Biocatalysis 2017; 3: 17–26

Communication Open Access

Gudrun Gygli, Willem J. H. van Berkel* Vanillyl alcohol oxidases produced in Komagataella phaffii contain a highly stable non- covalently bound anionic FAD semiquinone

DOI 10.1515/boca-2017-0002 [1]. FAD cofactors can attain three different redox states: Received October 3, 2016; accepted December 29, 2016 (i) the oxidised form, which is bright yellow in colour, Abstract: Vanillyl alcohol oxidase (VAO) from Penicillium (ii) the one-electron reduced semiquinone, a radical simplicissimum is a covalent flavoprotein that has species which can be blue or red depending on whether it emerged as a promising biocatalyst for the production of is in the neutral or anionic state, and (iii) the two-electron aromatic fine chemicals such as vanillin, coniferyl alcohol reduced form which is almost colourless (Fig. 1). VAO is and enantiopure 1-(4’-hydroxyphenyl) alcohols. The large- active with a wide range of 4-hydroxybenzylic compounds scale production of this eukaryotic enzyme in Escherichia (Fig. 2) and has emerged as a promising biocatalyst for the coli has remained challenging thus far. For that reason an production of aromatic fine chemicals, such as vanillin, alternative, eukaryotic expression system, Komagataella coniferyl alcohol and enantiopure 1-(4’-hydroxyphenyl) phaffii, was tested. Additionally, to produce novel VAO alcohols [1–3]. During catalysis, the FAD cofactor initially biocatalysts, we screened genomes for VAO homologues. becomes reduced through hydride transfer from the One bacterial and five fungal sequences were selected phenolic substrate, generating a quinone methide product for expression, using key active site residues as criteria intermediate [2]. The fate of this intermediate depends on for their selection. Expression of the putative vao genes the type of substrate [4]. With vanillyl alcohol, the aldehyde in K. phaffii was successful, however expression levels product (vanillin) is formed without the intermediate were low (1 mg per litre of culture). Surprisingly, all hydration of the quinone methide (Fig. 2). In the reactions purified enzymes were found to contain a highly stable, non-covalently bound anionic FAD semiquinone that FAD could not be reduced by dithionite or cyanoborohydride. FADH¯ Activity experiments revealed that VAO expressed in FAD°¯ 15 K. phaffii does not produce vanillin because the enzyme FADH°

suffers from oxidative stress. ) 1 − cm

Keywords: biocatalysis, flavoprotein, redox cycling, 1 −

oxidoreductase, Pichia pastoris (M 3 − x 10 1 Introduction ε

Vanillyl alcohol oxidase (VAO) is an homo-octameric flavoenzyme, which covalently binds its FAD cofactor 0 5 10 300 400 500 600 700 *Corresponding author: Willem J. H. van Berkel, Laboratory of Wavelength (nm) Biochemistry, Wageningen University & Research, Stippeneng 4, Wageningen 6708 WE, The Netherlands, Figure 1: Visible absorption properties of FAD redox states: yellow: E-mail: [email protected] oxidised; red: anionic semiquinone; blue: neutral semiquinone; Gudrun Gygli, Laboratory of Biochemistry, Wageningen University & black: anionic hydroquinone (modified from [32]) Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands

© 2017 Gudrun Gygli, Willem J. H. van Berkel, published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. 18 G. Gygli, W.J.H van Berkel

I HO O II OH III O O

O2 H2O2 O2 H2O2 O2 H2O2

CH3 CH3 O O H2O H2O CH3OH

OH OH OH OH OH OH

HO IV V H2N O

O2 H2O2 O2 H2O2

CH3 CH3 CH3 CH3 H2O O O H2O NH3

OH OH OH OH

Figure 2: The different reactions VAO catalyses are: the oxidation of vanillyl alcohol to vanillin (I), enantio-selective conversion of 4-ethyl- phenol to (R)-1-(4’-hydroxyphenyl)ethanol (II), oxidative demethylation of 4-(methoxymethyl)phenol to 4-hydroxybenzaldehyde (III), the oxidation of eugenol to coniferyl alcohol (IV), and the oxidative deamination of vanillylamine to vanillin (V). with 4-(methoxymethyl)phenol and eugenol, the quinone Ile238, Phe454, Glu502 and Thr505 [12]. Also, a loop ranging methide intermediate is hydrated at the Cα- or Cγ-atom, from residue 220 to 235 has been shown to be essential for generating 4-hydroxybenzaldehyde and coniferyl alcohol octamerisation of VAO [13]. as final products, respectively (Fig. 2). VAO also catalyses Over the past three decades, studies with VAO have the enantioselective hydroxylation of 4-alkylphenols to led to different experiences producing this enzyme the corresponding (R)-1-(4’-hydroxyphenyl) alcohols with in different expression systems [1,14]. VAO as it was an e.e. of 94% [3]. In the final step of the VAO reaction, first described in 1992 was purified from Penicillium the reduced flavin gets oxidised by molecular oxygen, simplicissimum [1]. Absence of a gene sequence until 1998 regenerating the oxidised enzyme [1]. [14] necessitated that the production of the enzyme relied VAO covalently binds its FAD cofactor via His422 [5–7]. on its natural host. Yields for VAO from P. simplicissimum The proposed sequence of events of this posttranslational were around 5% of total protein produced, leading to modification of VAO is as follows: Before any incorporation approximately 50 mg of protein per litre of culture. of FAD can occur, the VAO-polypeptide folds and VAO was then heterologously produced in E. coli (using oligomerises to the dimer. The produced apoprotein pBC11 [8]) and Aspergillus niger to yields of about then encapsulates FAD in its highly pre-organised 5-10 mg and greater than 50 mg of enzyme per litre of binding cavity. Non-covalent FAD binding promotes the culture, respectively [14]. Because the protein produced octamerisation of the enzyme. Next, the flavin becomes extracellularly in A. niger was prone to proteolysis, covalently bound via an autocatalytic process, involving E. coli was selected as host for subsequent protein the fully reduced form of FAD. Covalent incorporation engineering studies. The disadvantage of the relatively increases the redox potential of the flavin cofactor and low yield of the E. coli system compared to production stimulates efficient redox catalysis. The full process takes in P. simplicissimum was outweighed by the ease of site- approximately 30-60 minutes [5–7]. directed mutagenesis in E. coli systems. Active site residues which have been found to be Microbial systems previously used to produce other essential for the reactions and mechanisms described above VAOs are E. coli, to express VAO from Fusarium verticillioides are: (i) Asp170: essential for efficient redox catalysis and strains, as well as Byssochlamys fulva V107 to produce involved in the autocatalytic flavinylation [8] (ii) His422: VAO from B. fulva V107 [15,16]. The amino acid sequence covalently binds the FAD cofactor and thus enables efficient and crystal structure of VAO from P. simplicissimum are redox catalysis [9] (iii) His61: enables His422 to covalently available in sequence databases and the PDB under the bind FAD [5], (iv) Thr457: together with Asp170 directs the accession codes P56216 and 2VAO, respectively. Amino enantioselective hydroxylation of 4-alkylphenols of VAO to acid sequences for VAO from F. verticillioides strains are the R isomers [10] and (v) Tyr108, Tyr503 and Arg504: form annotated as EWG41284.1 and AFJ11909.11, of which the an anion-binding subsite which is hypothesised to stabilise second corresponds to the VAO described in [15]. No amino the phenolate form of VAO’s substrates [11]. Residues acid sequence is available for the VAO from B. fulva V107. remote from the active site that have been found through It is difficult to conclusively establish sequence- directed evolution to influence the enzyme reactivity are function relationships based on only two known Vanillyl alcohol oxidases produced in Komagataella phaffii contain... 19 characterised and sequenced VAOs. One can attempt CRG90285, M5FTW7, C7Z853, W9CKB3, V5FPK0, K0C1W8 to uncover such relationships through site-directed and P56216 (VAO from P. simplicissimum). Sequences with mutagenesis or directed evolution, as has been performed a longer N-terminus than in VAO from P. simplicissimum previously in our group [5,8,9,12]. Another approach is to were shortened and made to contain the same N-terminal tap natural evolution and screen genomes for VAO-like part as VAO from P. simplicissimum. Codon optimised sequences. We have chosen a combined approach, using synthetic genes for these sequences in pPICZαA vector the knowledge gained from mutagenesis experiments were ordered from Life Technologies. Transformation of K. to define a set of key VAO residues which are known to phaffii NRRL Y-48124 X33 strain was performed following be essential for function of VAO from P. simplicissimum the EasySelectTM Pichia Expression Kit instructions. and then use this criterion to select VAO-like sequences Growth of K. phaffii X33 and expression of VAO-like for expression in Komagataella phaffii (formerly known enzymes was as follows: a pre-pre culture of transformed as Pichia pastoris or Komagataella pastoris [17]). This X33 cells in 5 mL YPD medium was grown overnight in a 50 methylotrophic yeast was chosen for its ability to mL tube, then transferred into a 125 mL baffled flask with express eukaryotic proteins to high yields as well as its 20 mL BMGY medium (pre-culture), and grown overnight. use as an industrial system for protein expression [18]. The pre-culture was then transferred into a 1 L baffled All the selected putative VAOs as well as VAO from P. flask with 200 mL BMMY medium and grown for four simplicissimum produced in this way were found to days, with methanol being added to a final concentration be catalytically incompetent and to contain a highly of 0.5% twice a day. 50 mL of cultures that showed VAO- stable, non-covalently bound anionic FAD semiquinone activity with the horseradish peroxidase-coupled activity

(anionic FADSQ). assay (HRP assay) described below were transferred into 2 L baffled flasks with 500 mL BMMY medium and grown for another 5-6 days, with methanol being added to a final 2 Methods concentration of 0.5% twice a day. A negative control of non-transformed cells (X33 as present in the EasySelectTM 2.1 Chemicals Pichia Expression Kit from Invitrogen) was included and found to show no activity with the HRP assay described Vanillyl alcohol, eugenol and horseradish peroxidase below. Also, no intracellular expression of VAOs was (type II) were from Sigma-Aldrich. Peptone was from observed in samples where extracellular expression of Sigma-Aldrich (peptone from casein, pancreatic digest; for VAOs occurred. microbiology) and tryptone was from Duchefa Biochemie. Purification of enzymes produced in K. phaffii Catalase from beef liver and superoxide dismutase from was performed as follows (after [14]): The culture bovine erythrocytes were from Boehringer Mannheim supernatants were harvested by centrifugation (30 min, GmbH. All other chemicals were from commercial sources 4400 g, 4 °C). Subsequently, ammonium sulphate was and of the purest grade available. Growth media used added to 65% saturation at 4 °C and then the precipitate were: Yeast Extract Peptone Dextrose Medium (YPD) was removed by centrifugation (30 min, 4400 g, 4 °C). (1% yeast extract, 2% peptone, 2% glucose, 100 μg/mL The pellet was dissolved in 50 mM potassium phosphate Zeocin), Buffered Glycerol-complex Medium (BMGY) and buffer, 0.5 M ammonium sulphate, pH 7.0 and loaded Buffered Methanol-complex Medium (BMMY) (1% yeast onto a phenyl-Sepharose column pre-equilibrated with extract, 2% peptone, 100 mM potassium phosphate, pH the same buffer. The enzymes were eluted with a linear 6.0 or pH 7.0, 1.34% Yeast Nitrogen Base, 4 x 10-5% biotin, descending gradient from 0.5 M to 0 M ammonium 1% glycerol or 0.5% methanol for BMGY and BMMY sulphate in 50 mM potassium phosphate buffer, pH respectively). In BMMY, 2% peptone was replaced with 2% 7.0 using a Pharmacia Biotech Äkta system. Active tryptone or 0.1 mM FAD was added when testing different fractions, as determined using the HRP assay described growth conditions. below, were pooled, dialysed against 50 mM potassium phosphate buffer pH 7.0, containing 150 mM KCl and 2.2 Strains, expression of synthetic genes concentrated using Amicon 10kDa spin-filters. Samples and protein purification were then applied to a Superdex 200 10/30 GL column (GE Healthcare) running in 50 mM potassium phosphate Komagataella phaffii, NRRL Y-48124 X33 strain, as buffer pH 7.0, containing 150 mM KCl, at a flow rate of obtained from the EasySelectTM Pichia Expression Kit 1.0 mL/min. Fractions containing pure enzymes, as (Invitrogen) was used for extracellular expression of determined by SDS-PAGE, were pooled, concentrated 20 G. Gygli, W.J.H van Berkel and dialysed to a 50 mM potassium phosphate buffer pH potassium phosphate buffer pH 7.5 were determined using -1 -1 7.5 with 10% glycerol and stored at -80 °C. absorption values at 439 nm and ε 439=12.5 mM cm for VAOOX -1 -1 Analytical gel filtration was performed at room and assuming ε439=7.8 mM cm for VAOSQ, respectively. temperature using a Superdex 200 10/30 GL column (GE Reduction of the flavin cofactor was attempted by Healthcare) running in 50 mM potassium phosphate incubating 27 µM of enzyme with 10 mM dithionite or 30 buffer pH 7.0, containing 150 mM KCl, at a flow rate of 1.0 mM cyanoborohydride in 50 mM potassium phosphate mL/min. Analysis of the oligomeric state of the VAO-like buffer, pH 7.5 at room temperature for at least 1 h. After enzymes was done as described elsewhere [19]. the incubations, the redox state of the flavin cofactors was determined from their absorption spectra. To force the 2.3 Biochemical characterisation and release of their flavin cofactor, enzymes were denatured spectral properties using trichloroacetic acid (TCA) precipitation. For the TCA precipitation, cold 40% TCA was added to the enzyme A horseradish peroxidase-coupled activity assay (HRP sample to a final concentration of 5% on ice. Samples assay) was used to screen for presence of VAO-like enzymes were centrifuged at 4 °C, 18,000 g for 10 min to separate during production and purification. Reaction mixtures the denatured protein from the released flavin cofactor. containing 3 U/mL horseradish peroxidase, 0.1 mM Absorption spectra of neutralised (pH 7.0, using NaOH) 4-aminoantipyrine, 1 mM dichlorohydroxybenzenesulfonic supernatants were recorded as described above. acid, 0.2 mM vanillyl alcohol and an appropriate amount of enzyme sample in 50 mM potassium phosphate buffer, 2.4 Identification and quantification of FAD pH 7.5, were incubated at room temperature for at least using HPLC/MS 15 min. The appearance of the typical pink colour upon occurrence of a reaction was observed by eye. All activity To determine the identity of the flavin cofactor, it was measurements with the purified enzymes were performed separated from the enzymes by heat denaturation. with previously frozen enzymes. Without the addition of Samples were incubated for 10 min at 95 °C, followed glycerol to 10% before freezing the enzymes, it was found by removal of the protein by centrifugation (18,000 g that the activity detected in the HRP assay was lost. for 10 min). FAD was identified and quantified using a Specific activity was measured by following Shimadzu UPLC-triple quad mass spectrometer (LCMS- the conversion of vanillyl alcohol to vanillin or the 8040) using the following procedure: The purified flavin conversion of eugenol to coniferyl alcohol by monitoring cofactor from VAOSQ was freeze-dried and dissolved in 120 -1 the absorption of the products at 340 nm (ε340 = 14 mM µl H2O. 2 µL of this cofactor solution were injected onto a -1 -1 -1 cm at pH 7.5) and 296 nm (ε296= 6.8 mM cm at pH 7.5) Discovery HS F5-3 (2.1 mm I.D. x 150 mm, 3 µm particles) respectively. Reaction mixtures containing 0.26 µM of from Sigma Aldrich. Separation was performed at 40 °C

VAO-like enzyme and 0.5 mM vanillyl alcohol or 0.05 mM with a gradient from 100% H2O (with 0.1% formic acid) to eugenol in 50 mM potassium phosphate buffer, pH 7.5 35% acetonitrile (with 0.1% formic acid) in 15 min at a flow were incubated at 25 °C in quartz cuvettes. Production of rate of 250 µL/min. The triple quad mass spectrometer was vanillin or coniferyl alcohol was observed using a Hewlett- operating with 3 L/min nebulizing gas flow and 15 L/min Packard HP 8453 diode array spectrophotometer. drying gas flow. The inlet temperature was set to 250 °C Oxygen consumption was monitored using a and the heat block temperature to 400 °C. Electrospray Hansatech Oxytherm system (Hansatech Instruments, ionisation was used. FAD was detected in positive mode King’s Lynn, UK). Reactions were conducted at 25 °C with for the transition from 786.15 to 136.10 m/z and from 786.15 0.2 mM vanillyl alcohol in 50 mM potassium phosphate to 348.10 m/z at 8.53 min. buffer pH 7.5, and enzyme added to a final concentration of 0.4 µM VAO or 1.2 µM VAOSQ. Catalytic amounts of 2.5 Bioinformatics catalase, superoxide dismutase or horseradish peroxidase were added immediately after addition of VAO or VAOSQ to Novel, putative vao genes were identified through pblast the reaction mixture or towards the end of the reaction. using P56216 as query sequence in the non-redundant Flavin absorption spectra of the enzymes were sequence database. Sequences were aligned using clustal recorded using a Hewlett-Packard HP 8453 diode array omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and spectrophotometer and quartz cuvettes, or with a Thermo the obtained alignment was visualised with Jalview [20]. Scientific NanoDrop 2000c UV-Vis spectrophotomer. Six novel sequences were selected based on presence Protein concentrations of the purified enzymes in a or absence of residues corresponding to positions 61, Vanillyl alcohol oxidases produced in Komagataella phaffii contain... 21

108, 170, 220-235, 238, 422, 457 and 502-505 in P56216. thousand hits when blasting with the sequence of VAO from Visualisation of these positions out of context of the P. simplicissimum. For practical reasons, a maximum of alignment was performed using R version 3.2.2 (2015- six novel vao genes was selected for expression. Selection 08-14) [21]. A phylogenetic tree was created with the of these genes was based on the presence of residues Neighbour Joining Clustering method using percentage of that are important for the functioning of VAO. These key sequence identity option in Jalview and visualised using residues are His61, Tyr108, Asp170, Ile238, His422, Thr457, Dendroscope [20,22]. All final graphics were created using Glu502, Tyr503, Arg504 and Thr505 as well as a loop from Inkscape (https://inkscape.org/en/). residues 220 to 235 (Fig. 3). The alignment of the VAO-like sequences thus identified and their relationships to VAO from P. simplicissimum are depicted in Fig. S1. The only 3 Results bacterial sequence selected originates from Cycloclasticus, 3.1 Bioinformatics a proteobacterium, while the other sequences are of fungal origin, with the sequence from Dacryopinax being Nowadays, a vast amount of sequence data is available in the only one from a basidiomycete. Sequence identities to sequence databases, and when screening these databases VAO from P. simplicissimum were 49%, 45%, 62%, 63%, for homologues of a known protein one usually finds many 88% and 42% for CRG90285, M5FTW7, C7Z853, W9CKB3, more putative proteins than can be produced under non- V5FPK0 and K0C1W8, respectively. As can be seen from automated conditions. This is also the case for the genome Fig. 3B, all these sequences contain the key residues screening of the non-redundant sequence database for Tyr108, His422, Tyr503 and Arg504, and only K0C1W8 VAO-like sequences, where one finds more than three does not contain the critical Asp170.

A Thr457

Asp170 Ile238 Tyr503

Glu502 His422 Arg504

Thr505 Tyr108 His61

B 61 108 170 238 422 457 502 503 504 505 P56216 Penicillium simplicissimum H Y D I H T E Y R T CRG90285 Talaromyces islandicus M Y D S H A E Y R T M5FTW7 Dacryopinax sp. H Y D T H T E Y R T C7Z853 Nectria haematococca F Y D A H D E Y R T W9CKB3 Sclerotinia borealis Y Y D C H T E Y R T V5FPK0 Byssochlamys spectabilis H Y D T H T E Y R T K0C1W8 Cycloclasticus sp. Y Y A T H E E Y R T

Figure 3: Visualisation of key residues of VAO. (A) Functionally important residues are coloured according to (B). Also shown is the cova- lently bound FAD cofactor (in yellow). This figure was created using PyMOL (The PyMOL Molecular Graphics System, Version 1.7 Schrödinger, LLC.). (B) Summary of key residues in the selected sequences, as numbered in VAO. Data was extracted from the alignment in Fig. S1 and processed in R [21]. 22 G. Gygli, W.J.H van Berkel

3.2 Production of VAO-like enzymes in 3.3 Stability of the anionic FAD semiquinone K. phaffii Attempts to oxidise or reduce the flavin cofactor of VAO VAO from P. simplicissimum as well as the VAO-like enzymes with ferricyanide or dithionite or cyanoborohydride were successfully expressed extracellularly under the failed. VAOSQ persisted also against the addition of the control of an α-factor signal sequence in K. phaffii and substrates vanillyl alcohol and eugenol, as well as the purified as described in Methods. During production inhibitors isoeugenol and cinnamyl alcohol. Dialysis to and purification procedures, activity was tracked using remove eventually bound molecules, either in the absence the horseradish peroxidase coupled activity assay (HRP or presence of excess FAD, did not change the flavin redox assay) described in Methods. After purification, rather low state. Reoxidation of VAOSQ was only possible through yields were observed, ranging between 1 and 2 mg of pure denaturation of the protein through TCA precipitation or enzyme from 2 L of culture. During the third purification heating. This revealed that the VAOSQ cofactor was non- step, enzymes eluted at the volume of octameric VAO on a covalently bound, and the thus freed flavin was confirmed Superdex 200 column, see Fig. 4 [19]. SDS-PAGE showed a to be FAD through TLC and HPLC/MS. subunit molecular mass of about 70 kDa for all enzymes, Of note is that due to the low amounts of protein corresponding to the calculated mass of 74 kDa, as can be available, absorption spectra were recorded using a seen in Fig. 4. nanodrop 2000c machine, which gave reliable and quick Absorption spectra revealed the presence of an results (see Fig. 5). The fact that only 2 µL at most are anionic FADSQ in all the enzymes expressed in K. needed for a measurement reduced greatly the volume phaffii, compared to the oxidized FAD observed in VAO needed to test different conditions when trying to oxidise expressed in E. coli (see Fig. 5A compared to Fig. 5B). or reduce the anionic FADSQ in VAO. VAO from P. simplicissimum expressed in K. phaffii also contained anionic flavin semiquinone (VAOSQ). The use 3.4 Catalytic properties of different growth media (see Methods) did not change the semiquinone redox state of the purified enzymes. Tracking product formation by following absorption Changing the construct by removal of the c-terminal changes at 340 nm and 296 nm with the common VAO c-myc epitope and His-tag (6x) also lead to the production substrates vanillyl alcohol and eugenol indicated that of VAOSQ. Growth of K. phaffii and subsequent enzyme all enzymes expressed in K. phaffii did not produce purification under dark conditions also resulted in VAOSQ. the expected products vanillin and coniferyl alcohol,

280 nm 254 nm 450 nm Absorbance (mAU) 0 20 40 60 97 kDa

66 kDa

45 kDa

30 kDa 0510 15 20 25 Elution volume (mL)

Figure 4: Elution profile of VAO from P. simplicissimum as expressed in K. phaffii during purification on a Superdex 200 HR 10/30 column. An SDS-PAGE gel of collected fractions is shown below the curves. The big-framed box shows the subunit molecular masses of the marker proteins and the sample before purification on the Superdex 200 column. The small-framed box indicates which fractions were pooled and used for subsequent analysis. Vanillyl alcohol oxidases produced in Komagataella phaffii contain... 23

respectively. This is surprising as activity was detected again. In the case of PsVAO E, this oxygen exhaustion did using the HRP assay with purified enzymes. not occur because no more vanillyl alcohol was present.

To explain the discrepancy between the absorbance We can therefore conclude that with PsVAO K, the substrate measurements of vanillin production (at 340 nm) and acts as an effector uncoupling oxygen consumption from hydrogen peroxide formation (followed indirectly at 510 product formation. nm), we monitored oxygen consumption with VAO from P. simplicissimum as expressed in K. phaffii (PsVAO ) or K 4 Discussion E. coli (PsVAO E). Measurements for both enzymes with limiting amounts of vanillyl alcohol (0.2 mM), as described Many flavoprotein oxidases that form an anionic FADSQ are in Methods, revealed consumption of oxygen for both known: A transient anionic FADSQ is observed in D-amino enzymes. However, the rate of oxygen consumption with acid oxidase upon titration with methyl viologen at pH

PsVAO E was 10 times faster than with PsVAO K. Moreover, 8.5 and in sarcosine oxidase upon titration with L-proline

PsVAO E consumed 0.2 mM oxygen while PsVAO K used up or sarcosine at pH 7.0 [23,24]. Glycine oxidase was found all oxygen present in the reaction mixture. Absorbance to form a transient anionic FADSQ upon photoreduction measurements and olfactory analysis confirmed that [25]. Lactate oxidase forms a slowly decaying anionic vanillin was only formed with PsVAO E and not with FAD SQ, which is induced by reaction with pyruvate or

PsVAO K These observations hint at non-productive redox photoirradiation, and reoxidised by molecular oxygen cycling in the case of PsVAO K. Addition of catalase or within 24 h [26]. In methanol oxidase from K. phaffii anionic superoxide dismutase at the end or beginning of the FADSQ was found to make up approximately 30-35% of the reaction showed the production of hydrogen peroxide flavin after purification [27]. The anionic FADSQ of methanol during this redox cycling, but not superoxide. In the case oxidase was not reduced by methanol. A positively charged of PsVAO K, the oxygen produced from the reaction of residue near N(1) is present in many of these enzymes, hydrogen peroxide with catalase was completely used up mostly histidine or arginine, which could stabilise the

Figure 5: Illustration of the different spectra recorded for (A) VAOSQ with non-covalently bound FAD and (B) VAOOX with covalently bound FAD with Hewlett-Packard HP 8453 diode array spectrophotometer (HP) or Thermo Scientific NanoDrop 2000c UV-Vis spectrophotometer (nanodrop). 24 G. Gygli, W.J.H van Berkel

(transient) anionic FADSQ. In choline oxidase, the anionic expected to covalently bind FAD. Another unexpected

FADSQ is stabilised upon reduction in the wild-type enzyme finding is that all novel VAO variants present as octamers. and the H466A mutant but not in the H466D mutant. The Based on the absence of a loop that has been shown to be interaction of the negative charge of the N(1) of anionic essential for octamerisation in VAO, K0C1W8, CRG90285

FAD SQ with the introduced negative charge of Asp466 of and M5FTW7 are expected to be dimeric proteins [13]. the H466D mutant is suggested to be unfavourable for the The low yields of these enzymes as well as their formation of the anionic FADSQ. The absence of a positive unexpected behaviour when expressed in K. phaffii leaves charge near flavin N(1) in the H466A mutant is not enough open the question whether low yields are due to the to prevent formation of the anionic FADSQ in this enzyme unexpected behaviour or the use of an unsuited expression

[28]. Interestingly, the anionic FADSQ in choline oxidase is system. In E. coli and P. simplicissimum, VAO is produced unusually insensitive to oxygen or ferricyanide at pH 8, but intracelluarly, and in A. niger and K. phaffii it is secreted. It can be transformed into oxidised FAD by incubating the appears that the formation of VAOSQ is specific to K. phaffii, enzyme at pH 6 for several hours [29]. although the mechanism behind the formation of VAOSQ For VAO produced in its natural host, P. simplicissimum, is still unclear. Because the flavin semiquinone is non- the semiquinone state is not observed in rapid reaction covalently bound, we hypothesise that stabilisation of the kinetic experiments. This indicates that the semiquinone semiquinone state prevents the covalent flavinylation of is not kinetically stabilised during flavin reduction, in VAO by excluding the formation of the reduced flavin and agreement with the hydride transfer mechanism [11]. flavin iminoquinone methide that have been proposed to

A transient anionic FADSQ is observed upon anaerobic act as intermediates in the covalent flavinylation process reduction with dithionite, but it is easily reoxidised upon [30,31]. It is remarkable that the semiquinone state of FAD mixing with air. Photoreduction of VAO also leads to the persists despite the apparent oxygen consumption and transient formation of anionic FADSQ, while photoreduction hydrogen peroxide formation in reaction experiments. It in the presence of cinnamyl alcohol yields irreversibly appears that PsVAO K suffers from oxidative stress because bleached FAD [1]. In VAOH422A, a VAO variant that binds the substrate binding stimulates single electron flavin redox FAD cofactor non-covalently, a large portion of transient cycling, which uncouples oxygen reduction from aromatic neutral (blue) FADSQ is observed during xanthine oxidase product formation. mediated reduction [9]. In VAO, Arg504 is located near the N(1) locus of FAD. The above examples highlight that, independent of 5 Conclusion whether they bind their FAD cofactor covalently or not, It is of academic interest to further study the properties many flavoprotein oxidases are able to stabilise anionic of the persisting, non-covalently bound anionic FAD FAD , and most do form a transient anionic FAD upon SQ SQ SQ observed in VAO-like enzymes expressed in K. phaffii. From anaerobic reduction with dithionite. However, in all of a practical point of view however, the high stability of this these enzymes, the flavin radical is thermodynamically catalytically incompetent anionic FAD is an unfortunate unstable and can be easily transformed to FAD or FAD . SQ OX RED condition, which prevents the further characterisation or Here we found that expression of six novel VAO variants future applications of these enzymes as biocatalysts for as well as VAO from P. simplicissimum in K. phaffii results the production of aromatic fine chemicals such as vanillin, in the production of octameric enzymes that bind the coniferyl alcohol and enantiopure 1-(4’-hydroxyphenyl) anionic semiquinone form of FAD in a non-covalent mode. alcohols. Currently, we are working on expressing the Remarkably, the VAO-bound anionic FAD appeared to SQ VAO-like enzymes described here in E. coli. be extremely stable and could only be transformed to the oxidized state upon protein denaturation. Neither could it Acknowledgements: We thank Tom A. Ewing, Prof. be reduced with the strongest reducing agents dithionite Jacques J. M. Vervoort and Adrie H. Westphal for discussions or cyanoborohydride. Surprisingly, even VAO from P. and assistance in Oxytherm and MS experiments. This simplicissimum (P56216), which has been functionally work was supported by the European Union through the expressed in E. coli, A. niger and P. simplicissmum, presents INDOX project (FP7-KBBE-2013-7-613549). as catalytically incompetent VAOSQ. Given the presence of His422 and Arg504 in all sequences as well as His61 in Conflict of interest: Authors state no conflict of interest P56216, M5FTW7 and V5FPK0, these three enzymes are Vanillyl alcohol oxidases produced in Komagataella phaffii contain... 25

[16] Furukawa H, Wieser M, Morita H, Sugio T, Nagasawa T. References Purification and characterization of vanillyl-alcohol oxidase from Byssochlamys fulva V107. J Biosci Bioeng 1999; [1] de Jong E, van Berkel WJH, van der Zwan RP, de Bont JAM. 87:285–290. Purification and characterization of vanillyl-alcohol oxidase [17] Kurtzman CP. Biotechnological strains of Komagataella from Penicillium simplicissimum. Eur J Biochem 1992; (Pichia) pastoris are Komagataella phaffii as determined from 208:651– 657. multigene sequence analysis. J Ind Microbiol Biotechnol 2009; [2] Fraaije MW, Veeger C, van Berkel WJH. Substrate specificity 36:1435–1438. of flavin-dependent vanillyl-alcohol oxidase from Penicillium [18] Daniel B, Pavkov-Keller T, Steiner B, Gutmann A, Nidetzky B, simplicissimum: Evidence for the production of 4-hydroxy- Sensen CW, van der Graaff E, Wallner S, Gruber K, Macheroux cinnamyl alcohols from 4-allylphenols. Eur J Biochem 1995; P. Oxidation of monolignols by the berberine bridge enzyme 234:271–277. family suggests a role in plant cell wall metabolism. J Biol Chem [3] van den Heuvel RHH, Fraaije MW, Laane C, van Berkel WJH. 2015; 290:1877–18781. Regio- and stereospecific conversion of 4-alkylphenols by the [19] Fraaije MW, Mattevi A, van Berkel WJH. Mercuration of vanillyl- covalent flavoprotein vanillyl-alcohol oxidase. J Bacteriol 1998; alcohol oxidase from Penicillium simplicissimum generates 180:5646–5651. inactive dimers. FEBS Lett 1997; 402:33–35. [4] Fraaije MW, van Berkel WJH. Catalytic mechanism of the [20] Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. oxidative by vanillyl-alcohol oxidase: evidence for the Jalview Version 2 — a multiple sequence alignment editor and formation of a p-quinone methide intermediate. J Biol Chem analysis workbench. Bioinformatics 2009; 25:1189– 1191. 1997; 272:18111–18116. [21] R Development Core Team. R: A language and environment [5] Fraaije MW, van den Heuvel RH, van Berkel WJ, Mattevi A. for statistical computing 2016. R Foundation for Statistical Structural analysis of flavinylation in vanillyl-alcohol oxidase. J Computing, Vienna, Austria. URL https://www.R-project.org/ . Biol Chem 2000; 275:38654–38658. [22] Huson DH, Scornavacca C. Dendroscope 3 : An interactive tool [6] Jin J, Mazon H, van den Heuvel RHH, Heck AJ, Janssen DB, for rooted phylogenetic trees and networks. Syst Biol 2012; Fraaije MW. Covalent flavinylation of vanillyl-alcohol oxidase is 61:1061–1067. an autocatalytic process. FEBS J 2008; 275:5191– 200. [23] Kvalnes-Krick K, Jorns MS. Role of the covalent and noncovalent [7] Tahallah N, van den Heuvel RHH, van den Berg WAM, Maier flavins in sarcosine oxidase. In: Müller F (ed.), Chemistry and CS, van Berkel WJH, Heck AJR. Cofactor-dependent assembly Biochemistry of Flavoenzymes, vol. II. Boca Raton, FL: CRC of the flavoenzyme vanillyl-alcohol oxidase. J Biol Chem 2002; Press Inc; 1991:425–435. 277:36425–36432. [24] van den Berghe-Snorek S, Stankovich MT. Thermodynamic [8] van den Heuvel RHH, Fraaije MW, Mattevi A, van Berkel WJH. control of D-amino acid oxidase by benzoate binding. J Biol Asp-170 is crucial for the redox properties of vanillyl-alcohol Chem 1985; 260:3373–3379. oxidase. J Biol Chem 2000; 275:14799–14808. [25] Job V, Marcone GL, Pilone MS, Pollegioni L. Glycine oxidase [9] Fraaije MW, van den Heuvel RHH, van Berkel WJH, Mattevi A. from Bacillus subtilis: characterization of a new flavoprotein. J Covalent flavinylation is essential for efficient redox catalysis in Biol Chem 2002; 277:6985–6993. vanillyl-alcohol oxidase. J Biol Chem 1999; 274:35514– 35520. [26] Choong YS, Massey V. Stabilisation of lactate oxidase flavin [10] van den Heuvel RHH, Fraaije MW, Ferrer M, Mattevi A, van anion radical by complex formation. J Biol Chem 1980; Berkel WJH. Inversion of stereospecificity of vanillyl-alcohol 225:8672–8677. oxidase. Proc Natl Acad Sci U S A 2000; 97:9455–9460. [27] Geissler J, Ghisla S, Kroneck PMH. Flavin-dependent alcohol [11] Mattevi A, Fraaije MW, Mozzarelli A, Olivi L, Coda A, van Berkel oxidase from yeast: Studies on the catalytic mechanism and WJH. Crystal structures and inhibitor binding in the octameric inactivation during turnover. Eur J Biochem 1986; 160:93–100. flavoenzyme vanillyl-alcohol oxidase: the shape of the [28] Ghanem M, Gadda G. Effects of reversing the protein positive active-site cavity controls substrate specificity. Structure 1997; charge in the proximity of the flavin N (1) locus of choline 5:907–920. oxidase. Biochemistry 2006; 45:3437–3447. [12] van den Heuvel RHH, van den Berg WAM, Rovida S, van Berkel [29] Ghanem M, Fan F, Francis K, Gadda G. Spectroscopic and WJH. Laboratory-evolved vanillyl-alcohol oxidase produces kinetic properties of recombinant choline oxidase from natural vanillin. J Biol Chem 2004; 279:33492–33500. Arthrobacter globiformis. Biochemistry 2003; 42:15179–15188. [13] Ewing TA, Gygli G, van Berkel WJH. A single loop is essential [30] Heuts DPHM, Scrutton NS, Mcintire WS, Fraaije MW. What’s in a for the octamerization of vanillyl alcohol oxidase. FEBS J 2016; covalent bond ? On the role and formation of covalently bound 283:2546–2559. flavin cofactors. FEBS J 2009; 276:3405–3427. [14] Benen JAE, Sánchez-Torres P, Wagemaker MJM, Fraaije MW, [31] Mewies M, Mcintire WS, Scrutton NS. Covalent attachment of van Berkel WJH, Visser J. Molecular cloning, sequencing, and flavin adenine dinucleotide (FAD) and flavin mononucleotide heterologous expression of the vaoA gene from Penicillium (FMN) to enzymes: The current state of affairs. Protein Sci 1998; simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase. J 7:7–20. Biol Chem 1998; 273:7865– 7872. [32] Liu B, Liu H, Zhong D, Lin C. Searching for a photocycle of [15] van Rooyen N. Identification, cloning and heterologous the cryptochrome photoreceptors. Curr Opin Plant Biol 2010; expression of fungal vanillyl-alcohol oxidases, PhD thesis, 13:578–586. University of the Free State, 2012. 26 G. Gygli, W.J.H van Berkel 110 147 150 110 129 110 94 264 284 291 262 283 264 229 414 438 443 412 433 414 379 560 584 589 558 579 560 522 I I K K T K K K R V L V V L G G G G G G G T T T V T T T L G G G G G G G W W W W W W I Y Y Y Y Y Y Y V V V V V V K R N E S K S I I I I I I A V L L L L L L F G G G G G G S L F V V S F E E E E E E E G G G G G G G N N N N N N N Y L L F D E R T D D T M M M I I R R R R R R K N N N S N N N Y L T Y M S S S S S S S T D V T T T N G G G G G G G I I I I I - L T P P P P P P P L V P L Q Q Q Q Q Q Q M M I I I I I - S S S S S S S S S S T S S T V K E E V R E Q I I I I I - F F F F F F F F F F K C G G G G G G G W W W I I I P P P P P P T L L L L A S K T K D A S Q Q Q Q G G F H A C L L V R K S V E W W W W W G G G G G G G M M M M I I I L L L L V D D D D D D D T V T T T L H D A P H G G I I I P P P P P P P A L N N K R L L K K K S R R D P R R I I I I I F F Y F Y Y Y A D D A D A D V Y Y F Y E Y L M S K L E E S K P P P P P P A R R R R R R R R R S N Q G Q F Y Y Y F V Y V S S T V V Y S A R K A S K G G G G G G G K K K E E K N F F Y F F F R H H R D P P P P P P P Q W Q N N N N N N N L F L L L L G G G G G G G W W W W W W W W I I I I A A A C C A V Y Y Y Y Y Y Y V Y Y V V V V V V G I L L L L P P N P P K S D H L C S P W W Q G G G G G G G A R K R R A F F F F F F Y N N T N K N D S S N N N S G Q I V V V V V V L L S L L L L E P S N E E K K R K K K K G I I I I I I H P P P P K P K M Q Q Q Q Q G G G G G G G G I S T L A A A K A A T L V T R P P P P P P P W W W W G M I S T A C T T D S D D D D T A A S A A A S Q Q Q Q Q Q Q I I I V V V V V V K S P S K K K E T S E E E D L L L L L I I I I I I D E D E E D S S N N N N S P P P P P P D V Q - - A A A A E A E D P F D L F L F Y W G W G G G G G G G - - I V T T V V V V P E E P P Y F Y F Y H N N R K K N N - - - N S S D N N N F R L F F F F P P P P P P P Q Q Q Q - - - R S R R R H D D D K R R K K K K D D D D D D D G Q - - - P P P P P P P E H D E V A T A A V A V L L L L V L - - I - A Y Y T A A A P P P A A A A A A E G G G G G G G I I - - - V V C V V K P S K P P P P E P P N D D N N N D I I - - - I I I I I I V V V T A L A L L K K K K K K K G - - - - I I A A A A A A A K A T A S L L L L L G G G G Q - - - - I S S S S S S S E S F S S V R R T K A M M M G G - - - A A S S V A P T N V T F F F F F F F E E T E E H Q - - - L V V V V L A E P T E A E A A A T N N N N N N H Q - - - F F L F F F F P N P P D K R E D E D F F L L L F T - - Y E D Y Y Y D R A R K H K K K R R K K R K K D G Q Q - - I I I I D E S D D A K E N T R V V V L L W M M M M W - - I I E P K K D P K P P P T T V T V F L L F L Q Q Q Q D D P D E N D N N D D N E D K E D S A A A A S A G G Q Q L F T F T P A P P P P A L L S N N N D N M M W W W W M G I I V V V L T L L L L L T L K L L N N D N D N D M Q M G H Y H H H H N A A A A A A T V Y V V V N N N N N N N N Q H H F Y H Y D R A K D K K F M G G G G G G Q W W W W W W P A A P P R R R R R R N D N N N S M M M M M M M M M M G I I I I D D D D D D D F V Y Y Y Y Y Y F G G G G G G G M H H H H H H R T T T T T T T P H A P P P P T T T T T T T Dacryopinax sp. T T T A A T Y R R R R R R E E K A E K E E E G G G Q G G I I H N N K K H S L V V L V P P P P P P P A A A S A M M I I I I I I I T P P S S R R L V L L L V V G G G G G G G Sclerotinia borealis P H H P P L E E E T E E T Y Y Y Y Y Y Y Q Q Q Q Q Q Q Q Talaromyces islandicus Talaromyces - K E E D D D L S V L V L V D D D D D D D G G G G G G G - L T N T T D T N D A T A A A A S M M M M M M M M M M M Nectria haematococca - Penicillium simplicissimum Y Y Y Y Y Y A P P P P P A F L F L L V F G G G G G G G M5FTW7 M5FTW7 Byssochlamys spectabilis - S L S L L L L L L L Y Y Y Y Y Y Y A V V A A A S W W Q - I D E V V V V V V V F Y F F F F Y L L L L L L G G G G - W9CKB3 W9CKB3 D D D K K D V V V V V V V N L V N N N N H H H H H H H - V D S T H E E E E E E T T T T T T T Q Q W W W W W W W I I - I L L L L L L L R T S R R R R R R R R R R C7Z853 M M M M CRG90285 P56216 - P E E F C Y Y Y Y Y Y Y Q Q Q G G G G G G G G G G G G V5FPK0 V5FPK0 - - - - - D E E E D E S S C C C S C D E E E E E E E E Q - - - - - K D T K K K K H H H H H H Q G G G G G G G G G - - I S K K S C N L Y C L L L L Y Y Y M M M M M W W W W - - S S K S D A A L N N P N N D M M M M G G G G G G G G I I I I A N D F F L L L L L L L L N K A R N N L W W W W I I V F L V L H H H H H H H D K K K D A A R K D A A Q Q I E E V T E Y D D D D D D D K R A K K K K A A A A A A A I V V V V V V A V A A A A C A A A C C A G G G G G G G Q ------N N N N N Y Y Y Y Y Y Y Y A D D D D V D E W I I I I I I E E E E E E E P P P P P P E D D A D D D M Q I I I I I S A E S A C K T T T T T T T K K E A E K V V Q Y Y Y Y Y Y Y D R R D D D K L L L L L L G G G G G G G M I I V L L T V V L A V L L L R T A T E T G G G G G G G Q I I V L V V V V V V V A E L V E E E H R R R S R R A Q G I I I I R E E R K E N E K E E S F Q G G G G G G G G M M I I S R R E E R R R R R R H D D P D E D D L C C L L L L I I L L A A T E D D E E E E S T P T T S P A T K D W W W I I D E E A R E E V L C T T S L V L L L L Y H N H M Q Q D A S R A A T A T T A T P S P P P P V A A A A V A Q Q Q I I I I I I I N N N N N N N E H R E K N A R R K R G G G F A A F Y F A T P E D T K R E H R R K K G G G G G G G Q I E K A K E A L L L V L V R S V K K R K C K K K G G Q G I - - - - N F N H A N N V V V V V V D T S S E Q Q M Q Q I I F F F F F F L S S S S S S S F S D Y Y R S D D E G W I D T T V K E S Y T Y S Y L P P E L P Q G G G G G G G Q S P P T K S Y T S Y Y Y D D S D D D S G G G G G G G W Q L K R A S L L A S S A S S S S K T S E E K G G G G G G M S E D E T L T L L L L L L P R R R A K R R K R K R R R R I I I L V T L D D D D D D A K P S R K K S N D D D D N N - K D N P P P P P P A D K L T T D F F Y F F F F G G G G P P P P P V V V V T V C V V V V V V V Q Q G G G G G G M Cycloclasticus sp. I I P P P P P P P D D D D D D D L S T L S C L L L L M M I I I I L L L L L L L V V V L V L V V V C E E C C C M M T A V T A V P A A T A A A A V V V V V V F W W W W W W W I I I I I I I I L L P L L K L L L L L L L A L A A A A M - - P L P P P T K H K K K D A E D D D E H L V H H H H Q - R P P N D R D D D D D D L L L H H H H H H H M Q Q M W K0C1W8 K0C1W8 - - - - I F R E A N F R R D R L V L L L L A M M M M M Q - I I I I E K E E A E L Y L L L L S V A L E E E E E E D - R R S N N K N H H H S H H D R R R R R R R Q G M G G G - T P A N P T N R S N N N K R R R R R R V L S M M M M W - I I I K A S T E A A A A E S E L L V T A Q G G G G G G - I I I S S A T S E K R V K V T T S T V V V V Q Q Q Q M - I I T A L L L L L P A P P P P P T C C C T T L M G M M ------R Y A T Y Y Y Y V V V V V V A F F L F F F F ------N E E E D D N N N N N N N N T A T D T T E G ------L H Y H Y H Y E H P P W Q G Q Q Q G G G G G - - I - - I I I I I I I K P L L L L L L L L L L L L F F - - - - I I T T N D A S D D D A V V V A V F F F F F Y G - - - - A S P H Y F T F H Y N K D D D D D D D M M M M Q - I - - - T V Y F Y F F F Y N S N L L L F F L F G G Q G - - - - I I D S R T T T T T T S L T L L V G G G G G G G - - - - F H L V V V V V V V R R A R R R R A F F A A V Y - I - - - L A L L L L L L L E E E E E E E G G G G G G G 10 - - - - L L L P P P P P P P P P P P P P P A K E L E H Q - - - - - S L E E E E E E E R R R R R R L C A C C T C A - - - - - I I I I I I K K V V L V L V V S R R R R R R M - - - - - I I I I I I A S V L L L L L L L K S R R K K K - - - - - H E C C A A A C A D D S E D D D K R K K R E Q - - - - - I I E Y T S Y Y Y Y V V V V T T A C S T T Q M - - - - - I I I A A A H C A A A A L A A V V L L V G M M - - - - - K L D N H P E K T A T E E A K G G G G Q Q Q G - - - - - D P E D E D D E K K A P H P K E Y Y Y Y Y Y W - I - - - - A V P V T V E L L Y L L L L L L G Q Q Q Q Q - - - - - L L N N D D N N N D D S D D D D N K A K L N M - - - - - I R S V L V V V V A E E T D H T K V T G M M M - - - - - E P E D K K E E E D E E D D D E A A V A A A S - - - - P P P L L L L L L L K E E R K R N D D H D D D S - I I - - - - I I V V V V V P P A P P P E E P E D E E K - - - - - R K R K K R R L F F L F L L G G G G G G G G G - - - - - S S N N N N N N N T T T T T T T S R T S S D G - - - - - I I I I I I H H Y L V V T V V A M M M M M M M - - - - - K S N R R H N N L L L L L K P P K K K A M M M - - - - - A E K R K K K K K Y Y Y Y Y Y F A S C A S A S ------I I I I I I S S S S S P S P V Q G G G G G G - - - - - L T L L L L E T T P P P P P P P M M M Q Q Q Q - - - - - T T D D D E D N D Y Y H Y Y Y Y S S S S S S S - - - - - I I - P P L L L L L F F F F F F F G G G G G G - - - - I T T T V V V V R P F A A F F F N G G G Q G G G - - - - I I I I I I R P V V L P P T P P P P L V L L L G - - - - P T T S S S S S S Y N N A N N N E H H H H H H H - I - I - - L P P P P P P P A A A A A G G G G G G G G G - - I - - D T S R R P P S P M M M M M M M G G G G G G G - - - - K T S V V V V V V K L L L L L L L N N N N N N G - - - - P E P R R R R R R R T P P P P P P W W W W W W G - - - - I I I A A P P P P P P P V V L Y V L L V V M M M - - - - - A A A A A A A A M G G G G G G G W W W W W W W ------I A A A A A A P L L D N A K D D N M M M M M 1 1 1 1 1 1 1 95 111 148 151 111 130 111 265 285 292 263 284 265 230 415 439 444 413 434 415 380 P56216 Penicillium simplicissimum/1-560 islandicus/1-584 CRG90285 Talaromyces M5FTW7 Dacryopinax sp./1-589 C7Z853 Nectria haematococca/1-558 W9CKB3 Sclerotinia borealis/1-579 V5FPK0 Byssochlamys spectabilis/1-560 K0C1W8 Cycloclasticus sp./1-522 P56216 Penicillium simplicissimum/1-560 islandicus/1-584 CRG90285 Talaromyces M5FTW7 Dacryopinax sp./1-589 C7Z853 Nectria haematococca/1-558 W9CKB3 Sclerotinia borealis/1-579 V5FPK0 Byssochlamys spectabilis/1-560 K0C1W8 Cycloclasticus sp./1-522 P56216 Penicillium simplicissimum/1-560 islandicus/1-584 CRG90285 Talaromyces M5FTW7 Dacryopinax sp./1-589 C7Z853 Nectria haematococca/1-558 W9CKB3 Sclerotinia borealis/1-579 V5FPK0 Byssochlamys spectabilis/1-560 K0C1W8 Cycloclasticus sp./1-522 P56216 Penicillium simplicissimum/1-560 islandicus/1-584 CRG90285 Talaromyces M5FTW7 Dacryopinax sp./1-589 C7Z853 Nectria haematococca/1-558 W9CKB3 Sclerotinia borealis/1-579 V5FPK0 Byssochlamys spectabilis/1-560 K0C1W8 Cycloclasticus sp./1-522 A B : Bioinformatic analysis of selected sequences: (A) Multiple sequence alignment, showing consensus sequence. Note that a proposed variant of the PTS1 sequence, WKL-COOH instead instead WKL-COOH sequence, the PTS1 of variant a proposed that Note sequence. consensus alignment, showing sequence (A) Multiple sequences: selected of analysis S1 : Bioinformatic Figure 1998; Letters WJH. FEBS Berkel van M, Veenhuis KA, Sjollema MW, . Fraaije simplicissimum in Penicillium oxidase vanillyl-alcohol of localization Subcellular in: described (as SKL-COOH of the by indicated as substitutions, of the number to proportional length is Branch sequences. of tree (B) Phylogenetic genes. vao novel in the other, not in P56216 but present only 422:65-68), is position). per sequence (10 substitutions bar