Báo cáo khoa học: Probing the determinants of substrate specificity of a feruloyl esterase, AnFaeA, from Aspergillus niger doc

Feruloyl esterases EC 3.1.1.73 release ferulic acid FA Keywords active site specificity; Aspergillus niger; ferulic acid; feruloyl esterase; plant cell wall Correspondence C.. Here we de

feruloyl esterase, AnFaeA, from Aspergillus niger

Craig B Faulds1, Rafael Molina2, Ramo´n Gonzalez3, Fiona Husband1, Nathalie Juge1,4,

Julia Sanz-Aparicio2and Juan A Hermoso2

1 Institute of Food Research, Colney, Norwich, UK

2 Grupo de Cristalografia Macromolecular y Biologia Estructural, Instituto de Quı´mica Fı´sica ‘Rocasolano’, CSIC, Madrid, Spain

3 Departamento de Microbiologia, Instituto de Fermentaciones Industriales, CSIC, Madrid, Spain

4 Institut Me´diterrane´en de Recherche en Nutrition, Universite´ Paul Ce´zanne, Marseille, France

The plant cell wall is a complex mixture of

rides, proteins, phenolics and lipids The

polysaccha-rides form the skeleton of the plant cell wall, and are

composed of cellulose microfibrils embedded within a

matrix of hemicellulose or pectin, depending on the

plant tissue Hemicelluloses are the most abundant

renewable polymers after to cellulose and they are the

key components in the degradation of plant biomass

However, this degradative process is often inefficient

because most polymers of cellulose and hemicellulose

are either insoluble or simply too closely associated with the insoluble matrix In cereals, the main hemi-cellulosic polymer is arabinoxylan, which is composed

of a b-(1,4) glycosidic-linked d-xylopyranosyl units, substituted at positions O-2 or O-3 with arabinose To deconstruct or modify arabinoxylans, plants or micro-organisms require a battery of glycoside hydrolases (xylanases, a-arabinofuranosidases, b-xylosidases, glu-curonidases) and esterases (feruloyl, acetyl) Feruloyl esterases (EC 3.1.1.73) release ferulic acid (FA)

Keywords

active site specificity; Aspergillus niger;

ferulic acid; feruloyl esterase; plant cell wall

Correspondence

C B Faulds, Institute of Food Research,

Norwich Research Park, Colney,

Norwich NR4 7UA, UK

Fax: +44 160 350 7723

Tel: +44 160 325 5152

E-mail: craig.faulds@bbsrc.ac.uk

(Received 10 March 2005, revised 27 May

2005, accepted 7 July 2005)

doi:10.1111/j.1742-4658.2005.04849.x

Feruloyl esterases hydrolyse phenolic groups involved in the cross-linking

of arabinoxylan to other polymeric structures This is important for open-ing the cell wall structure makopen-ing material more accessible to glycoside hydrolases Here we describe the crystal structure of inactive S133A mutant

of type-A feruloyl esterase from Aspergillus niger (AnFaeA) in complex with a feruloylated trisaccharide substrate Only the ferulic acid moiety of the substrate is visible in the electron density map, showing interactions through its OH and OCH3 groups with the hydroxyl groups of Tyr80 The importance of aromatic and polar residues in the activity of AnFaeA was also evaluated using site-directed mutagenesis Four mutant proteins were heterologously expressed in Pichia pastoris, and their kinetic properties determined against methyl esters of ferulic, sinapic, caffeic and p-coumaric acid The kcatof Y80S, Y80V, W260S and W260V was drastically reduced compared to that of the wild-type enzyme However, the replacement of Tyr80 and Trp260 with smaller residues broadened the substrate specificity

of the enzyme, allowing the hydrolysis of methyl caffeate The role of Tyr80 and Trp260 in AnFaeA are discussed in light of the three-dimen-sional structure

Abbreviations

AnFaeA, an A-type feruloyl esterase from Aspergillus niger; AXE, acetylxylan esterase; diFA, diferulic acid; FA, ferulic acid; FAE_XynY, Clostridium thermocellum celulosomal xylanase Y domain that displays feruloyl esterase activity; FAE_XynZ, Clostridium thermocellum celulosomal xylanase Z domain that displays feruloyl esterase activity; FAXX, O-[5-O-[(E)-feruloyl]-a- L -arabinofuranosyl}-(1fi3)-O-b- D

-xylopyranosyl-(1fi4)- D -xylopyranose; MCA, methyl caffeate (methyl 3,4-dihydroxycinnamate); MFA, methyl ferulate (methyl 4-hydroxy-3-methyoxycinnamate); MpCA, methyl p-coumarate (methyl 4-hydroxycinnamate); MSA, methyl sinapate (methyl

3,5-dimethoxy-4-hydroxycinnamate).

(Fig 1) from arabinose-substituted xylans and

rhamnogalacturonans [1] While most of the feruloyl

esterases to date have been grouped into the

carbo-hydrate esterase family 1 [2] (for more information, see

http://www.afmb.cnrs-mrs.fr/CAZY/), a

complement-ary classification based on amino acid sequence

simi-larities and substrate specificity has putatively grouped

feruloyl esterases into four types, A–D [3] AnFaeA

is a type-A feruloyl esterase isolated from Aspergillus

niger[4]

From protein sequence homology, feruloyl esterases

belong to the same family as the serine proteases,

est-erases and lipases, with a serine residue acting as the

nucleophile in a catalytic triad comprising the hydroxyl

group of the active serine, the imidazole side chain

of histidine and a buried carboxylic acid chain [5]

Although the mechanism of deferuloylation has not

been reported, it is probable, based on the general

hydrolytic mechanism of esterases [6], that the basic

His248 (AnFaeA numbering) removes a proton from

the hydroxyl group of Ser133 and that the nucleophilic

oxygen attacks the carbonyl carbon of the feruloyl

group to form a tetrahedral intermediate

Amino acids with aromatic side chains play a

pro-minent role in binding carbohydrates [7] The

hydro-phobic patch of a sugar moiety, resulting from the

disposition of the equatorial and axial hydroxyls to

one side of the pyranose ring of a sugar monomer,

aligns itself upon binding with the aromatic ring face

of the amino acid to contribute to selectivity of fit of

the substrate to the binding site of the enzyme [8]

Tryptophan has been shown to be essential for

sub-strate binding in most of the glycoside hydrolases

stud-ied to date, such as cellulases [9], xylanases [8,10,11]

and a-amylases [12] The protein sequence of AnFaeA,

showed the presence of four tryptophan residues in the

molecule [13] and chemical modification of the mature

protein with N-bromosuccinimide (NBS) demonstrated

that one tryptophan essential for activity was exposed

on the surface of the enzyme [14]

The structure of AnFaeA has recently been solved

(PDB accession numbers 1USW, 1UZA, 1UWC)

[15,16] The enzyme displays an a⁄ b hydrolase fold [17] similar to that found in fungal lipases, such as those from Thermomyces lanuginosa [18] and Rhizo-mucor miehei [19] This structure is different from that reported for the feruloyl esterases from Clostridium thermocellum [20,21], although the catalytic triads can

be superimposed allowing direct extrapolation of the position of the oxyanion pocket Crystallography and point replacement of the nucleophilic serine of An-FaeA, Ser133, allowed the identification of the active site, confined by a lid (residues 68–90) and a loop (resi-dues 226–244) which confers plasticity to the substrate binding site [15] While structurally resembling lipases, AnFaeA does not exhibit lipase activity [22] From these studies we postulated that Tyr80 could play an essential role in substrate binding and specificity In addition, Trp260, located at the C terminus and near the surface is the closest tryptophan to the active centre

In this study, we used site-directed mutagenesis and X-ray crystallography to give insights into the specifi-city and affinity of AnFaeA for methyl hydroxycin-namic acid substrates

Results and Discussion

Crystal structure of the S133A AnFaeA–FAXX complex and design of AnFaeA mutants

To determine which residues are important for the interaction of ferulate and ester-linked carbohydrates with AnFaeA, the crystal structure of the inactive S133A nucleophilic mutant of AnFaeA complexed to the feruloylated trisaccharide O-[5-O-[(E)-feruloyl]- a-l-arabinofuranosyl}-(1(q)3)-O-b-d-xylopyranosyl-(1fi4)-d-xylopyranose (FAXX) was solved at 2.5-A˚ resolution AnFaeA requires both the hydroxycinna-mate as well as a carbohydrate grouping as part of the substrate for optimal activity, and a feruloylated trisac-charide consisting of the linkage of sugars found in FAXX has been shown to be the optimal size of sub-strate [23] Three molecules of S133A AnFaeA mutant are present in the asymmetric unit and show no signifi-cant differences between the native and mutant struc-tures, as revealed by the low r.m.s.d deviation of their backbones (0.47 A˚) after superimposition of both structures The electron density maps revealed the presence of a FA moiety bounded at the active site in the three molecules of the asymmetric unit (not shown) However, the remaining groups of the sub-strate (i.e the arabinose and the two xylose units) were not visible in the 2Fo–Fc map, neither difference map indicated the presence of carbohydrate groups As the

HO

OH OCH3

O

H3CO

HO

OH

O

D C

HO O

OH OH

B

HO

OH

OCH3

O

A

Fig 1 Hydroxycinnamic acids (A) Ferulic acid (B) Caffeic acid (C)

Sinapic acid (D) p-Coumaric acid.

S133A mutant is inactive [15], the substrate should be

complete and therefore the carbohydrate moiety is

probably disordered The substrate is placed in a long

and narrow cavity (Fig 2A) The active site cavity is

mainly confined by the flap (residues 68–80) and the

226–244 loop (Fig 2B) As shown in Fig 3, the

arrangement of the FA in the active site is essentially

the same to that observed in the high-resolution

struc-ture of the AnFaeA–FA complex determined by

McAuley et al (PDB code 1UWC) [16] The FA

inter-acts (Fig 2B,C) through the OH group at C4 with the

hydroxyl group of Tyr80 in the enzyme substrate

com-plex Despite the apparently long distance found in the

present structure (3.8 A˚) the hydroxyl group of Tyr80

probably interacts with the OCH3 group at C-3 as it

also occurs in the high resolution structure of 1UWC

[16] Tyr80 is one of the residues that takes part in the

formation of the substrate cavity and its arrangement

delimitates the long substrate cavity where the

aroma-tic ring of the FA is placed (Fig 4A) Moreover, the

global arrangement of residues in the substrate cavity

provides a molecular surface in which OCH3 group fits

perfectly (Fig 4A) The carboxylate moiety is located

at the oxyanion hole defined by the Leu134-N

main-chain and both the backbone N atom and the OH

group from Thr68 Leu199, Val243 and Ile196 provide

the hydrophobic environment to stabilize the aromatic

and the hydrocarbon chain of the FA The importance

of the interaction between an aromatic tyrosine and

the phenolic ring of the substrate is consistent with the

biochemical specificity of this enzyme [4,24] The role

of Tyr100 was previously probed by site-directed

muta-genesis; mutating the Phe100 (AnFaeA numbering) of

the lipase from Thermomyces lanuginose to Tyr was

essential to confer ferulate ester-hydrolysing activity

[25] Of the four tryptophan residues in the sequence

of AnFaeA, only one is located near the surface, as

demonstrated by chemical modification, and is

essen-tial for activity [14] Trp260 is the terminal residue,

located on a flexible loop [15] and although far from

the active site, this residue is the closest Trp in the

vicinity of the active site, and is thus a probable

candi-date for substrate specificity (Fig 4B) This residue is

buried in a hydrophobic cavity surrounded by Met253,

Thr19 and Ala23 side chains In the present work,

site-directed mutagenesis is used to probe the role of

polar-ity and⁄ or hydrophobicity in the environment of Tyr80

and Trp260

The active site of AnFaeA is placed in a long and

narrow cavity that connects two crevices at the

molecular surface [15], displaying hydrophobic residues

that stabilizes the aromatic moiety of the substrate

As with the structure of the C thermocellum feruloyl

Fig 2 Crystal structure of the S133A AnFaeA mutant in complex with FAXX (A) Molecular surface of S133A AnFaeA mutant The catalytic triad and the Y80 and W260 residues are labelled Ferulic acid molecule is coloured in cyan (B) Environment of FA (green) in the active site of S133A The flap region of AnFaeA is highlighted

in dark blue (C) Proposed interactions of FA with residues at the substrate cavity of AnFaeA.

esterase in complex with FAXX, FAE_XynZ-FAXX

(1JT2) [21], the FA moiety is clearly visible in the

act-ive site but the carbohydrate parts of the substrate are

not visible, suggesting that tight binding of the

carbo-hydrate is not required for catalysis

Production and characterization of AnFaeA

variants

All four mutants (Y80S, Y80V, W260S, W260V) were

efficiently produced in Pichia pastoris as confirmed by

SDS⁄ PAGE and western blot analysis with anti-FaeA

polyclonal antibodies Purified recombinant variants

were obtained in yields ranging from 163 mgÆL)1

(Y80V) to 628 mgÆL)1(W260V) using a single

chroma-tographic step (hydrophobic interaction: HIC) While

wild-type AnFaeA was purified using the phenyl

seph-arose HIC column [26], the mutants were retained on

the column, even by reducing the hydrophobicity of

the buffer Due to this, these four variants of AnFaeA

were then purified using a butyl sepharose column

To evaluate the consequence of altering the

hydro-phobicity or the bulking effect in the active site of

AnFaeA around the Tyr80 mutation, and the effect of

altering the only surface exposed tryptophan residue,

Trp260, on activity, the four variants were tested on

the methyl esters of hydroxycinnamic acids: methyl

ferulate (MFA) and compared to wild-type AnFaeA

All of the variants displayed feruloyl esterase activity

albeit at a reduced value compared to the wild-type

enzyme The effects of these mutations on the secon-dary structure of the Y80V, Y80S, W260V, W260S were tested by CD All mutants show an increase

in a-helix content, reflecting possible small local structural rearrangements (Table 1) However, as the kinetic values for Y80S and Y80V and for W260S and W260V were similar, such changes in structure did not duly affect the catalytic arrangement of the enzyme

Kinetic analyses and substrate specificity

of AnFaeA variants The kinetic parameters (kcat and Km) of the Y80V, Y80S, W260V, W260S mutants were determined

Fig 3 Superimposition of the complex S133A AnFaeA–FA

(orange), native structure (red) and AnFaeA–FA complex

deter-mined by McAuley [16] (blue) In the case of AnFaeA–FA complex

determined by McAuley [16], apart from the active histidine

confor-mation (blue) the inactive histidine conforconfor-mation form (grey) is also

found.

Fig 4 Local environment of Tyr80 and Trp260 (A) Arrangement of the Tyr80 residue Ferulic acid is shown in blue, Tyr80 is shown in red and the residues that participate in the substrate cavity are shown in green (B) Arrangement of Trp260 residue (red) The resi-dues that bury Tpr260 are shown in blue.

against MFA, methyl sinapate (MSA), methyl caffeate

(MCA) and methyl p-coumarate (MpCA) (Fig 1) All

variants showed a significant decrease in the hydrolytic

rate compared to the wild-type enzyme in addition to

a slight increase in Kmfor all the substrates, except for

MCA (Table 2) The decrease in the hydrolytic rate

was between 1.5- and 4-fold, with the largest changes

occurring with MFA and MSA as substrates Both

Tyr80 and Trp260 variants were able to hydrolyse

MCA The type of substitution on the phenolic ring of

the substrate is important for defining the type of

feru-loyl esterase [3,24] Previous inhibition studies showed

that AnFaeA binds MCA but does not hydrolyse it,

suggesting that the enzyme possesses a fairly

nonspe-cific binding site [24] In the present study, replacement

of Tyr80 or Trp260 by a nonaromatic amino acid

resulted in the reduction in the activity and broadened

the specificity of AnFaeA for phenolic acids, in

partic-ular for MCA

From the close up view of the phenolic binding

pocket (Fig 2) it is clear that two tyrosine residues,

Tyr80 and Tyr100, are closely located near the

substit-uent groups around the phenolic ring, in agreement

with the results from the mutagenesis study It is

pos-sible that the removal of the bulky tyrosine from the

pocket in the mutant variants must result in a local

realignment allowing the accommodation of a hydro-xyl group at O-3 of FA instead of the methohydro-xyl group

In comparison, the structures of the two feruloyl esterases from C thermocellum, FAE_XynY (PDB accession code 1GKK) [20] and FAE_XynZ (1JJF) [21] show that ferulate binds in a small blunt-ended surface depression, with the hydroxyl group interacting with an Asp residue and the methoxyl group with

a Trp, instead of Tyr as in AnFaeA The tryptophan did not form a direct stacking interaction with the phenolic ring of FA, instead contributing to the hydro-phobic environment by forming a small cavity with a leucine residue on one side of the binding depression [20]

The structural implication of Trp260 in binding of the substrate is less clear Although relatively far from the active site (
14 A˚) (Fig 2a), biochemical evidence demonstrated that Trp260 interacted with the active site pocket, as modification of AnFaeA with 4500-fold excess of N-bromosuccinimide (a chemical oxidizer of Trp residues) resulted in an 80% loss of activity against MSA [14] This is not due to this residue hav-ing a role in enzyme stability, as joinhav-ing a bacterial dockerin domain to the C-terminal end of AnFaeA through Trp260 did not significantly affect the activity

of the feruloyl esterase [27] One hypothesis is that Trp260 may be in a position to interact with the car-bohydrate moiety of a feruloylated polysaccharide In FAE_XynZ, the C-terminal tryptophan, Trp265, is located in a hydrophobic pocket of primarily aromatic residues adjacent to the binding pocket [21] whereas it

is absent in FAE_XynY [28] However, the interactions between Trp260 and the sugar moieties of the substrate could not be directly demonstrated due to both the lack of resolved sugar interactions in the AnFaeA– FAXX complex, and the nature of the methyl

Table 1 Secondary structure of wild type and mutant AnFaeA,

from circular dichroism and SELCON analysis.

Table 2 Kinetic parameters of the wild-type and mutated AnFaeA determined against the methyl esters (1 m M ) of ferulate (MFA), sinapate (MSA), caffeate (MCA) and p-coumarate (MpCA) nd, Activity not detected.

MFA kcat(molÆs)1Æmol)1) 70.74 (± 1.44) 1.56 (± 0.04) 2.56 (± 0.08) 20.06 (± 0.67) 18.33 (± 0.44)

MSA kcat(molÆs)1Æmol)1) 84.95 (± 2.26 3.48 ± 0.11) 7.85 (± 0.28) 27.76 (± 0.62) 28.28 (± 0.49)

MpCA kcat(molÆs)1Æmol)1) 0.73 (± 0.05) 0.10 (± 0.003) 0.26 (± 0.01) 0.49 (± 0.02) 0.29 (± 0.02)

hydroxycinnamates used as substrates Alternatively,

from the 3D structure and the measured effects on the

kinetic parameters, it is possible to hypothesize that

this tryptophan affects the mobility of the catalytic

his-tidine [29] Such a shift was reported in the side chain

position of His260 and His247 in the FAE_XynZ–

FAXX [21] and the AnFaeA–FA complexes [16],

respectively While in the free enzyme His247 is present

in a single conformation corresponding to the active

orientation for a catalytic histidine residue, in the case

of the enzyme–product complex His247 can move

pro-viding an inactive form As it was described in the high

resolution AnFaeA–FA complex [16], in the complex

structure His247 can present two histidine

conforma-tions which easily interconvert from an active to

an inactive form However in our case, the S133A

AnFaeA–FA electron density maps did not reveal any

difference between the arrangement of His247 in the

complexed and free enzyme structures, as His247

is always in the active conformation (Fig 3) In

FAE_XynZ, Trp265 is only 4 A˚ from the catalytic

his-tidine (His247) [21], allowing direct interaction, which

is not the case in AnFaeA In other

carbohydrate-active esterases, the exposed catalytic His187 residue of

the acetylxylan esterase, AXE-II, of P purporogenum

forms a hydrogen bond with a sulphate ion forcing the

histidine to adopt an altered conformation [30] This

has also been observed with a cutinase from Fusarium

solani[31] With AXE-II, transition of histidine from a

resting state to an active state necessitated the

rear-rangement of other residues of the active site, most

notably the movement of Tyr177 which moved 2 A˚

away to accommodate the catalytic histidine in the

act-ive state While no change in the position of His247

was determined when the free and complexed

struc-tures were compared, Trp260 still can influence both

catalytic rate and specificity The role of Trp260 in the

catalytic mechanism of AnFaeA requires further

exam-ination The above differences in reported structures

and activities of feruloyl esterases are reflected in the

cladogram for carbohydrate-active esterases with

known 3D structures (Fig 5) While AnFaeA closely

resembles the lipases of Rhizomucor meihei and

Thermomyces lanuginosa, of the feruloyl esterases,

FAE_XynZ from C thermocellum shows the closest

homology AnFaeA releases 5,5¢ diFA from

cereal-derived material [32] and the 3D structure shows how

the dimer can be accommodated within the active site

[15] The structure of FAE_XynZ suggested that the

open and solvent-exposed FA binding site can interact

with diFA [15], while FAE_XynY could not

accommo-date such a substrate This is in agreement with the

closeness demonstrated in the phylogenic analysis

(Fig 3) FAE_XynY, on the other hand, is further removed from AnFaeA and may resemble more the acetylxylan esterases of Penicillium purpogenum (1BS9) [30] and Trichoderma reesei (1QOZ) [33] Further bio-chemical characterization of these enzymes is required

to test these hypotheses

Experimental procedures

Site-directed mutagenesis

In vitro site-directed mutagenesis of the faeA gene on plas-mid pFAE-W was performed by using the QuickChangeTM

Site-Directed Mutagenesis Kit from Stratagene (La Jolla,

CA, USA) following the manufacturer’s instructions with two exceptions: DH5a Escherichia coli cells where used instead of Epicurian Coli XL1-Blue, and the elongation step in each thermal cycle was extended from the recom-mended 18 s (2 s per kb) to 25 s Alanine, serine and valine replacement codons were chosen taking into account codon usage in yeast Two complementary oligonucleotides were used for replacement of S133A, W260V or W260S, how-ever, following the observations of Makarova et al [34], a single primer was successfully used for Y80V or Y80S replacements The plasmids carrying the resulting mutant faeA alleles were called pFAE-S133A, pFAE-W260V, pFAE-W260S, pFAE-Y80V and pFAE-Y80S, respectively Table 3 shows the sequence of all the oligonucleotides used

in this work In all cases, the plasmid region containing the faeA gene, as well as the AOX1 promoter and terminator, was sequenced completely to rule out the presence of any additional mutation Spheroplasts from Pichia pastoris strain GS115 were transformed with these plasmids, by using the Pichia expression kit from Invitrogen (Carlsbad,

CA, USA) and His+MutS strains were selected for the expression of the mutated versions of AnFaeA

CtXYNZ AnFAEA RmLipase TiLipase CtXYNY PpAXEll TrAXE PpAXEl

Fig 5 Cladogram of feruloyl esterases and related enzymes of known 3D structure Enzyme names are shown on the right hand side of the tree: CtXYNZ, Clostridium thermocellum FAE_XynZ (M22624); AnFAEA, Aspergillus niger FaeA (AF361950); RmLipase, Rhizomucor miehei lipase (P19515); TiLipase, Thermomyces lanugi-nosus lipase (O59952); CtXYNY, Clostridium thermocellum FAE_XynY (X83269); PpAXEII, Penicillium purporogenum acetylxy-lan esterase-II (AF015285); TrAXE, Trichoderma reesei acetylxyacetylxy-lan esterase I (S71334); PpAXEI, Penicillium purporogenum acetylxylan esterase-I (AAM93261).

Purification of the AnFaeA mutants

AnFaeA mutants were purified from the P pastoris cultures

by HIC based on previously described protocols [26] Apart

from the wild-type and S133A mutant, the other mutants

were retained on a phenyl-sepharose column (Amersham

Biotech, Little Chalfont, Bucks, UK), even after a water

elution A butyl-sepharose column (Amersham Biotech)

was used to purify these mutants

Crystallization, data collection and processing

Co-crystallization of S133A AnFaeA–FAXX complex was

performed by the hanging-drop, vapour-diffusion method

at 291 K, testing the conditions obtained for the native

pro-tein (1.8 m ammonium sulphate, 0.1 m Hepes, pH 7.5)

FAXX was purified from Driselase-hydrolysed de-starched

wheat bran, as described previously [35] After preliminary

trials, crystals suitable for X-ray studies were obtained by

mixing 4 lL of a well solution (1.7 m ammonium sulphate),

1 lL of FAXX substrate (10 mm) and 2 lL of the mutant

enzyme solution at 12 mgÆmL)1 The crystals were tested

on an in-house MAR Research IP area detector with CuKa

X-rays (k¼ 1.5418 A˚) generated by an Enraf-Nonius

rotating anode generator, but diffraction data were of low

resolution Consequently, synchrotron radiation was used

Data sets were collected at ESRF (ID14-4 beamline), with

k¼ 0.9184 A˚ All data were processed and scaled using

mosflm [36] and scala from CCP4 package software [37]

Data processing statistics are given in Table 4 The crystals

belong to space group P21, with unit cell dimensions a¼

46.74 A˚, b¼ 130.75 A˚, c ¼ 76.51 A˚ and b ¼ 98.14
A˚

Spe-cific volume calculations yielded three molecules of S133A

AnFaeA in the asymmetric unit, with a solvent content of

55.3% (v⁄ v) (VM¼ 2.75 A˚3ÆDa)1)

Structure determination

The structure of the S133A AnFaeA–FAXX complex was

determined by the molecular replacement method using the

program amore [38,39] The atomic coordinates of the

AnFaeA (PDB code 1USW) were used as the search model

for a rotational and translational search in the 49–3.5 A˚ resolution range We obtained a good solution for three molecules in the asymmetric unit and the values of the final correlation coefficient and Rfactor were 0.70 and 21.6%, respectively The structure was refined with cns [40] up to 2.5 A˚ resolution using strict ncs refinement, and restrained ncs refinement in the last stages Refinement statistics are given in Table 4

Table 3 Sequences of the oligonucleotides used for site directed mutagenesis.

Table 4 Data collection and refinement statistics for the S133A AnFAEA mutant in complex with FAXX Values in parentheses cor-respond to the highest resolution shell R factor ¼ P

h ||F obs | – |F calc || ⁄ P

|Fobs|, eEh||Fobs| – |Fcalc|| ⁄ O ´ |Fobs|, where Fobs and Fcalc are observed and calculated structure factor amplitudes, respectively.

R free calculated for 7% of data excluded from the refinement.

Unit cell parameters

Refinement statistics

r.m.s.d from ideal

Model quality and accuracy

The final model consists of three molecules of S133A

AnFaeA (A, B, C), three FA molecules (one per S133A

AnFaeA molecule) and 375 water molecules As the native

structure, the S133A AnFaeA is glycosylated at Asp79 and

two molecules of N-acetyl glucosamine residue were built at

each glycosylation site In the complex, the electron density

maps in this region reveal a carbohydrate structure larger

than only two units of N-acetyl glucosamine but this could

not be modelled because of the poor electron density

defini-tion The stereochemical quality of the model was checked

with the program procheck [41] The figures were

gener-ated with molscript [42], raster 3d [43] and grasp [44]

The atomic coordinates and structure factors for S133A

AnFaeA–FA complex have been deposited in the Protein

Data Bank, with accession number 2BJH

Gel electrophoresis and immunoblotting

SDS⁄ PAGE was carried out on a 10% Bis ⁄ Tris precast

NuPAGE gel (Invitrogen) with wild-type AnFaeA as a

marker Proteins were transferred to nitrocellulose

mem-branes by semidry blotting (Bio-Rad, Hercules, CA, USA)

The blotted membranes were probed with a 1000-fold

dilu-tion of polyclonal antiserum raised in rabbits against

AnFaeA [45] Immunoreactive proteins were visualized

using alkaline phosphatase-conjugated anti-rabbit

secon-dary antibody (Sigma, St Louis, MO, USA; 1 : 2000)

Circular dichroism

Circular dichroism spectra were collected using a JASCO

710 spectropolarimeter (Great Dunmow, Cambs, UK) Far

UV CD spectra were recorded at 0.5 mgÆmL)1 with a

0.2-mm path length cell The spectra shown are an average

of four accumulations, with a scan speed of 100 nmÆmin)1,

band width 1 nm, response 1 s, data pitch 0.2 nm and

range 260–190 nm Analysis of the spectra was estimated

using selcon [46]

Enzyme assays

Feruloyl esterase activity, assayed with hydroxycinnamate

methyl esters, was determined by HPLC for all the AnFaeA

mutants as described previously [26] All measurements

were carried out in 100 mm Mops pH 6.0 at 37
C In all

measurements, the free acid present in samples pretreated

with glacial acetic acid was subtracted from that in the test

assays One unit of esterase activity was defined as the

amount of enzyme required to release 1 lmol

hydroxy-cinnamic acidÆmin)1Æmg protein)1 at 37
C, pH 6.0 The

kinetic results obtained from the hydrolysis of a range of

0.2–2 mm methyl hydroxycinnamates was interpreted using

the Michaelis–Menten kinetic model, using grafit [47] For each variant and each substrate, at least 10 substrate concentrations were measured in duplicate

Phylogenetic analyses

Multiples alignment of sequences encoding feruloyl ester-ases and related enzymes such as lipester-ases, and construction

of neighbor-joining cladogram [48], were performed with clustal w(http://www.ebi.ac.uk/clustalw/) [49]

Acknowledgements

This work was funded by the Biotechnology and Bio-logical Research Council (BBSRC), UK through an ISIS travel grant to CBF and by the BBSRC and the Department of Trade and Industry (DTI), UK, through the award of an Applied Biocatalysts Link award (grant number ABC11741)

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