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Supplementary Data

Material and methods

SDS-PAGE

SDS-PAGE was performed using 10% polyacrylamide gels. Proteins were visualized by staining with Coomassie Brilliant Blue. Molecular masses of bands were estimated by Bio-Rad Precision Plus Protein Standards All Blue (Bio-Rad,

#161-0373) molecular mass marker and Kodak 1D analysis software.

Veratryl alcohol oxidation using laccases and mediators

Oxidation reactions of veratryl alcohol to veratraldehyde were performed in duplicate in vials by the addition of laccase from commercial sources and fungal cell free crude extracts to a 500 l reaction mixture of 5 mM veratryl alcohol and

4.5 mM mediator in McIlvaine buffer at appropriate pH containing 20% tBuOH.

Reactions were incubated in screw-cap vials at 30°C, 240rpm for 24hr and stopped by addition of an equal volume of DMSO. Control samples were incubated without the enzyme or a mediator. Supplementary Table 1. Oxidation of veratryl alcohol to veratraldehyde catalyzed by crude laccase enzyme preparations with a variety of mediators in 20% tert-butanol.1 ATCC 64658 ATCC 20869 NRRL 25150 NRRL 1163 NRRL 6413 NRRL 20956 ID Mediator Con Con Conv2 SD3 Conv2 SD3 Conv2 SD3 Conv2 SD3 v2 SD3 v2 SD3 Conv2 SD3 Conv2 SD3 trac 2 n-HPA1 trace e trac 3 SYRald trace trace trace e trace trace 4.5 0.7 4 MB29 trace trac 5 VIO1 11.1 0.0 55.4 2.2 33.3 2.2 86.6 0.6 e trace 4.8 0.7 trac 7 HBA trace e trace 8 AtQ trace trac 9 ASA trace trace e trac 10 CR1 trace trace trace e trac 11 CR2 trace 4.3 0.3 trace trace e trace trac 12 DCIP trace trace trace trace e trace trace trace trac 13 CPR 9.6 0.5 trace 5.9 1.2 e trace trace trac 14 ATCA trace trace trace e trace trace 15 PR trace 4.0 0.3 trace trace trace 9.2 0.8 trac 16 ABTS 16.1 0.3 28.7 0.3 13.0 1.5 28.1 2.7 e trace trace trac 17 HAA trace trace trace e trace trace 18 PPt trace trace trac 19 Que trace trace e trace trace trac 20 MPA trace trace trace e trace trace 21 HB 14.9 1.5 trace 4.4 0.6 5.5 1.0 12.8 0.9 9.8 0.3 23.8 1.9 22 DMOA 14.5 0.2 trace trace 6.7 1.9 11.2 1.5 trace 6.4 0.1 19.3 1.8 trac 23 HA trace e trace trac 24 AZ trace e trace 25 HBS trace trace 26 LccM 10.6 1.7 trace trace trace 10.1 0.3 trace 11.9 0.8 18.7 3.5 28 nPA trace 29 HOBT trace 65.7 2.9 14.5 0.3 91.0 0.0 9.6 0.6 trace trace trac 30 SYRaz trace trace e trace trace trace 31 oxTEMPO trace trace 32 TEMPO 50.6 1.3 100.0 0.0 16.3 0.8 98.8 0.7 42.1 0.1 4.2 0.1 trace 8.3 0.3 33 ADMP trace 34 None trace 1 trace represents conversion of veratryl alcohol to veratraldehyde of <4% 2 conversion of veratryl alcohol to veratryl aldehyde in percent 3 standard deviation of duplicate experiment Supplementary Table 2. Oxidation of veratryl alcohol to veratraldehyde catalyzed by commercial laccase enzymes with a variety of mediators in 20% tert-butanol.1 Fluka Fluka 38429 LCC10 Laccase A Laccase C 5373 9 Laccase P I Mediator Conv Conv Con D 2 SD3 2 SD3 v2 SD3 Conv2 SD3 Conv2 SD3 Conv2 SD3 Conv2 SD3 Conv2 SD3 2 n-HPA1 trace trace 35.0 2.2 trace 3 SYRald trace trace trace trace trace trace trace 7.2 0.8 4 MB29 trace trace 5 VIO1 87.1 1.9 23.9 1.4 11.7 0.4 25.5 2.9 5.0 0.9 trace 12.5 0.4 7 HBA trace trace trace 8 AtQ trace trace 9 ASA trace trace trace trace 10 CR1 6.4 0.9 trace trace trace trace 11 CR2 7.0 2.7 trace trace trace trace trace 12 DCIP 4.3 1.0 trace trace trace trace trace 13 CPR 18.3 2.8 trace trace trace trace trace 14 ATCA trace trace trace trace trace trace trace 15 PR 9.3 3.5 trace trace trace trace trace trace 16 ABTS 40.7 1.4 trace 13.4 2.9 4.7 0.3 4.7 0.4 trace 13.4 0.6 17 HAA trace trace trace trace trace 18 PPt trace trace trace trace 19 Que trace trace trace trace trace trace 20 MPA trace trace trace 21 HB 7.7 2.1 20.3 0.4 7.7 0.7 7.1 1.0 11.7 0.0 10.7 2.0 14.8 1.9 24.8 0.6 22 DMOA 6.3 2.8 15.1 0.8 5.9 0.7 8.1 0.5 12.4 0.9 10.8 1.6 16.1 1.2 18.7 0.4 23 HA trace trace trace 24 AZ trace trace 4.0 0.2 trace 25 HBS trace trace 26 LccM trace 12.0 0.6 10.0 1.6 9.0 1.3 5.9 0.8 9.4 0.9 14.6 0.2 15.0 1.8 28 nPA trace trace 29 HOBT 67.6 1.8 62.4 3.3 11.2 1.3 15.0 1.0 11.7 0.4 trace 87.2 1.0 30 SYRaz trace trace trace trace trace trace 31 oxTEMPO trace trace trace 32 TEMPO 99.4 0.9 96.7 0.1 8.4 1.4 98.9 0.7 97.3 1.5 trace 98.5 0.1 33 ADMP trace trace 34 None trace trace 1 trace represents conversion of veratryl alcohol to veratraldehyde of <4% 2 conversion of veratryl alcohol to veratryl aldehyde in percent 3 standard deviation of duplicate experiment Supplementary Figure Legends

Fig 1 SDS-PAGE of commercially available laccases (A) and crude enzyme preparations from laccase induced fungal cultures (B) used in veratryl alcohol model experiments. Molecular size markers (M) are indicated in kilodaltons. A. Lanes: M marker, 1 laccase from Trametes versicolor (Fluka 53739), 2 laccase from Trametes versicolor (Fluka 38429), 3 laccase from Rhus vernificera (Sigma L2157), 4 Americos Laccase P (Americos Industries, Inc.), 5 Ecostone LCC10

(AB Enzymes), 6 EcoFade LT100 (Genencor), 7 Laccase C (ASA Spezialenzyme GmbH), 8

Laccase A (ASA Spezialenzyme GmbH). B. Lanes M marker, 1 ATCC 64658 (Phlebia radiata),

2 ATCC 200478 (Pycnoporous cinnabarinus), 3 NRRL 1163 (Trichoderma reesei), 4 ATCC

20869 (Trametes versiciolor), 5 ATCC 11235 (Trametes versicolor), 6 NRRL 6413 (Fusarium proliferatum), 7 NRRL 25150 (Trichoderma atroviride) 8 NRRL 20956 (Fusarium verticillioides). Figure 1