Báo cáo khoa học: Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure pdf

According to the crystal structure of MaL, the four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site.. reesei; ScdelDSGL559, Melan

albomyces laccase for enzyme production, catalytic

properties and structure

Martina Andberg1, Nina Hakulinen2, Sanna Auer1, Markku Saloheimo1, Anu Koivula1,

Juha Rouvinen2and Kristiina Kruus1

1 VTT Technical Research Center of Finland, Finland

2 Department of Chemistry, University of Joensuu, Finland

Introduction

Laccases (EC 1.10.3.2; p-dihenol dioxygen

oxidoreduc-tases) are copper-containing metalloenzymes that

oxidize various phenolic compounds, anilines and even

some nonaromatic compounds by a one-electron

removal mechanism, which usually generates radicals

Oxidation of reducing substrates occurs concomitantly with the reduction of molecular oxygen to water Lac-cases are ubiquitous enzymes found in various micro-organisms, insects, and plants They share structural similarities with other blue multicopper oxidases,

Keywords

ascomycete; C-terminal plug; multicopper

oxidase; mutants; site-directed mutagenesis

Correspondence

M Andberg, VTT Technical Research Center

of Finland, P.O Box 1000, FIN-02044 VTT,

Finland

Fax: +358 20 722 7071

Tel: +358 20 722 5124

E-mail: martina.andberg@vtt.fi

Website: http://www.vtt.fi/research/bic/

?lang=en

Database

The atomic coordinates and structure

factors have been submitted to the

Protein Data Bank under the accession

number 3DKH

(Received 2 July 2009, revised 17 August

2009, accepted 28 August 2009)

doi:10.1111/j.1742-4658.2009.07336.x

The C-terminus of the fungal laccase from Melanocarpus albomyces (MaL)

is processed during secretion at a processing site conserved among the ascomycete laccases The three-dimensional structure of MaL has been solved as one of the first complete laccase structures According to the crystal structure of MaL, the four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site The C-terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper In order to analyze the role of the processed C-terminus, site-directed mutagenesis of the MaL cDNA was performed, and the mutated proteins were expressed in Tricho-derma reesei and Saccharomyces cerevisiae Changes in the C-terminus of MaL caused major defects in protein production in both expression hosts The deletion of the last four amino acids dramatically affected the activity

of the enzyme, as the deletion mutant delDSGL559was practically inactive Detailed characterization of the purified L559A mutant expressed in

S cerevisiae showed the importance of the C-terminal plug for laccase activity, stability, and kinetics Moreover, the crystal structure of the L559A mutant expressed in S cerevisiae showed that the C-terminal muta-tion had clearly affected the trinuclear site geometry The results in this study clearly confirm the critical role of the last amino acids in the C-terminus of MaL

Abbreviations

2,6-DMP, 2,6-dimethoxyphenol; ABTS, 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid); BsL, Bacillus subtilis laccase; MaL,

Melanocarpus albomyces laccase; rMaL, recombinant MaL expressed in T reesei; Sc(delDSGL559), Melanocarpus albomyces laccase delDSGL559 mutant expressed in Saccharomyces cerevisiae; Sc(L559A), Melanocarpus albomyces laccase L559A mutant expressed in Saccharomyces cerevisiae; ScMaL, Melanocarpus albomyces laccase expressed in Saccharomyces cerevisiae; Tr(delDSGL559),

Melanocarpus albomyces laccase delDSGL 559 mutant expressed in Trichoderma reesei; Tr(L559G), Melanocarpus albomyces laccase L559G mutant expressed in Trichoderma reesei.

including ascorbate oxidase, ceruloplasmin, CueO, and

Fet3p For catalytic activity, all four copper atoms are

needed: one type 1 (T1) copper forming a mononuclear

site, and one type 2 (T2) copper and two type 3 (T3

and T3¢) coppers forming a trinuclear site

Melanocarpus albomyces is a thermophilic fungus

expressing a laccase with substantial thermal stability

and a pH optimum with phenolic substrates in the

neutral pH region [1] These unusual properties, as

compared with most reported fungal laccases, makes

M albomyceslaccase (MaL) an interesting enzyme for

many applications The three-dimensional structure of

MaL has been solved as one of the first complete

laccase structures (Protein Data Bank codes: 1GW0,

laccase from M albomyces; 2IH8, a low-dose crystal

structure of a recombinant MaL; 2IH9, a high-dose

crystal structure of a recombinant MaL; and 2Q9O,

near-atomic resolution structure of recombinant MaL)

[2–4] The enzyme is composed of three cupredoxin

domains, A, B, and C (or 1, 2, and 3), which all have

a similar Greek key b-barrel structure The

mononu-clear site is located in domain C, whereas the

trinucle-ar site is between domains A and C Electrons trinucle-are

withdrawn from the mononuclear site and further

transferred about 13 A˚ along a conserved Cys–His

pathway into the trinuclear site, where dioxygen is

reduced to water The MaL structure was the first

solved three-dimensional structure showing dioxygen

binding [2] Since then, dioxygen binding has also been

found for Bacillus subtilis laccase (BsL) [5] and

cerulo-plasmin [6] Other solved laccase crystal structures

have shown only one oxygen atom between the two T3

coppers [7–10] The crystal structure of MaL also has

a chloride ion attached to the T2 copper, whereas

other crystal structures of multicopper oxidases have

hydroxyl ion⁄ water in this position The role of the

chloride ion is unknown A number of anions, i.e

CN), N3), and F), are known to act as effective

lac-case inhibitors [11,12] However, chloride ion does not

act as an inhibitor for MaL, as shown by Kiiskinen

et al [1] Instead, azide is a well-known inhibitor of

MaL According to spectroscopic measurements, the

binding of azide has been suggested to bridge the T2

copper and one of the T3 coppers [13,14], or bind to

one T3 copper, as observed in the crystal structure of

ascorbate oxidase [15] Recently, the azide was found

to bind between two T3 coppers in the crystal

struc-ture of BsL [5]

The three-dimensional structure of MaL revealed

that the C-terminus of the enzyme penetrates to a

tunnel leading to the trinuclear site (Fig 1) This

unique feature has not been observed in any other

published laccase crystal structures Instead, in other

known laccases, this cavity is open, and it is thought

to provide access to the fresh oxygen molecules needed in the catalytic cycle The C-terminus of MaL blocks this route, as the packing of the C-terminus against the tunnel is extensive, and there is no space for dioxygen or any other molecules to enter Further-more, the C-terminal carboxylate group in MaL is hydrogen-bonded to the side chain of His140, which

is one of the His residues coordinating the T2 copper

in the trinuclear center

C-terminal sequencing of the mature MaL showed that the C-terminus is post-translationally processed after Leu559, leading to removal of the last 14 amino

Fig 1 (A) Surface model of M albomyces laccase; a few of the last amino acids are represented as a purple worm (Protein Data Bank code: 2Q9O) (B) The C-terminus of M albomyces (in purple) penetrates to the tunnel leading towards the trinuclear site Coppers are represented as orange balls, water atoms as red balls, and dioxygen as a red stick.

acids of the mature protein [16] Similar C-terminal

processing has also been reported for other ascomycete

laccases, from Neurospora crassa [17],

Podospori-na anseriPodospori-na [18], and Myceliophthora thermophila [19]

The processing site for all of these laccases is

Asp-Ser-Gly-Leu Interestingly, a similar type of sequence is

found in the C-termini of other ascomycete laccases

that do not undergo the C-terminal processing, e.g

Borytris cinerea, Cryptonectria parasitica, and

Gau-emannomyces graminis var tritici The last four amino

acids in these laccases are Asp, Ser, Gly, and Leu⁄ Ile ⁄

Val The C-terminal end seems to be conserved among

all ascomycete laccases The conserved C-terminus of

ascomycete laccases might have a special role in the

enzyme

In order to analyze the role of the C-terminus,

site-directed mutagenesis of MaL cDNA was performed,

and the mutated proteins and wild-type enzyme were

expressed in Trichoderma reesei and Saccharomyces

cerevisiae We report here the characterization of the

C-terminal mutants and the three-dimensional structure

of the L559A mutant expressed in S cerevisiae

[Sc(L559A)]

Results

Production and characterization of MaL mutants

expressed in T reesei

Two mutations were made in the MaL gene, and the

mutated proteins were expressed in T reesei The

mutated laccase constructs were produced by

site-directed mutagenesis on the plasmid pLLK8, a T

ree-seiexpression vector containing the cDNA coding for

MaL between the cbh1 promoter and terminator In

the L559G mutant expressed in T reesei [Tr(L559G)],

Leu559 was replaced with a Gly to change the

pro-cessing site to prevent C-terminal cleavage, and in

the delDSGL559 mutant expressed in T reesei

[Tr(delDSGL559)], an Asp at position 556 was replaced

by a stop codon to delete the last four amino acids

(Asp-Ser-Gly-Leu) of the mature MaL protein The

mutated laccases, as well as two wild-type recombinant

MaLs (rMaLs) (produced by the pLLK13 and the

cbh1-negative pMS176 strains), were expressed in

T reesei in shake flask cultures, and laccase

pro-duction was analyzed by activity determination

with 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid)

(ABTS) and by immunoanalysis of culture

superna-tants For the mutant Tr(delDSGL559), a transformant

in which the cbh1 gene had been replaced by the

expression construct was found and used The

C-termi-nal mutations in Tr(L559G) and Tr(delDSGL559) had

a substantial effect on the production level in T reesei,

as very low expression levels were observed for the mutants according to the activity assays and western blot analysis, as compared with the production of wild-type rMaL (Fig S1) The production level in the

T reesei culture supernatant of the two mutants was estimated to be 10 mgÆL)1, which was significantly lower than the production level of the pLLK13 wild-type rMaL (200 mgÆL)1) The activities on ABTS, 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine in the culture supernatants of the mutants were considerably reduced In fact, no activity could be detected for the Tr(delDSGL559) mutant, although amounts detectable

by western blot analysis were expressed into the super-natant The ABTS activity in the culture supernatant

of the Tr(L559G) mutant was several hundred-fold lower than that of the wild-type rMaL Comparison of the production level and the activity in the culture supernatant showed the specific activity of the mutants

to be considerably lower than that of the wild-type enzyme In addition to having low expression levels, the Tr(L559G) and Tr(delDSGL559) mutants were partly degraded (Fig S1, lanes 2 and 3) Some laccase degradation products were also detected in the culture supernatants of the wild-type rMaL produced in the two strains, but the ratio of degraded laccase to full-length laccase was much higher in the mutant strains Changes in the original C-terminus thus caused major defects in protein production in T reesei as well as changes in the protein properties

The T reesei strain producing the Tr(delDSGL559) protein was also cultivated in a laboratory-scale bioreactor (20 L), and the protein was purified from the culture supernatant by applying the procedure optimized for the wild-type rMaL [20] The purifica-tion protocol contained three chromatographic steps: anion exchange chromatography, hydrophobic interac-tion chromatography and, finally, anion exchange chromatography with a high-resolution resin The Tr(delDSGL559) mutant started to degrade during purification, and the mutant laccase could not be obtained from the T reesei culture filtrate

Production and characterization of MaL in

S cerevisiae Owing to the difficulties in producing the mutant laccases in T reesei, S cerevisiae was chosen as

an expression host for the designed mutants The full-length gene of MaL containing the C-terminal extension, i.e the last 14 amino acids that are cleaved from the mature protein, was expressed in S cerevisiae, and the protein was purified from this source The

C-terminus of the purified laccase was analyzed by

C-terminal sequencing to determine whether yeast is

also able to process the C-terminus properly MaL has

previously been showed to be processed at its

C-termi-nus during secretion, both in Melanocarpus and in

Trichoderma [16,20] The results from the sequencing

clearly showed that the yeast was not able to process

MaL correctly, and that the additional 14 amino acids

were present in the protein Therefore, all further work

was performed with another construct, pMS175, where

mature MaL cDNA, with a stop codon, was

intro-duced after the C-terminal processing site [16]

The effects of using S cerevisiae as expression host

on the properties of M albomyces laccase (ScMaL)

were also studied The conditions for production of

ScMaL in shake flask cultures were optimized in terms

of CuSO4 concentration in the medium, temperature,

culture medium, and induction conditions The

opti-mal culture conditions were found to be as follows:

synthetic complete medium (SC-URA) buffered to pH

6 with succinate and supplemented with 1 mm CuSO4

at 250 r.p.m and 30C Yeast cells were grown on

raffinose (20 gÆL)1), and a washing step prior to a

change to induction medium containing galactose

(20 gÆL)1) was shown to have a positive effect on

lac-case production The production level in shake flask

cultures was about 4.5 nkatÆmL)1 (ABTS activity),

roughly corresponding to 7 mgÆL)1 ScMaL, when

calculated from the specific activity for ScMaL

(610 nkatÆmg)1)

ScMaL was heavily overglycosylated (Fig 2)

Iso-electric focusing combined with activity staining of the

culture supernatant demonstrated that ScMaL had

sev-eral pI forms (pI 3.5–5), whereas the rMaL produced

in T reesei only had one pI form (pI 4.0) (data not

shown) Purification of ScMaL was performed in two

chromatographic steps, including weak and strong

anion exchange resins Owing to the heterogeneous

overglycosylation of ScMal, the laccase was

fraction-ated into two separate pools: pool 1, containing the

more heavily overglycosylated laccase; and pool 2,

with less overglycosylated laccase showing a major

laccase band at 100 kDa as analyzed by SDS⁄ PAGE

(Fig 2) The ABTS activity for pool 1 (430 nkatÆmg)1)

was consistently slightly lower than that for pool 2

(520 nkatÆmg)1) Pool 2 was therefore used for

terization Table 1 presents a summary of the

charac-teristics of purified ScMaL in comparison to the rMaL

produced in T reesei The specific activity of ScMaL

(520 nkatÆmg)1) was lower than the reported specific

activity of rMaL (840 nkatÆmg)1) However, the Km

values for ABTS showed practically no difference

between the MaL preparations expressed in S

cerevi-siae and T reesei Also, the temperature stability was similar for ScMaL and rMaL The N-terminal sequencing verified correct processing of the yeast a-prepro sequence (KEX2 cleavage site) Removal of the glycans by enzymatic deglycosylation with endo-b-N-acetylglucosaminidase F1 slightly lowered the specific ABTS activity of ScMaL, but had no effect on the specific activity of rMaL Deglycosylation of over-glycosylated ScMaL resulted in one pI isoform of the enzyme, in contrast to the several isoforms seen with the enzyme still having the glycans attached The results confirmed that MaL can be expressed in

S cerevisiae and that the protein properties are com-parable to those of the wild-type laccases and the lac-case expressed heterologously in T reesei

Production and purification of the Sc(delDSGL559) and Sc(L559A) mutants

Two C-terminal mutants of MaL, Sc(delDSGL559) and Sc(L559A), were expressed in S cerevisiae, and the proteins were purified to homogeneity from the yeast culture supernatant In the Sc(delDSGL559) mutant, the last four amino acids (Asp-Ser-Gly-Leu) were deleted from the protein [equivalent to the Tr(del-DSGL559 mutant], and in the Sc(L559A) mutant, the C-terminal Leu was replaced with a smaller Ala, in order to prevent the hydrogen bonding of the carbox-ylate group to a side chain of His140 Production of the two mutated proteins in S cerevisiae was carried out in shake flasks using conditions optimized for

Fig 2 SDS ⁄ PAGE gel and western blot analysis of purified ScMaL The samples from a Resource Q run were separated in a 12% SDS ⁄ PAGE gel In lanes 1–3, a Coomasssie-stained gel is shown, and in lanes 5–7, the gel has been blotted using antibodies against MaL The samples are rMaL (purified from T reesei) in lanes 1 and 7, ScMaL pool I, containing the more heavily overgly-cosylated laccase, in lanes 2 and 5, and the less overglyovergly-cosylated laccase ScMaL pool II in lanes 3 and 6.

ScMaL production The total cultivation volume was

4 L for both mutated proteins The culture

superna-tant was concentrated prior to purification Similarly

to what was found for the Sc(delDSGL559) mutant

expressed in T reesei, laccase activity on ABTS

sub-strate was not detectable in the Sc(delDSGL559) culture

supernatant, or in the concentrated culture filtrate,

although the expression of the mutant laccase was

confirmed by western blot analysis The result clearly

confirms that the last four amino acids are essential

for enzyme activity In the culture supernatant of

Sc(L559A), the activity on ABTS was 1.8 nkatÆmL)1,

which was 2.5-fold lower than the activity for ScMaL

(4.5 nkatÆmL)1)

The Sc(delDSGL559) mutant was purified in four

subsequent chromatographic steps As the

Sc(del-DSGL559) mutant was not active in the culture

super-natant or after the first purification steps, the pooling

of the laccase-containing fractions was based on

anti-body detection on dot blots In the pooled fractions

after the third hydrophobic interaction step, very low

but detectable laccase activity on ABTS could be

observed The specific activities in two separate pools

were 0.21 and 0.45 nkatÆmg)1 These values are over

1000-fold lower than the specific activity of the

wild-type ScMaL (520 nkatÆmg)1) The activity results for

the Sc(delDSGL559) mutant, together with the activity

results for the corresponding mutant Tr(delDSGL559)

(see above), clearly indicate the essential role of the

last four amino acids for the function of MaL

The Sc(L559A) mutant was also produced in shake

flasks for the initial characterization studies From 4 L

shake flask cultures, overall 11.1-fold purification and

activity recovery of 24% were achieved Altogether,

8.7 mg of purified Sc(L559A) mutant was recovered,

with a specific ABTS activity of 102 nkatÆmg)1 In

order to produce enough protein for crystallization purposes, the Sc(L559A) mutant was also produced in

a laboratory-scale bioreactor (20 L), and the enzyme was purified to homogeneity The protein yield was

22 mg, and the specific activity of the final Sc(L559A) sample was 184 nkatÆmg)1 Thus, the specific activity

of the Sc(L559A) mutant was three-fold to four-fold lower than that of the wild-type ScMaL (520 nkatÆmg)1)

Characterization of the Sc(delDSGL559) and Sc(L559A) mutants

The two purified MaL mutant proteins expressed in yeast were characterized and compared with the wild-type ScMaL In order to determine whether the muta-tions had affected the overall structure of the protein,

CD spectra of the Sc(delDSGL559) and Sc(L559A) mutants were measured and compared with that of the wild-type laccase (ScMaL) (Fig 3A) The general shapes of the spectra were the same for the Sc(del-DSGL559) and Sc(L559A) mutants and wild-type ScMaL, which suggests that no major changes in the conformation of the mutated enzymes had occurred The thermal unfolding profiles measured with CD (Fig 3B) were broad, with no clear folded–unfolded transition for ScMaL and the two mutants The Sc(L559A) mutant starts to unfold at a lower tempera-ture as compared with ScMaL, suggesting slightly reduced thermal stability The difference between the Sc(delDSGL559) mutant and ScMaL is even greater; the Sc(delDSGL559) mutant exhibited an unfolding behavior without any transition state, and started to unfold at relatively low temperatures (40–50C) The

Tmvalues were estimated from the graph to be 71C,

69C and 65 C for ScMaL, the Sc(L559A) mutant,

Table 1 Characterization of the M albomyces laccase produced in S cerevisiae (ScMaL) and in T reesei (rMaL).

Overglycosylation (on SDS ⁄ PAGE

and western blot)

Effect of deglycosylation

(endo-b-N-acetylglucosaminidase F1)

on isoelectric point

and the Sc(delDSGL559) mutant, respectively Both

mutants were expected to be stable at 25C, and all of

the following kinetic analyses were therefore performed

at this temperature

The redox potential of the mononuclear (T1) copper center for the Sc(L559A) mutant was measured using the ferrocyanide⁄ ferricyanide redox buffer system (E0,Fe= 0.433 V) [21] in 20 mm Tris⁄ HCl (pH 7.5) The laccase concentration used in the redox measure-ments was estimated from the 600 nm absorbance, using an extinction coefficient of 5700 m)1Æcm)1 The redox potential of the Sc(L559A) mutant was deter-mined to be 0.43 V, which is in agreement with the value measured for ScMaL and also for the rMaL expressed in T reesei (0.47 V)

As the deletion mutant Sc(delDSGL559) was practi-cally inactive, no detailed kinetic characterization could be performed, and the kinetic characterization was performed only with the Sc(L559A) mutant For analysis of the purified Sc(L559A) mutant in more detail, three different substrates were used The kinetic constants presented in Table 2 show that the mutation had a two-fold increased Kmvalue on the nonphenolic substrate ABTS (Km= 900 lm) as compared with wild-type ScMaL (Km= 400 lm) The Sc(L559A) mutant also exhibited a four-fold decreased catalytic constant on ABTS (kcat= 394 min)1) as compared with ScMaL (kcat= 1686 min)1) Consequently, the specificity constant on ABTS dropped about 10-fold from 4.2 lm)1Æmin)1 for ScMaL to 0.44 lm)1Æmin)1 for the Sc(L559A) mutant However, for the two phenolic substrates 2,6-DMP and syringaldazine, the L559A mutation did not greatly influence the catalytic parameters (Table 2) The Km values for the Sc(L559A) mutant on 2,6-DMP and syringaldazine were 16 and 31 lm, and the corresponding Km values for ScMaL were 11 and 37 lm, respectively The L559A mutation had decreased the turnover number

on syringaldazine about two-fold (kcat= 1263 min)1)

as compared with wild-type ScMaL (kcat= 2410 min)1) With 2,6-DMP, no significant changes in the kinetic parameters were observed

In order to determine whether the L559A mutation affects the trinuclear site, inhibition constants for sodium azide were also determined Azide inhibits the

Fig 3 (A) CD spectra of wild-type ScMaL and the Sc(delDSGL 559 )

and Sc(L559A) mutants The CD spectra of wild-type ScMaL (solid

line) and the Sc(delDSGL559) (dotted line) and Sc(L559A) (double

dotted line) mutants were recorded from 240 to 190 nm at 25 C in

10 m M sodium phosphate buffer (pH 7.1) (B) Temperature-induced

unfolding of wild-type ScMaL and the Sc(delDSGL559) and

Sc(L559A) mutants measured by CD spectroscopy Changes in

ellipticity of ScMaL and the Sc(delDSGL 559 ) and Sc(L559A) mutants

were recorded at 202 nm upon heating from 30 C to 90 C in

10 m M sodium phosphate buffer at pH 7.1 The data were

smoothed with ORIGIN 7.5 (OriginLab).

Table 2 Kinetic parameters for wild-type ScMaL and the Sc(L559A) mutant ABTS activity was measured in 25 m M succinate buffer at pH 4.5 and 25 C, and 2,6-DMP and syringaldazine activities were measured in 40 m M MES buffer at pH 6 and 25 C For determination of inhi-bition constants for the sodium azide, the enzyme was preincubated for 2 min with NaN3prior to addition of substrate The Kivalue was obtained from Dixon plots The error in all measurements was estimated to ± 15% ND, not determined.

laccase activity by binding to the trinuclear centrer In

the crystal structure of ascorbate oxidase, azide is

sug-gested to bind to one T3 copper [15], and in the crystal

structure of BsL, between two T3 coppers [5]

Spectro-scopic findings have suggested that azide bridges the

T2 copper and one of the T3 coppers [13,14] The

inhibition constants (Ki) of sodium azide for the

Sc(L559A) mutant and ScMaL were determined using

ABTS or 2,6-DMP as substrate The results indicated

that sodium azide was a mixed inhibitor with respect

to both ABTS and 2,6-DMP; that is, the inhibitor

binds at a location distinct from the reducing

sub-strate-binding site When the nonphenolic substrate

ABTS was used, the Ki value of sodium azide was

increased approximately 11-fold for the Sc(L559A)

mutant as compared with that of the wild-type ScMaL,

and was calculated (from Dixon plots) to be 85 lm

(Table 2) The corresponding value for the wild-type

enzyme was 7.9 lm However, the difference in the Ki

values for ScMaL and the Sc(L559A) mutant was only

two-fold when the phenolic substrate 2,6-DMP was

used

The pH optima of the purified Sc(L559A) mutant

were determined using ABTS and 2,6-DMP as

sub-strates On ABTS, both wild-type ScMaL and the

Sc(L559A) mutant had optimal activity at pH 4, but

the pH activity profile of the Sc(L559A) mutant was

more narrow than that of the wild-type enzyme, and

had shifted to the alkaline side (Fig 4) At pH 3, the

relative laccase activity was 88% for ScMaL, whereas

for the Sc(L559A) mutant it had dropped to below

3% On 2,6-DMP, the pH activity profile of the

Sc(L559A) mutant was similar to that of the wild-type

enzyme, the mutant having a slightly broader pH

activity in an alkaline pH range (Fig 4)

The stability of the purified Sc(L559A) mutant was

also analyzed as a function of pH and temperature

The Sc(L559A) mutant remained stable within the pH

range 5.5–8 after 330 h of incubation at 4C (data not

shown) At pH < 5, the enzyme started to lose its

activity, the residual activity being 40% at pH 5, and

5% at pH 4 after 330 h No activity was observed at

pH 3 and pH 2 after 330 h In addition, it was shown

that the Sc(L559A) mutant was not stable at

tempera-tures higher than 50C during prolonged incubations

(at pH 6) The thermal stability was clearly reduced in

comparison to ScMaL As an example, the half-life

(T1 ⁄ 2) of wild-type ScMaL at 60C was 4.5 h, whereas

the half-life of the Sc(L559A) mutant at this

tempera-ture was only a few minutes (Table 3) The results are

consistent with the CD spectrum as a function of

temperature, which also indicated lowered thermal

stability of the Sc(L559A) mutant

Deglycosylation of ScMaL and the Sc(L559A) mutant

The protein properties of wild-type ScMaL were com-parable to the properties of rMaL (Table 1), although ScMaL was heavily overglycosylated Thus, the yeast was a suitable host for production of the MaL vari-ants for structural analysis As the long glycan chains attached in yeast to the laccase protein most probably disturb crystallization, optimization of conditions for removing the N-glycans was performed Enzymatic deglycosylation of ScMaL and the Sc(L559A) mutant was performed with endo-b-N-acetylglucosaminidase

Fig 4 The pH optima of the wild-type (h) and Sc(L559A) mutant (d) laccases measured at 22 C, using ABTS (A) or 2,6-DMP (B) as substrate The enzymes were incubated in McIlvaine’s buffer.

Table 3 The apparent half-life values, T1⁄ 2, of the Sc(L559A) mutant and ScMaL for ABTS at 40 C, 50 C, and 60 C.

F1 (Sigma-Aldrich, St Louis, MO, USA), which is an

enzyme suitable for deglycosylation of native proteins

Endo-b-N-acetylglucosaminidase F1 generates a

trun-cated sugar molecule with one N-acetylglucosamine

residue remaining attached to the Asn The effect of

deglycosylation was analysed by SDS⁄ PAGE and

activity measurements A single band at 90 kDa was

detected in the deglycosylated laccase samples, in

con-trast to the major band at about 100 kDa with an

additional smear of larger proteins observed for the

nontreated enzyme (Fig S2) The removal of the

gly-cans reduced the laccase activity by approximately

5% (data not shown)

The secondary structure of the deglycosylated

ScMaL was also measured and compared with that

of the nonglycosylated ScMaL by CD spectroscopy

The spectra of ScMaL and deglycosylated ScMaL

showed very little difference, indicating no major

changes in the protein fold (data not shown) The

thermal stability of the enzymes was also analyzed by

CD measurement The results indicated that the

ther-mostability of the deglycosylated enzyme was slightly

improved in comparison with the glycosylated

enzyme

The three-dimensional structure of the Sc(L559A)

mutant

In order to determine the structural effects of the

C-terminal mutation, the Sc(L559A) mutant was

crystallized for X-ray analysis A crystal was diffracted

to 2.4 A˚, and the crystal structure was solved by

molecular replacement The electron density map

clearly confirmed that the last residue of the mutant

was an Ala instead of a Leu (Fig 5A)

Superimposi-tion of the rMaL (Protein Data Bank code: 2Q9O)

and Sc(L559A) mutant (Protein Data Bank code:

3KDH) structures showed that an additional water

molecule was present in the mutant structure (Fig 5B)

Owing to the lower steric limitations of the side chain

of the Ala than of the Leu, water may occupy the

space Furthermore, the side chain of His140, which is

coordinated to the T3 copper, rotated slightly and

formed a hydrogen bond with the new water In the

structure of wild-type MaL, His140 was

hydrogen-bonded to the carboxylate group of the C-terminus,

with a distance of 3.1 A˚ In the Sc(L559A) mutant, the

distance between the ND1 atom of His140 and the

ter-minal oxygen OXT atom of Ala559 increased to 4.0 A˚

In addition, a nearby Asn109 adopted a different

conformation in the Sc(L559A) mutant structure

(Fig 5B) The C-terminal mutation clearly affected the

trinuclear site geometry

In addition, the B-value of the T2 copper was clearly higher than the B-values of the two T3 coppers in the trinuclear site (Table 4) This was observed in both molecules in an asymmetric unit, thus verifying the phenomenon Furthermore, no electron density was observed for the chloride ion in molecule A A chlo-ride ion is coordinated to the T2 copper in the wild-type MaL [4] In molecule B, some electron density was observed, but the refined chloride showed a very high B-value On the basis of this structure solved at 2.4 A˚ resolution, it is impossible to say whether there

is a hydroxide or chloride ion, but we decided to refine

a chloride ion, because our near-atomic resolution structure has confirmed that MaL has a chloride ion

in this position It is likely that the occupancy of the chloride ion was less in molecule B and that it was totally lost in molecule A Therefore, it seems that the

Fig 5 (A) 2F o ) F c electron density map of the C-terminus in the crystal structure of the Sc(L559A) mutant (Protein Data Bank code: 3KDH) (B) Superimposition of the native enzyme (Protein Data Bank code: 2Q9O) (green) and the Sc(L559A) mutant structure (in blue).

Sc(L559A) mutant at least partly loses its chloride ion

and probably also the T2 copper, as the B-value of the

T2 copper was rather high On the basis of the

anoma-lous signal, the estimated occupancy of the T2 copper

was about 0.5 T2 copper depletion is known to be

common for laccases The first solved laccase structure

lacked T2 copper [22], and several published structures

seem to have only partial T2 occupation, on the basis

of their high B-values So far, MaL has been found to

be particularly stable, and before this mutant structure,

no signs of partial occupation of the T2 copper had

been observed

Dioxygen was refined inside the trinuclear site of the

Sc(L559A) mutant Dioxygen has earlier been observed

to bind to coppers in the trinuclear site, with all

copper–oxygen distances being 2.2–2.6 A˚ [2] or slightly

more close to one of the type 3 coppers as observed in

a near-atomic resolution structure [4] In the present

Sc(L559A) mutant structure, dioxygen was again

refined slightly differently The copper–ligand distances

are presented in Table 4 In molecule B, the distance

between dioxygen and the T2 copper has extended to

3.1 A˚ However, our previous studies with MaL have

shown that the trinuclear site is sensitive to X-rays, and

the observed structure may depend on the data

collec-tion strategy or intensity of the beam [3] Electrons are

extracted in the mononuclear site by the T1 copper and

further transferred to the trinuclear site, where

dioxy-gen acts as a terminal electron acceptor Therefore, it is

difficult to draw any conclusions about the effect of the

C-terminal mutation on the binding of dioxygen In addition, it should be noted that the B-values of oxygen atoms are very low, especially in molecule A

Discussion

The ascomycete M albomyces produces a thermostable and alkaline laccase that undergoes C-terminal processing The processing site has been shown to be conserved, but the C-terminal extension is not present

in all ascomycete-type laccases The laccase sequences from Chaetomium globosum, P arenaria, N crassa,

My thermophila, Thielaviae arenaria and Magnapor-the grisea contain the extension (Fig 6) Processing has been reported in the literature for only some laccases [16–19,23] The amino acid preceding the C-terminal extension in the laccases undergoing processing has been shown to be a Leu C-terminal processing has also been shown for the basidomycete laccase from Coprinus cinereus, but the processing is distinct from that of the ascomycete laccases, because the C-terminus of C cinereus laccase does not contain the conserved ascomycete cleavage site [24]

The role of C-terminal processing of the ascomycete laccases is not known, but it has been suggested to be involved in the activation of the laccase [17,19] Screening a My thermophila laccase mutant library, Zumarraga et al found a laccase variant with better kinetics than the parental type, with two mutated amino acids in the C-terminal extension The mutations

Table 4 Distances and B-values of atoms at the trinuclear site The Sc(L559A) mutant structure is compared with published native rMaL structures: a low-dose structure (Protein Data Bank: 2IH8), a high-dose structure (Protein Data Bank: 2IH9) and a near-atomic structure (Protein Data Bank: 2Q9O) A and B are two molecules in the asymmetric unit.

L559A mutant

rMaL, low-dose structure

rMaL, high-dose structure

rMaL, near-atomic structure

B-values (A ˚ 2 )

in My thermophila laccase (75% identical to MaL)

resulted in a protein with disturbed T1 copper

geome-try and reduced redox potential, as well as an altered

trinuclear copper site, as shown by reduced oxygen

uptake [25] Surprisingly, the mutations in the

C-termi-nal extension affected the protein properties, although

the extension was cleaved from the mature protein

The mutations were suggested [25] to affect the folding

of the protein during the post-translational processing,

and thereby the function of the mature laccase

Our structural analysis of wild-type MaL indicated

that the last four amino acids of the mature protein

penetrate the tunnel leading from the surface to the

trinuclear site and form a plug [2] C-terminal blocking

might be a general feature of ascomycete laccases In

fact, we have determined a low-resolution structure

(3.1 A˚) of Th arenaria laccase (unpublished results),

and it clearly shows that the C-terminus similarly

blocks the tunnel leading towards the trinuclear site

This is strong evidence that C-terminal blocking is a

common feature of ascomycete laccases

The deletion of the last four amino acids in MaL

dramatically affected the specific activity of the

enzyme, as the deletion mutant delDSGL559, expressed

both in yeast and in T reesei, was practically inactive

Also, the kinetic parameters were altered The change

of only one amino acid in the C-terminus of the

lac-case, giving the Tr(L559G) and Sc(L559A) mutants,

reduced the turnover of the mutant proteins For the

substrate ABTS, the Michaelis constant of the

Sc(L559A) mutant increased two-fold, and the turnover

decreased four-fold, yielding an enzyme with 10-fold

reduced catalytic efficiency, indicating that the last

resi-due, Leu559, has a role in the catalysis of ABTS

oxida-tion The catalytic constants for the phenolic substrates

2,6-DMP and syringaldazine were not greatly affected

by the L559A mutation For the substrate 2,6-DMP,

the mutation led to a slightly increased Km value without significantly altering the kcat

The thermal stability of the yeast mutants Sc(del-DSGL559) and Sc(L559A) was determined by monitor-ing the CD spectrum as a function of temperature, because the kinetic stability of the inactive Sc(del-DSGL559) mutant was impossible to determine by activity measurements Although the thermal unfolding profiles for the mutant and wild-type laccases were broad, with no clear folded–unfolded transition, it was evident that the mutants were less thermostable than the wild-type ScMaL However, the similarities between the far-UV CD spectra of the mutants and that of the wild-type ScMaL do not support any major conformational changes of the deletion mutant The reduced thermostability of the Sc(L559A) mutant was also confirmed by residual activity measurements at different temperatures The crystal structure of the Sc(L559A) mutant revealed T2 copper and chloride ion depletion in the trinuclear site It has been observed that the first step in the denaturation of laccases, before actual denaturation, is the loss of one copper atom [26] The clearly lowered protein stability

of the Sc(L559A) mutant protein is probably due to T2 copper depletion

A well-known laccase inhibitor, azide, has been shown to bind to the trinuclear site in the crystal structure of BsL [5], thus preventing binding of oxygen

We analyzed the azide inhibition and determined the inhibition constants for sodium azide, in order to see the effect of the mutation (L559A) on the binding properties in the trinuclear center The inhibition of MaL by azide was determined to be mixed inhibition

in which both specific and catalytic effects are present Thus, azide can bind both to the free laccase and

to the laccase–substrate complex By comparing the inhibition constants for azide of wild-type ScMaL and

Fig 6 Multiple sequence alignment of the C-termini of some ascomycete laccases Alignment of the C-terminal amino acid sequneces of MaL (Q70KY3) with the laccases or putative laccases of My thermophila (MtL, from patent CN1157008), Th arenaria (TaLcc1, from patent US2006063246), Ch globosum (CgL1, XP_001228806; and CgL2, XP_001230068), Ma grisea (MgL, XP_362544), P anserina (PaL, P78722),

N crassa (NcL, XP_956939), Cr parasitica (CpL, Q03966), G graminis var tritici (GgL, CAD10749), and Botryotinia fuckeliana (BfL, AAK77953) The protein abbreviation and the protein accession numbers are in parentheses.