Supplementary Data s8

Supplementary data

Table S1. Amino acid sequence comparisons of the ferredoxin reductases, ferredoxin and CYP enzymes from N. aromaticivorans with PdR, Pdx and P450 enzymes from the CYP101 and CYP111 families.

Ferredoxin Pdx Identities Positives Score Arx (Saro_1477) 40/102 (39%) 64/102 (62%) 81.2 Ferredoxin reductase PdR Identities Positives Score ArR (Saro_0216) 166/410 (40%) 241/410 (58%) 287 bphA4 (Saro_3838) 132/411 (32%) 208/411 (50%) 213 P450 CYP101A1 Identities Positives Score CYP101B1 173/395 (43%) 236/395 (59%) 340 CYP101C1 173/395 (43%) 231/395 (58%) 331 CYP101D1 187/418 (44%) 258/418 (61%) 366 CYP101D2 184/398 (46%) 251/398 (63%) 363 P450 CYP111A1 Identities Positives Score CYP111A2 253/401 (63%) 304/401 (75%) 501

Table S2. Steady state NADH turnover activity of CYP101D2 and CYP101A1 with camphor in the presence of 200 mM KCl, with different electron transfer proteins from Pseudomonas putida and Novosphingobium aromaticivorans. ArR: ferredoxin reductase; Arx: ferredoxin; PdR: putidaredoxin reductase; Pdx: putidaredoxin. Turnover rates are given in nmol (nmol-CYP)–1 min–1. The data are reported as meanS.D. (n  3).

Product formation rates and coupling were estimated using the 5-exo-hydroxycamphor analogue 2-hydroxy-3-pinanone as a standard and adjusted assuming 100% coupling for the PdR/Pdx/CYP101A1 turnover with camphor.

CYP101D2 CYP101A1 Electron transfer NADH Product Coupling NADH Product Coupling chain consumption rate formation rate % consumption rate formation rate % ArR/Arx 74546 74128 97 24.63.1 0.70.1 3 PdR/Arx 1564.6 1534.5 98 – – – ArR/Pdx – – – 17.90.9 6.30.3 35 PdR/Pdx 16.04.7 11.13.4 70 75316 75316 100 Figure S1. Amino acid sequence alignment of the ferredoxin reductases ArR, PdR and PuR

(palustrisredoxin reductase from Rhodopseudomonas palustris CGA009).

10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| ArR MASEVQAERADVVIVGAGHGGAQAAIALRQNGFEGRVLVIGREPEIPYERPPLSKEYLAR 60 PuR ------MNDTVLIAGAGHAGFQVAVSLRQAKYPGRIALINDEKHLPYQRPPLSKAYLKS 53 PdR -----MNANDNVVIVGTGLAGVEVAFGLRASGWEGNIRLVGDATVIPHHLPPLSKAYLAG 55 Clustal Consensus . *:*.*:* .* :.*..** : *.: ::. :*:. ***** ** 26

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| ArR EKTFERICIRPAQFWEDKAVEMKLGAEVVSLDPAAHTVKLGDGSAIEYGKLIWATGGDPR 120 PuR GGDPNSLMFRPEKFFQDQAIELIS-DRMVSIDREGRKLLLASGTAIEYGHLVLATGARNR 112 PdR KATAESLYLRTPDAYAAQNIQLLGGTQVTAINRDRQQVILSDGRALDYDRLVLATGGRPR 115 Clustal Consensus : : :*. . : : ::: .:.::: : : *..* *::*.:*: ***. * 54

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| ArR RLSCVG---ADLAGVHAVRTKEDADRLMAELDAGAKNAVVIGGGYIGLEAAAVLTKFGVN 177 PuR MLDVPN---ASLPDVLYLRTLDESEVLRQRMPD-KKHVVVIGAGFIGLEFAATARAKGLE 168 PdR PLPVASGAVGKANNFRYLRTLEDAECIRRQLIA-DNRLVVIGGGYIGLEVAATAIKANMH 174 Clustal Consensus * . .. .. :** :::: : .: :. ****.*:**** **. .:. 78

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| ArR VTLLEALPRVLARVAGEALSEFYQAEHRAHGVDLRTGAAMD--CIEGDGTKVTGVRMQDG 235 PuR VDVVELAPRVMARVVTPEISSYFHDRHSGAGIRMHYGVRAT--EIAAEGDRVTGVVLSDG 226 PdR VTLLDTAARVLERVTAPPVSAFYEHLHREAGVDIRTGTQVCGFEMSTDQQKVTAVLCEDG 234 Clustal Consensus * ::: .**: **. :* ::. * *: :: *. : : :**.* .** 105

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| ArR SVIPADIVIVGIGIVPCVGALISAGASGGNGVDVDEFCRTSLTDVYAIGDCAAHANDFAD 295 PuR NTLPCDLVVVGVGVIPNVEIAAAAGLPTAAGIIVDQQLLTSDPHISAIGDCALFES-VRF 285 PdR TRLPADLVIAGIGLIPNCELASAAGLQVDNGIVINEHMQTSDPLIMAVGDCARFHS-QLY 293 Clustal Consensus . :*.*:*:.*:*::* :** *: ::: ** . : *:**** . . 134

310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| ArR GAVIRLESVQNANDMATAAAKDICGAPVPYKATPWFWSNQYDLKLQTVGLSTGHDNAVLR 355 PuR GETMRVESVQNATDQARCVAARLTGDAKPYDGYPWFWSDQGDDKLQIVGLTAGFDQVVIR 345 PdR DRWVRIESVPNALEQARKIAAILCGKVPRDEAAPWFWSDQYEIGLKMVGLSEGYDRIIVR 353 Clustal Consensus . :*:*** ** : * * : * .. *****:* : *: ***: *.*. ::* 166

370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| ArR GDPATRSFSVVYLKGGKVVALDCVNMVKDYVQGKKLVEARAQIAPEQLADAGVPLKEMLA 415 PuR GSVAERSFSAFCYKAGKLIGIESVNRAADHVFGRKILPLDKSVTPEQAADLSFDLKKAAA 405 PdR GSLAQPDFSVFYLQGDRVLAVDTVNRPVEFNQSKQIITDRLPVEPNLLGDESVPLKEIIA 413 Clustal Consensus *. * .**.. :..:::.:: ** :. .:::: : *: .* .. **: * 191

....|.... ArR ------415 PuR ------405 PdR AAKAELSSA 422 Clustal Consensus 191 Figure S2. Amino acid sequence alignment of the ferredoxins Pdx, Arx and Pux

(palustrisredoxin from Rhodopseudomonas palustris CGA009)(Bell et al. 2008).

10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| Pdx MSKVVYVSHDGTRRELDVADGVSLMQAAVSNGIYDIVGDCGGSASCATCHVYVNEAFTDK 60 Pux MPSITFILPDGERRTTEAAVGDTAMYAALSLGLDGVVAECGGNAVCATCHVYVEHGL-EK 59 Arx MTAILVTTRDGTRTEIQAEPGLSLMEALRDAGIDELLALCGGCCSCATCHVLVAPAFADR 60 Clustal Consensus *. : ** * :. * : * * . *: ::. *** . ****** * .: :: 27

70 80 90 100 ....|....|....|....|....|....|....|....|....|.. Pdx VPAANEREIGMLECVTAELKPNSRLCCQIIMTPELDGIVVDVPDRQW 107 Pux LPAVAADEDDLLDGTAAERLPNSRLSCQIKLSSDLDGLILRIPDRQV 106 Arx LPALSGDENDLLD-SSDHRTPHSRLSCQITINDKLEGLEVEIAPED- 105 Clustal Consensus :** * .:*: : . *:***.*** :. .*:*: : :. .: 51 Figure S3. Amino acid sequence alignment of the CYP101 and CYP111 family proteins from P. putida (P450cam, CYP101A1), P. putida strain PpG777 (CYP111A1, P450lin) and N. aromaticivorans.

10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| P450cam ----MTTETIQSNANLAPLPPHVPEHLVFDFDMYNPSN-LSAGVQEAWAVLQESNVPDLV 55 CYP101C1 ------MIPAHVPADRVVDFDIFNPPG-VEQDYFAAWKTLLDG--PGLV 40 CYP101D2 -LATNFDEAVRAKV---ERPANVPEDRVYEIDMYALNG-IEDGYHEAWKKVQHPGIPDLI 55 CYP101D1 MNAQTSTATQKHRV---APPPHVPGHLIREIDAYDLDG-LEQGFHEAWKRVQQPDTPPLV 56 CYP101B1 MLPHDRGQNSTRRITAMEAPAHVPADRVVDIDIYMPPGLAEHGFHKAWSDLS-AGNPAVV 59 CYP111A1 ---MERPDLKNPDLYTQQVPHDIFARLRREEPVYWNPE---SDGSGFWAVLRHKDIIEVS 54 CYP111A2 -----MLDLKNPDTYQGGVPYAALQDLRAEGPVHWNPE---SDGAGFWAVLGHDEIVAVS 52 Clustal Consensus * : . . * : : 5

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| P450cam WTRCNGGHWIATRGQLIREAYEDYRHFSSECPFIPREAGEAYDFIPTSMDPPEQRQFRAL 115 CYP101C1 WSTANGGHWIAARGDVVRELWGDAERLSSQCLAVTPGLGKVMQFIPLQQDGAEHKAFRTP 100 CYP101D2 WTPFTGGHWIATNGDTVKEVYSDPTRFSSEVIFLPKEAGEKYQMVPTKMDPPEHTPYRKA 115 CYP101D1 WTPFTGGHWIATRGTLIDEIYRSPERFSSRVIWVPREAGEAYDMVPTKLDPPEHTPYRKA 116 CYP101B1 WTPRNEGHWIALGGEALQEVQSDPERFSSRIIVLPKSVGEMHGLIPTTIDPPEHRPYRQL 119 CYP111A1 RQPLLFSSAYENGG------HRIFNENEVGLTNAGEAAVGVPFISLDPPVHTQYRKV 105 CYP111A2 RQPDLFSSAFENGG------HRIFNENQVGLTGAGESAIGIPFISRDPPSHTQYRKF 103 Clustal Consensus . * :... :. . * . : :* 12

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| P450cam ANQVVGMPVVDKLENRIQELACSLIESLRPQGQCNFTEDYAEPFPIRIFMLLAGLPEEDI 175 CYP101C1 VMKGLASRFVVALEPKVQAVARKLMESLRPRGSCDFVSDFAEILPLNIFLTLIDVPLEDR 160 CYP101D2 LDKGLNLAKIRKVEDKVREVASSLIDSFAARGECDFAAEYAELFPVHVFMALADLPLEDI 175 CYP101D1 IDKGLNLAEIRKLEDQIRTIAVEIIEGFADRGHCEFGSEFSTVFPVRVFLALAGLPVEDA 176 CYP101B1 LNAHLNPGAIRGLSESIRQTAVDLIEGFAAQGHCNFTAQYAEQFPIRVFMALVGIEASEA 179 CYP111A1 IMPALSPARLGDIEQRIRVRAEALIERIPLGEEVDLVPLLSAPLPLLTLAELLGLDPDCW 165 CYP111A2 VMPALSPARLQGIEERIAKRVERLFAQVPLGETVNILPLLTVPLPLLTLAELLGVPADLW 163 Clustal Consensus : : :. : . :: . :: : :*: : * .: . 27

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| P450cam PHLKYLTDQMTRPDG------SMTFAEAKEALYDYLIPIIEQRRQKPGTDAISIVANGQV 229 CYP101C1 PRLRQLGVQLTRPDG------SMTVEQLKQAADDYLWPFIEKRMAQPGDDLFSRILSEPV 214 CYP101D2 PVLSEYARQMTRPEGNTPEEMATDLEAGNNGFYAYVDPIIRARVGGDGDDLITLMVNSEI 235 CYP101D1 TKLGLLANEMTRPSGNTPEEQGRSLEAANKGFFEYVAPIIAARRGGSGTDLITRILNVEI 236 CYP101B1 PRIRHWAECMTRPG------MDMTFDEAKAVFFDYVGPLVDARRETPGEDMISAMINADL 233 CYP111A1 YELYNWTNAFVGEDDPEFRKSPEDMAKVLGEFMGFCQELFESRRANPGPDIATLLANAEI 225 CYP111A2 PDLHRWTDAFVGEDDPDFRQSPEAMQAVLAEFMGFATALFEDRRANPGPDIASLLANTEI 223 Clustal Consensus : :. . : :. * * * : : . : 37

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| P450cam N-GRPITSDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELIERPERIPAACEE 288 CYP101C1 G-GRPWTVDEARRMCRNLLFGGLDTVAAMIGMVALHLARHPEDQRLLRERPDLIPAAADE 273 CYP101D2 N-GERIAHDKAQGLISLLLLGGLDTVVNFLSFFMIHLARHPELVAELRSDPLKLMRGAEE 294 CYP101D1 D-GKPMPDDRALGLVSLLLLGGLDTVVNFLGFMMIYLSRHPETVAEMRREPLKLQRGVEE 295 CYP101B1 GDGRRLTRDEALSVVTQVLIAGLDTVVNVLGFIMRELAGNPALRADLRQRGADILPVVHE 293 CYP111A1 N-GQPVALRDFIGNLTLTLVGGNETTRNSISHTIVTLSQQPDQWDILRQRPELLKTATAE 284 CYP111A2 R-GEPAPLRDFIANLILALVGGNETTRNSINHTMIALAENPGQWDILRADPSLMTAAVKE 282 Clustal Consensus *. . *..* :*. :. *: * : : * 49 310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| P450cam LLRRFS-LVADGRILTSDYEFHGVQLKKGDQILLPQMLSGLDERENACPMHVDFSR--QK 345 CYP101C1 LMRRYP-TVAVSRNAVADVDADGVTIRKGDLVYLPSVLHNLDPASFEAPEEVRFDRGLAP 332 CYP101D2 MFRRFP-VVSEARMVAKDQEYKGVFLKRGDMILLPTALHGLDDAANPEPWKLDFSR--RS 351 CYP101D1 LFRRFA-VVSDARYVVSDMEFHGTMLKEGDLILLPTALHGLDDRHHDDPMTVDLSR--RD 352 CYP101B1 LFRRFG-LVSIAREVRRDIEFHGVHLKAGDMIAIPTQVHGLDPRVNPDPLAIDPSR--KR 350 CYP111A1 MVRHASPVLHMRRTAMEDTEIGGQAIAKGDKVVLWYASGNRDESVFSDADRFDVTR--TG 342 CYP111A2 MVRFASPVIHMRRTAMRDTQLGQQAICKGDKVVIFYPAGNRDPAVFENPDRFEITR--PV 340 Clustal Consensus :.* : * * : : ** : : . * . . * 62

370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| P450cam VSHTTFGHGSHLCLGQHLARREIIVTLKEWLTRIPDFSIAPGAQIQHKSGIVSGVQALPL 405 CYP101C1 IRHTTMGVGAHRCVGAGLARMEVIVFLREWLGGMPEFALAPDKAVTMKGGNVGACTALPL 392 CYP101D2 ISHSTFGGGPHRCAGMHLARMEVIVTLEEWLKRIPEFSFKEGETPIYHSGIVAAVENVPL 411 CYP101D1 VTHSTFAQGPHRCAGMHLARLEVTVMLQEWLARIPEFRLKDRAVPIYHSGIVAAVENIPL 412 CYP101B1 ARHSTFGSGPHMCPGQELARKEVAITLEEWLRRIPDFALGPNSDLSPVPGIVGALRRVEL 410 CYP111A1 VQHVGFGSGQHVCVGSRLAEMQLRVVFEILSTRVKRFELCS-KSRRFRSNFLNGLKNLNV 401 CYP111A2 RQHLAFGSGAHVCVGSRLAEMQLRLAFAEMARHVRAFEVVG-EPSRVRSNFINGFKRLEV 399 Clustal Consensus * :. * * * * **. :: : : : * . . : . : : 79

430 ....|....| P450cam VWDPATTKAV 415 CYP101C1 VWRA------396 CYP101D2 VWPIAR---- 417 CYP101D1 EWEPQRVSA- 421 CYP101B1 VWNT------414 CYP111A1 VLVPK----- 406 CYP111A2 RLLV------403 Clustal Consensus 79

Sequence alignments were performed using ClustalW (http://www.ebi.ac.uk/clustalw/) and annotated using BioEdit (Hall 1999; Chenna et al. 2003). Figure S4. Spectra of FeIII (black), FeII (blue) and FeII(CO) (red) forms of CYP101B1, CYP101C1, CYP101D1, CYP101D2 and CYP111A2.

CYP101B1 CYP101C1

0.75 1

0.75 e

e 0.5 c c n n a a b b r

r 0.5 o o s s b

b 0.25 A A 0.25

0 0 350 400 450 500 550 600 350 400 450 500 550 600 wavelength (nm) Wavelength (nm) CYP101D1 CYP101D2 0.75 1.25

1 e 0.5 e c c n a n 0.75 a b r b r o s o s b 0.5

0.25 b A A 0.25

0 0 350 400 450 500 550 600 350 400 450 500 550 600 Wavelength (nm) Wavelength (nm)

CYP111A2

0.75 e c n a b

r 0.5 o s b A 0.25

0 350 400 450 500 550 600 Wavelength (nm) Figure S5. Spectroscopic titration of camphor binding by CYP101D2 (top) and fit of the peak-to-trough absorbance differences to a hyperbolic function (bottom) for binding constant determination.

4.00E-02

3.00E-02

2.00E-02

1.00E-02 e c n

a 0.00E+00 b

r 300 350 400 450 500 550 o -1.00E-02 s b

A -2.00E-02

-3.00E-02

-4.00E-02

-5.00E-02 Wavelength (nm)

7.00E-02

6.00E-02

5.00E-02

4.00E-02 A D 3.00E-02

2.00E-02

1.00E-02

0.00E+00 0 20 40 60 80 100 camphor (mM)

References

Bell SG, Xu F, Forward I, Bartlam M, Rao Z, Wong LL (2008) Crystal structure of CYP199A2, a para-substituted benzoic acid oxidizing cytochrome P450 from Rhodopseudomonas palustris. J Mol Biol 383: 561-574. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31: 3497-3500. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Sym Series 41: 95-98.