDVADGVSLMQAAVSNGIYDIVGDCGGSASCATCHVYVNEAFTDK Clustal Consensus *..: :: * . :. * : * **: .: .: .:*** . ********:. * 70 80 90 100 ....|....|....|....|....|....|....|....|....|.. Pux LPAVAADEDDLLDGTAAERLPNSRLSCQIKLSSDLDGLILRIPDRQV HaPux LPPVDANEDDLLDGTASDRLPNSRLSCQIKIAPELDGLVLRLPERQT Pdx VPAANEREIGMLECVTAELKPNSRLCCQIIMTPELDGIVVDVPDRQW Clustal Consensus : * .:*: : *****.*** :: :***::: *:**



Figure S3. Sequence alignment of CYP199A4, CYP199A2 and CYP101A1 from R.

palustris HaA2, R. palustris CGA009 and P. putida respectively.

10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| CYP199A2 --MTTAPSLVPVTTPSQHGAGVPHLGIDPFALDYFADPYPEQETLREAGPVVYLDKWNVY CYP199A4 --MISNSSAESISAPPND-STIPHLAIDPFSLDFFDDPYPDQQTLRDAGPVVYLDKWNVY CYP101A1 MTTETIQSNANLAPLPPHVPEHLVFDFDMYNPSNLSAGVQEAWAVLQESNVPDLVWTRCN Clustal Consensus : * ::. . . . : :* : . : : :: : . * * . 70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| CYP199A2 GVARYAEVYAVLNDPLTFCSSRGVGLSDFKKEKPWRPPSLILEADPPAHTRTRAVLSKVL CYP199A4 GVARYAEVHAVLNDPTTFCSSRGVGLSDFKKEKPWRPPSLILEADPPAHTRPRAVLSKVL CYP101A1 GGHWIATRGQLIREAYEDYRHFSSECPFIPREAGEAYDFIPTSMDPPEQRQFRALANQVV Clustal Consensus * * ::.:. . . : :* : . *** : : **: .:*: 130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| CYP199A2 SPATMKRLRDGFAAAADAKIDELLARGGNIDAIADLAEAYPLSVFPDAMGLKQEGRENLL CYP199A4 SPATMKTIRDGFAAAADAKVDELLQRG-CIDAIADLAEAYPLSVFPDAMGLKQEGREHLL CYP101A1 GMPVVDKLENRIQELACSLIESLRPQG-QCNFTEDYAEPFPIRIFMLLAGLPEED----I Clustal Consensus . ..:. :.: : * : ::.* :* : * **.:*: :* ** :*. : 190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| CYP199A2 PYAGLVFNAFGPPNELRQSAIERSAPHQAYVAEQCQR-PNLAPGGFGACIHAFSDTGEIT CYP199A4 PYAGLVFNAFGPPNELRQTAIERSAPHQAYVNEQCQR-PNLAPGGFGACIHAFTDTGEIT CYP101A1 PHLKYLTDQMTRPDGSMTFAEAKEALYDYLIPIIEQRRQKPGTDAISIVANGQVNGRPIT Clustal Consensus *: : : : *: * :.* :: : ** : ....:. :. : ** 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| CYP199A2 PEEAPLLVRSLLSAGLDTTVNGIAAAVYCLARFPDEFARLRADPSLARNAFEEAVRFESP CYP199A4 PDEAPLLVRSLLSAGLDTTVNGIGAAVYCLARFPGELQRLRSDPTLARNAFEEAVRFESP CYP101A1 SDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELIERPERIPAACEELLRRFSL Clustal Consensus .:** : ** .****.** :. :: **: * . .* * * ** :* * 310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| CYP199A2 VQTFFRTTTRDVELAGATIGEGEKVLMFLGSANRDPRRWDDPDRYDITRKTSGHVGFGSG CYP199A4 VQTFFRTTTREVELGGAVIGEGEKVLMFLGSANRDPRRWSDPDLYDITRKTSGHVGFGSG CYP101A1 VADG-RILTSDYEFHGVQLKKGDQILLPQMLSGLDERENACPMHVDFSRQKVSHTTFGHG Clustal Consensus * * * : *: *. : :*:::*: :. * *. * *::*:. .*. ** * 370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| CYP199A2 VHMCVGQLVARLEGEVVLAALARKVAAIEIAGPLKRRFNN-TLRGLESLPIQLTPAZ--- CYP199A4 VHMCVGQLVARLEGEVMLSALARKVAAIDIDGPVKRRFNN-TLRGLESLPVKLTPA---- CAMC SHLCLGQHLARREIIVTLKEWLTRIPDFSIAPGAQIQHKSGIVSGVQALPLVWDPATTKA Clustal Consensus *:*:** :** * * * ::. :.* : :.:. : *:::**: ** . CYP199A2 - CYP199A4 - CYP101A1 V Clustal Consensus



Figure S4. Electrospray mass spectra of (a) HaPuR (b) HaPux and (c) CYP199A4.

(a)

41000 42000 43000 44000 45000

%

0

100 43674.0000

43543.0000

41424.0000 42683.000045191.0000

44629.0000

(b)

10000 10500 11000 11500 12000 12500 1

%

0

100 11298.0010

11281.0010 11329.0010

(c)

42000 44000 46000

%

12

44404.0000

43727.0000

43443.0000

42380.000046704.000044431.0000 49204.0047532.0000

Calc. 43532.1 Da (43663.2 Da including Met)

Calc. 11296.5 Da

Calc. 44405.8 Da



Figure S5. Construction of the plasmids of the whole cell oxidation system.



Figure S6. Example of NADH turnover data using CYP199A4 (0.5 μM), HaPux and HaPuR in a 1:10:1 ratio.

-0.5

0

0.5

1

1.5

2

0 0.5 1 1.5 2 2.5 3

Time (min)

Abs

orba

nce

(340

nm

)

4-methoxybenzoicacid

veratric acid

NADH added

Substrate added



cyp199a4/hapux/hapur cyp199a2/pux/pur

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