Báo cáo khoa học: Characterization of the flavin association in hexose oxidase from Chondrus crispus pot

A homology model of hexose oxidase was constructed using the crystal structure of glucooligosaccharide oxidase from Acremonium strictum as template.. Abbreviations ABTS, 2,2¢-azinobis3-e

oxidase from Chondrus crispus

Thomas Rand1, Karsten B Qvist1, Clive P Walter1and Charlotte H Poulsen1,2

1 Danisco A ⁄ S, Brabrand, Denmark

2 Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Denmark

Hexose oxidase (HOX) catalyses the oxidation of a

variety of hexose sugars with concomitant reduction of

molecular oxygen to hydrogen peroxide The oxidation

product is the corresponding lactone, which

subse-quently hydrolyses to the respective aldobionic acid

The reaction is exemplified using glucose below:

d-glucose + O2fi d-d-gluconolactone + H2O2

d-d-gluconolactone + H2Ofi d-gluconic acid

The structural gene of HOX from the marine algae Chondrus crispus has previously been expressed suc-cessfully in both Pichia pastoris [1] and Hansenula polymorpha [2] Purified enzyme from both of these expression systems has been shown to be active and to contain FAD [1,2] The FAD in HOX from C crispus has been shown to be intricately involved in the cata-lytic mechanism of the enzyme [3], but the mechanism

Keywords

FAD; flavin; hexose oxidase; mass

spectroscopy; modeling

Correspondence

T Rand, Danisco A ⁄ S, Edwin Rahrs Vej 38,

8220 Brabrand, Denmark

Fax: +45 8625 1077

Tel: +45 8943 5000

E-mail: thomas.rand@danisco.dk

(Received 20 January 2006, revised 17 April

2006, accepted 20 April 2006)

doi:10.1111/j.1742-4658.2006.05285.x

Hexose oxidase (EC 1.1.3.5) from Hansenula polymorpha was found to exhibit a dual covalent association of FAD with His79 via an 8a-histidyl linkage as well as a covalent association between Cys138 and C-6 of the isoalloxazine moiety of FAD Spectral properties of the wild-type enzyme exhibited maxima at 364 nm and 437 nm as well as a distinct shoulder at

445 nm An H79K mutant enzyme exhibited only one maximum at

437 nm The difference absorption spectrum between an oxidized and a substrate-reduced enzyme preparation showed maxima at 360 nm and

445 nm corresponding to an apparent novel type of association Hexose oxidase showed a low, pH-independent fluorescence at 525 nm when exci-ted at 450 nm Flavin was released from the holoenzyme by treatment with trypsin Sequencing of the flavopeptide revealed two peptides comprising positions 74–91 and 132–157 associated with FAD in equimolar amounts

A homology model of hexose oxidase was constructed using the crystal structure of glucooligosaccharide oxidase from Acremonium strictum as template The model placed both of the sequences found above in the close vicinity of the FAD cofactor, and suggests covalent bonds between both His79 and Cys138 and FAD, in accordance with the chemical evidence Based on the results, hexose oxidase is identified as incorporating FAD with a double covalent association with His79 and Cys138 in the holo-enzyme A reaction mechanism involving the concerted action of Tyr488 and Asp409 in hexose oxidase is suggested as the initiator of the proton abstraction from the substrate molecule in the active site

Abbreviations

ABTS, 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid; COX, choline oxidase; Fl(), position on the isoalloxazine ring; GOOX,

glucooligosaccharide oxidase; HOX, hexose oxidase; MeCN, acetonitrile; MSOX, monomeric sarcosine oxidase; MTOX, N-methyltryptophan oxidase; PCMH, p-cresol methylhydroxylase; TADH, thiamine dehydrogenase; TMDH, trimethylamine dehydrogenase; VAO, vanillyl alcohol oxidase.

of this action is not known The main reason for this

is the lack of a crystal structure and the lack of

infor-mation regarding the association and placement of

FAD in the holoenzyme FAD in HOX has been

shown to be covalently associated by several methods,

including acid precipitation (unpublished results) and

illuminating an acid-fixated denatured protein in

SDS⁄ PAGE by UV light In this case, a fluorescent

band was observed [4] The oligomer substrates of

HOX include the b-d-forms of lactose, maltose,

cello-biose and maltotriose [5], as well as

glucooligosaccha-rides with up to seven glucose residues when

immobilized on a solid support [6] In addition, HOX

has been shown to have a high affinity for the

mono-mer sugars glucose and galactose [5]

According to the Pfam database (http://www.sanger

ac.uk/Software/Pfam/), HOX contains an

FAD-bind-ing 4 domain, which places it in the p-cresol

methyl-hydroxylase (PCMH) FAD-binding group This group

of enzymes is named after PCMH, the first enzyme in

the family to be crystallized [7] These enzymes are

typ-ically composed of two major domains, one of which

binds the FAD (the F-domain) and one of which binds

the substrate (the S-domain) A recently published

novel type of flavin association found in

glucooligosac-charide oxidase (GOOX) from Acremonium strictum

shows FAD to have a double covalent association with

both a Cys and a His residue [8] With a sequence

identity of about 24%, this enzyme has the highest

identity with HOX from C crispus, among enzymes

with an experimentally determined three-dimensional

structure HOX shows a somewhat peculiar

spectro-scopic profile and, based on the sequence homology to

GOOX, we set out to investigate whether wild-type

(WT) HOX, expressed recombinantly in H polymor-pha, also contains a His–FAD–Cys association, and if that was the case, to develop a method of characteriz-ing this type of association without havcharacteriz-ing a crystal structure A homology model of the active site of the enzyme is reported, based on the GOOX structure, and an H polymorpha HOX variant with a mutation

of His79 to Lys (H79K) is analysed and compared to the WT enzyme The biochemical evidence for the FAD association in combination with the model will

be used to make deductions on the specific reaction mechanism of HOX

Results

Absorbance properties of the flavin component

of HOX

On initial viewing, the absorbance spectrum of oxidized HOX exhibits a profile similar to that of other flavinyl-ated proteins (Fig 1) [9,10] Using an enzyme concen-tration of 0.005 mm, a first absorbance maximum is observed at 364 nm (0.119) and a second at 437 nm (0.123) Furthermore, the spectrum shows a distinct shoulder at 450 nm (0.122) The spectrum did not change significantly after incubation in 0.3% sodium dodecyl sulfate, with 1 mm dithiothreitol (data not shown) The molecular mass of HOX has been deter-mined by gel filtration to be between 110 000 [1] and

130 000 Da [11] cDNA was previously isolated from HOX, corresponding to a polypeptide of
62 000 Da, which was confirmed by SDS⁄ PAGE [1] Assuming a homodimeric structure for HOX, these observations suggest a 1 : 2 enzyme⁄ FAD ratio in the WT enzyme

0

5

10

15

20

A

1

1

2

3 2

3

Wavelength (nm)

-1 )

B

-1 )

0 5 10 15 20

Wavelength (nm)

C

-10 -5 0 5 10

Wavelength (nm)

Fig 1 Absorbance properties of the covalently bound flavin in hexose oxidase (HOX) Absorption spectra were recorded in 0.1 M sodium phosphate buffer, pH 6.3 Enzyme concentrations were standardized to 0.005 m M according to a 1 : 2 enzyme ⁄ FAD stoichiometry For other details, see Experimental procedures (A) (1) The spectrum of the wild-type (WT) oxidized HOX (2) The spectrum of the H79K mutant HOX (3) The absorbance spectrum of an equimolar amount of free FAD (0.01 m M ) is shown in comparison (B) (1) The spectrum of the WT HOX anaerobically substrate-reduced enzyme using 2.5 m M glucose (2) The spectrum of the WT HOX aerobically substrate-reduced enzyme using 2.5 m M glucose (3) The spectrum of the WT oxidized HOX (C) The difference spectrum between WT HOX before and after sub-strate-mediated reduction (curve 3–2 in (B)) The second absorption maximum of the difference spectrum is hypsochromically shifted in rela-tion to free FAD The peak maximum is observed at 360 nm, indicating covalent FAD in the holoenzyme.

This stoichiometry in HOX is further substantiated by

reports of extinction coefficients (e445–e448) for 8a- and

6-substituted flavins in the range 11.3–12.3 mm)1Æcm)1

[12] Assuming a 1 : 2 enzyme⁄ FAD ratio, the

extinc-tion coefficients, with respect to the covalently bound

flavins in HOX, are e364¼ 11.9 mm)1Æcm)1, e437¼

12.3 mm)1Æcm)1and e450¼ 12.2 mm)1Æcm)1

When HOX is reduced with substrate, its spectrum

changes significantly The resulting reduced spectrum

exhibits a maximum at 420 nm (pH 6.3), possibly due

to the bond between the Fl(6)C atom and a Cys

resi-due, as observed for trimethylamine dehydrogenase

(TMDH) [13,14] The apparent lack of a semiquinone

species upon substrate reduction is similar to what has

been observed for VAO [15] Such an intermediate

probably occurs unobserved in HOX, as is common

with oxidase- and monoxygenase-type flavoenzymes

[16] The possible contribution of the Fl(8a)C–His

bond to the spectrum of the flavin component is

visu-alized by the difference spectrum between the oxidized

and the reduced enzyme (HOXox–HOXred), in the same

way as done previously [3] This spectrum shows two

distinct maxima at 360 nm and at 450 nm The second

absorption maximum is clearly more intense than

the first The difference spectrum displays a

hypso-chromic shift of the first absorption peak in

compar-ison to free FAD of about 12 nm to 360 nm This shift

is indicative of a modification at the Fl(8a)C atom,

although quantitatively different from the shifts

previ-ously observed in other 8a-bound flavin species [12]

The absorbance spectrum of a mutant enzyme (H79K),

in which the putative FAD-binding His had been

chan-ged to a Lys residue, showed only a single absorbance

peak at 437 nm The fact that the near-UV flavin band

was missing from this protein can be explained by the

single bond to a cysteine residue at the Fl(6) position

This behaviour was previously reported for TMDH

[14] and other 6-S-substituted flavins [17]

Fluorescence properties of the flavin component

in HOX

Surprisingly HOX fluorescence quantum yield was

sig-nificantly less than the fluorescence of choline oxidase

(COX) from Arthrobacter globiformis, which contains

an 8a-N1-histidyl FAD (see Fig 2) [18] When excited

at 450 nm at pH 8, both HOX and COX showed a

distinct fluorescence emission peak at 525 nm The

fluorescence was considerably lower than that of free

FAD (about a factor of 3) for all the species measured

The WT forms of both HOX and COX were buffered

in solutions of decreasing pH Whereas the

fluores-cence emission of COX was clearly higher at lower pH

values, the fluorescence emission of HOX was largely

pH independent An equimolar amount of purified flavopeptide released from the holoenzyme using tryp-sin showed the same distinct, but low, fluorescence emission as HOX and COX, but was pH independent, like HOX HOX was treated with 6 m HCl (see Experimental procedures) under conditions that were previously shown to preserve both the thioether Fl(6)C–S bond of TMDH [14] and the Fl(8a)C–N1 bond of thiamine dehydrogenase (TADH) [19] and COX [18] The fluorescence was measured on the resulting hydrolysate The HOX hydrolysate fluores-cence was shown to increase about three-fold, similar

to that of COX when the pH was reduced to 3 The H79K mutant showed no measurable fluorescence

Kinetic parameters The kinetic parameters determined with d-glucose as substrate for the WT HOX were as follows: Km¼ 4.2 mm, Vmax ¼ 140 mU These are similar to those reported for the native enzyme [5,11] The H79K mutant was not active Furthermore according to the

0 20 40 60 80 100 120

pH

Fig 2 pH-dependent fluorescence emission at 525 nm recorded using an excitation wavelength of 445 nm Concentration with respect to flavin was 0.06 l M in all samples Wild type (WT) hexose oxidase (HOX) from H polymorpha (o) shows low, pH-independent fluorescence emission at 525 nm, as does a trypsin-released flavopeptide from HOX (), contrary to COX from

A globiformis (D), which shows a clear pH dependence with an apparent pKaof 5.8 Flavin released from WT HOX by acid hydro-lysis (o) shows the same pH dependence (pKa5.8) as COX At

pH 8, each sample showed a similar, low, but distinct fluorescence emission maximum at 525 nm, when excited at 445 nm.

apparent extinction coefficient of the flavin in the

mutant enzyme (e437¼ 5.5 mm)1Æcm)1), only half of

the flavin is incorporated in the mutant holoenzyme as

compared with the WT enzyme A unit (U) is defined

as the production of 1 lmol H2O2Æmin)1 at 25
C,

pH 6.3 [3], according to the reaction catalysed by

HOX as mentioned above

Identification of the tryptic flavin–peptide

complex

A flavopeptide was isolated, based on the absorbance

at 450 nm, by RP-HPLC in three sequential steps (see

Experimental procedures) A fraction of the purified

flavopeptide complex was sequenced using standard

Edman sequencing(Figs 3 and 4) The sequencing of

the peptide fraction of the complex yielded two

separ-ate sequences corresponding to positions 74–91 and

132–157 in the primary sequence of HOX There

were two deviations, however: Cycle 6 of the Edman

degradation yielded no His, indicative of a

modifi-cation of His79 Cycle 7 yielded only one equivalent of carbamidomethylated Cys, implying that either Cys80

or Cys138 was chemically different from the carbami-domethylated phenylhydantoin standard These find-ings, together with the fact that His79 and Cys80 are

in the same peptide, suggest that Cys80 was alkylated and Cys138 is covalently associated with FAD

Mass determination of the peptide–flavin complex was carried out simultaneously with the chromatogra-phy steps using an LC-MS⁄ MS setup (see Experimen-tal procedures) Flavopeptide-related ions in the MS spectrum were observed at m⁄ z 893.57, 1071.81 and 1339.49 (Fig 4A) Indicative water loss (data not shown) predicted that these signals would correspond

to the (M + 6H)6+ (M + 5H)5+ and (M + 4H)4+ ions of the flavin–peptide complex, where M is the mass of the neutral complex with two carbamidome-thylations (see Fig 3B) The singly charged ion spe-cies (M + H)+ was confirmed using a MALDI ion source with a TOF analyser Two signals that were clearly related, based on their resolution pattern, were

Putative covalent attachment site Pos H79

N

A

C

I74 VSG G H CYED FVFD ECV K91

V132 LPG GS C YSVG LGG HIVGGG DGILA R15 7

B

N

10

N

5

9

8 8α

2

N

C

H 3

O

O

C

2

2

O

O

OH

P

O

OH

H 2

N

N

N H 2

OH OH

OH

OH

OH

Putative covalent attachment site Pos C138

Fig 3 (A) Overview of the peptides sequenced from the isolated flavin complex Edman degradation of a purified trypsin-released flavin assigned two peptide sequences to the complex The peptides were copurified with the flavin moiety through three consecutive gradients The first peptide is assigned to I74–K91 of the primary sequence No His was detected in cycle 6 The second peptide stems from V132– R157 in the primary sequence Only one carbamidomethylated Cys could be detected in cycle 7 (B) The deduced identity of the peptide complex linked by FAD Cysteines marked by an asterix are modified by a carbamidomethyl group The theoretical mass of the neutral com-plex is 5354.129.

observed at m⁄ z 5008.52 and 5354.05 (Fig 4B) The

deduced mass of the singly charged ion from

the LC-MS⁄ MS was calculated to be 5355.48 Da

The mass discrepancy of 1.43 Da is attributed to the

relatively poor resolution of the signals The mass

observed at 5008.52 Da corresponds to the

flavopep-tide complex with the loss of AMP () 347 Da)

MS⁄ MS of the 893.57 and the 1071.81 signals yielded

major fragment ions of m⁄ z 1001.78 and 1252.53, respectively (data not shown) corresponding to (M-AMP + 5H)5+ and (M-AMP + 4H)4+ The deduced m⁄ z of the singly charged ion species (M-AMP + H)+ is 5006.01; a mass discrepancy of 2.51 Da is observed in this case, again attributable to the poor signal resolution MS⁄ MS of the signals observed at m⁄ z 893.57 and 1071.81 both yielded an

0 10 20 30

A

Intens.

mAU

00010000.D: UV Chromatogram, 215 nm 00010000.D: UV Chromatogram, 450 nm

1071.72

1339.26 2073.52 893.57

+MS, 40.7min #616

0.0 0.5 1.0 1.5 2.0 2.5 x10 6

Intens.

200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

879.98 1070.45

B

1200

1000

800

600

400

200

m/z

5-N N O

O P O

OH

O C2

N N

NH2

OH OH

Fig 4 Determining the mass of the

pep-tide–flavin complex isolated from a hexose

oxidase (HOX) trypsin digest by RP-HPLC.

(A) The LC-MS chromatogram of a trypsin

digest of HOX The absorbance was

con-tinually monitored at 215 nm (red) and

450 nm (green) The trypsin-released FAD

elutes from 40 to 46 min MS was

per-formed on the most intense peak at

40.7 min The signals observed at m⁄ z

893.57, 1071.81 and 1339.49 correspond to

the (M + 6H) 6+ (M + 5H) 5+ and the

(M + 4H) 4+ ions of the peptide–FAD

com-plex (B) Part of the MALDI mass spectrum

of a purified fraction of the complex

show-ing signals at m ⁄ z 5354.05 and 5008.52.

The signals correspond to the singly

charged species of the complex (M + H) 1+

The difference between the two peaks

corresponds roughly to the loss of AMP

(347 Da), as shown in the insert.

intense signal at m⁄ z 347.96 corresponding to singly

charged AMP (348 Da), confirming that both of these

ions are FAD related

Taken together with the Edman degradation results,

the MS data suggest that the isolated flavo component

is the peptide–FAD–peptide complex shown in

Fig 3B The neutral theoretical mass of this complex

is calculated to be 5354.13 Da

Homology modelling

The model of HOX accounts for the first 451 of the

456 residues, without any gaps, and has a root

mean square (RMS) value, relative to the template,

GOOX, of 0.92 A˚, based on 388 Ca-atoms

Accord-ing to the model, the N-1 atom of His79 is located

2.4 A˚ from the 8a C atom of the isoalloxazine ring

in FAD, and the S atom of Cys138 is located 1.8 A˚

from the C-6 atom The distance between possible

covalent attachment points in FAD [10] and other

His, Cys or Tyr residues in HOX were in all other

cases larger than 4.5 A˚, suggesting covalent bonds

between both His79 and Cys138 and the FAD, in

accordance with the chemical evidence shown above

To explore the possibility that dual covalent binding

of FAD may be common, although unnoticed until

very recently [8], we made a multiple sequence

align-ment of HOX, GOOX and the 50 sequences most

closely related to GOOX (see Experimental

proce-dures) In this alignment, 42 of the 50 GOOX

homologues had a conserved His and Cys, at

posi-tions 70 and 130, respectively (GOOX numbering),

which suggests that dual covalent binding through a

His and a Cys is not uncommon To illustrate the

point, Fig 5 shows the alignment of a subset of the

42 sequences, thinned such that no two sequences

are more than 70% identical, and with the sequences

of GOOX and HOX added

Since interaction between amino acids and the pyro-phosphate of FAD usually plays an important role in anchoring FAD, amino acids with atoms near the O3P atom of pyrophosphate were determined, and Val75, Ser76, Gly77, Gly78, His79, Cys80, Val85, Val141, Gly142, Gly144, Gly145 and His146 were found within

a distance of 6 A˚ In this list, the residues appearing in italics are structurally aligned with the seven residues found to interact with pyrophosphate in GOOX [8], which strongly suggests that FAD is anchored in HOX

in much the same way as it is in GOOX (Fig 6) For

Fig 5 Truncated multiple sequence alignment of hexose oxidase (HOX), glucooligosaccharide oxidase (GOOX), and nine GOOX homologues found and selected as described in the text Covalent binding of His and Cys is indicated with red stars P93762, HXO, Chondrus crispus; Q5Z7I6, putative CPRD2, Oryza sativa; Q9AYM8, CPRD2 protein, Vigna unguiculata; Q9SVG7, hypothetical protein F21C20.150, Arabidopsis thaliana; Q9ZPP5, berberine bridge enzyme, Berberis stolonifera; Q9SVG3, reticuline oxidase-like protein, Arabidopsis thaliana; O64743, puta-tive berberine bridge enzyme, Arabidopsis thaliana; Q4HVL0, hypothetical protein, Gibberella zeae PH-1; Q9SA86, T5I8.16 protein, Arabidop-sis thaliana; Q6PW77, GOOX, Acremonium strictum; Q8GTB6, tetrahydrocannabinolic acid synthase, Cannabis sativa.

Fig 6 Residues 75–80 and 85 (blue) and 141–142 and 144–146 (red) in hexose oxidase (HOX), all having atoms within 6 A ˚ of the O3P atom of FAD (indicated) Also shown are covalent links between the isoalloxazine ring of FAD (yellow) and His79 and Cys138 (green) The figure is based on a homology model of HOX, using glucooligosaccharide oxidase (GOOX) as a template, and gen-erated as described in the text.

GOOX, a catalytic mechanism, which involves Tyr429

acting as a general base, and Asp355 facilitating the

necessary abstraction of a proton from Tyr429, was

suggested [8], and used to explain the optimum pH of

about 10 Interestingly, in the HOX model, Tyr488

and Asp409 are structurally aligned with Tyr429 and

Asp355 in GOOX, thus suggesting a similar reaction

mechanism in the two enzymes

Discussion

WT HOX expressed in H polymorpha appears to

con-tain the same rare dual covalent flavin association in

the holoenzyme as the recently published GOOX from

A strictum [8] Based on the GOOX crystal structure

and our understanding of the reaction mechanism of

this enzyme, it has been possible to make analogous

observations on HOX as detailed in Results In this

section a series of procedures will be highlighted that

may be generally useful in identifying this type of

fla-vin association in enzymes

Spectral properties

As the Cys–FAD–His binding mode is novel, few

spec-tral properties have been reported to date Summing

up some of these characteristics for HOX yields the

following The absorbance spectrum of WT HOX

exhibits maxima at 364 and 437 nm The extinction

coefficients with respect to flavin are 11.9 mm)1Æcm)1

and 12.3 mm)1Æcm)1, respectively (Fig 1) The

differ-ence spectrum between an oxidized and a

substrate-reduced enzyme shows maxima at 360 nm and

450 nm The hypsochromic shift of the first absorption

maximum is less than that of other 8a-histidine-bound

flavoproteins, i.e COX [18], VAO [15], and the shift is

larger than that observed for the 8a-cysteine-bound

flavoproteins, i.e monoamine oxidases A and B

(MAO A and B) [20,21], monomeric sarcosine oxidase

(MSOX) and N-methyltryptophan oxidase (MTOX)

[22] Flavoproteins with FAD bound to cysteine at the

Fl(6) carbon have a very different absorbance profile,

with a single maximum at 437 nm [14] We consider

the second maximum of WT HOX observed at 437 nm

to be a specific feature of the C–S bond at the Fl(6)

position, an observation that is backed up by the fact

that the His79K mutant shows only one maximum at

437 nm, as expected for a 6-S-cysteinyl flavin,

although no apparent shoulder at 380 nm, as observed

for TMDH [14] Furthermore, we consider the

hypso-chromic shift of the first maximum of the difference

spectrum to 360 nm a unique feature of the

6-S-cystei-nyl, 8a-N1-histidyl FAD Together, these observations

support the fact that FAD in WT HOX is indeed a 6-S-cysteinyl, 8a-N1-histidyl FAD

The observation made in this article of inducing pH-dependent fluorescence of the flavin in HOX by acid hydrolysis might provide a novel and quick method to distinguish between this type of flavin-bind-ing motif and other covalent-bindflavin-bind-ing motifs The fol-lowing line of reasoning may be employed in this case HOX exhibits a distinctly lower fluorescence at pH 8 than an equimolar amount of COX, which contains the same linkage to a His but lacks a linkage to a Cys The same was evident for the denatured forms of the enzymes, as observed when equimolar amounts of the enzymes were separated by SDS⁄ PAGE and illumin-ated under a UV lamp (data not shown) In compar-ison, 6-S-cysteinylriboflavin is virtually nonfluorescent, both in the isolated form and when bound in enzyme (as discussed above) unless treated with performic acid [14] Flavins in enzymes exhibiting the 8a-Cys flavin-binding motif are only weakly fluorescent and show no fluorescence pH dependency when enzyme bound or when released as aminoacylriboflavin by acid hydrol-ysis [12]

The reason for the sudden boost in fluorescence of the HOX hydrolysate is not fully understood, but the most likely reason is that some of the C–S bonds were broken, yielding a population of N1-histidylriboflavin

It was previously shown that the C–S bond is stable under these conditions [14], but this was done using isolated 6-S-cysteinylriboflavin as the starting material and not enzyme-bound 6-S-cysteinyl, 8a-N1-histidyl-riboflavin The pKa value of the hydrolysate of HOX containing the histidyl–cysteinylriboflavin moiety was determined to be 5.8, which is slightly higher than the 5.2 found previously for a synthetic isomer [23] This observation, combined with the modelling data of the FAD surroundings (Fig 6), leads to the conclusion that His79 is bound at the N-1 position of histidine The higher pKa value is attributed to the influence of the remaining free amino acids in the hydrolysate

HOX, GOOX comparison According to the model of FAD in HOX presented in this article, the surroundings of the coenzyme in the two enzymes are highly similar in spite of only about 25% overall sequence identity In a structural align-ment of the model of HOX and the structure of GOOX, the FAD-binding amino acids His79 and Cys138 are superimposed on the two amino acids cov-alently linked to FAD in GOOX, His70 and Cys130, respectively The differences in substrate specificity and kinetic parameters, along with the fact that the

sequence identity of the F-domain is considerably

higher (33%), suggest that the two enzymes probably

evolved a somewhat different mode of binding to the

substrate but kept the overall structure of the

F-domain [1,5,11] It is quite possible that this is

nat-ure’s way of reusing a good concept (the F-domain) in

combination with a variable S-domain to specialize

oxidases of this type Although the two enzymes are

very similar, several pieces of biological and

biochemi-cal evidence separate the two The substrate

specifici-ties of the two enzymes are similar, but whereas

GOOX displays a distinct preference for

glucosaccha-rides, HOX is more promiscuous in its substrate

bind-ing, resulting in a Kmof 3.6 mm for galactose in spite

of the axial OH4 group of the sugar [5] It has indeed

been proposed that HOX is generally nonspecific for

the C-4 position in its substrates [24] In comparison,

GOOX did not show any measurable activity on

galac-tose when tested against a range of sugars and sugar

derivatives [8] Both enzymes oxidize the substrates

glucose, lactose, maltose, cellobiose and

glucooligosac-charides of up to five glucose residues in solution

GOOX is reported to be able to utilize a heptamer of

glucose [8], whereas this has only been reported for

HOX when the enzyme is immobilized on a solid

sup-port [6] The Km and kcat values of the two enzymes

differ markedly, in that GOOX appears to have higher

affinity for longer-chain gluco- and cello-oligomers,

while HOX exhibits higher affinities for the monomer

and dimer sugars One could hypothesize that the

pro-cessing of longer-chain sugars in the F-domain of

GOOX is enhanced by docking of a multimer of

dis-crete length in the S-domain substrate-binding groove

This does not appear to be the case with HOX, where

Km increases significantly with longer-chain oligomers,

indicating that the longer-chain substrates are less

firmly anchored [5]

With the similarities of the two enzymes thus

con-firmed on the basis of similar substrate specificities and

a novel FAD-binding mode, it may be speculated that

a large family of oligosaccharide oxidases exists that

may be screened for, based solely on the FAD-binding

motif Indeed, our results from the multiple sequence

alignment against the GOOX sequence pinpointed 42

putative oligosaccharide oxidases, all of which have

conserved Cys and His at the FAD-binding positions

The dual flavin linkage found in HOX and GOOX

may serve to provide stability, especially in the active

site of the enzyme Protection against hydroxylation at

the C-6 position of flavin has been suggested to be a

result of the 6-S-linkage [8] and the 8a-N1-linkage was

found to affect the activity of HOX as well as flavin

incorporation in the holoenzyme

Experimental procedures

Purification

WT and mutant HOX were expressed in H polymorpha and extracted using a recently patented process ([2], example 9) The enzyme was isolated using ion exchange chromato-graphy at ambient temperature The ionic strength of 2 L

of the crude sample was reduced to 11 mS with 20 mm

FF (XK50) column (200 mL) was prepared as described

by the manufacturer (Amersham Biosciences, Hillerød, Denmark) The column was equilibrated with 20 mm

(pH 6.3) Active fractions were pooled and concentrated The purification yielded more than 3 g of pure enzyme, cor-responding to total activity of 450 000 U as judged by hydrogen peroxide-dependent horseradish peroxidase reduc-tion of 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid) (ABTS) [25] The specific activity of the WT recombinant

analysis and western blotting (data not shown) Assuming that the extinction coefficient of the 8a-, 6-dual covalent FAD is in the same range as other 8a- or 6-modified flavins

0.005 mm solution of the recombinant WT enzyme (0.115)

The H79K mutant enzyme was purified using a slightly different procedure The ionic strength of 1.7 L of the crude

pH was adjusted to 6.3 A Q-Sepharose FF (XK50) column (200 mL) was prepared as described by the manufacturer (Amersham Biosciences) The column was equilibrated with

at ambient temperature using a linear gradient of 0–1 m

subsequent western blotting using antibodies raised against HOX was used to isolate relevant fractions These were subsequently pooled The purification yielded 50 mg of inactive mutant enzyme The final purity of the mutant

ana-lysis and western blotting (data not shown) A 0.005 mm solution of the H79K mutant enzyme was found to exhibit

an A437 nmof 0.055 Judging by the extinction coefficient of

mutant incorporates flavin in a 1 : 0.9 relationship

Substrate-mediated FAD reduction

Aerobic absorption spectra (310–500 nm) were recorded at ambient temperature and pressure in 0.1 m sodium

spectrophotometer with a 96-well ELISA reader (Molecular

Devices, Sunny Vale, CA) Plates were transparent All

con-centrations of HOX used were 0.005 mm, resulting in a

0.01 mm concentration with respect to flavin content

holoenzyme Substrate sugar (glucose) was added in excess

glu-cose solution was used following incubation at ambient

temperature in the dark for 24 h to allow equilibrium

muta-rotation to reach completion

Anaerobic absorption spectra (310–500 nm) were recorded

in 1 mL quartz cuvettes at ambient temperature and pressure

in 0.1 m phosphate buffer containing glucose (2.5 mm),

pH 6.3, which had been bubbled through and flushed with

sealed before measuring Spectra of 2.5 mm solutions of

reaction products, hydrogen peroxide, gluconic acid–lactone

and gluconic acid were recorded (Sigma-Aldrich, St Louis,

MO) These were found to exhibit negligible absorbance at

these concentrations in the 300–500 nm UV range

Fluorescence

Fluorescence experiments were carried out using a

Gemi-niEM spectrofluorometer from SPECTRAMAX with a

96-well ELISA reader (Molecular Devices) Plates were

opaque All solutions were standardized in 0.05 m

and 8, so that each well (300 lL) had a flavin concentration

of 0.06 lm The fluorescence emission intensity at 525 nm

was recorded using an excitation wavelength of 445 nm

Native protein samples, peptide and hydrolysate showed

the same low but distinct fluorescence emission maximum

at 525 nm at pH 8

Acid hydrolysis

Samples (0.5 mg ) of pure HOX were lyophilized and

0.5 mL of 6 m HCl was added to the lyophilized protein in

sealed glass vials The vials were incubated in the dark for

1 mL of deionized water and diluted to a final

respectively; 10 lL of each sample was used in the

fluores-cence experiment

Isolation and mass determination of flavopeptide

component from a trypsin digest

A sample of purified HOX was digested using modified

tryp-sin V5111 of sequencing grade (> 99% purity) (Promega

Gmbh, Mannheim, Germany) The manufacturer’s protocol

was followed, using iodoacetamide as the alkylating agent

The digest was separated in three consecutive RP-HPLC

runs using a different gradient of acetonitrile (MeCN) in

was split and MS was performed simultaneously using an ESQUIRE ion trap mass spectrometer (Bruker, Rheinstet-ten, Germany) The initial sample injection was loaded onto

Agilent Technologies, Naerum, Denmark) in 0.1% trifluoro-acetic acid for salt removal, and the flow was reversed in 0.1% formic acid when loading from the guard column onto the analytical column (Zorbax 300 SB-C18 RP-HPLC,

trifluoro-acetic acid causes severe disturbances in the MS (Buffer A: 0.1% formic acid in HPLC grade water Buffer B: 0.1% formic acid, in MeCN.) The fraction absorbing most inten-sely at 450 nm was collected from each run and reapplied to the analytical sample column The conditions used for the three consecutive runs were as follows:

Run 1: Start 2% (isocratic) B; 5 min, 2% B; 7 min, 12.5% B; 150 min, 45% B; 170 min (isocratic), 80% B;

180 min, 80% B; 181 min, 2% B

Run 2: Start 2% (isocratic) B; 5 min, 2% B; 7 min, 12.5% B; 60 min, 35% B; 70 min (isocratic), 80% B;

73 min, 80% B; 73.1 min, 2% B

Run 3: Start 2% B (isocratic); 5 min, 2% B; 7 min, 12.5% B; 60 min, 28% B; 70 min, 80% B (isocratic);

73 min, 80% B; 73.1 min, 2% B

MALDI-TOF using an AUTOFLEX mass spectrometer (Bruker) was used to confirm the singly charged ion species

of the isolated flavopeptide from HOX The sample (0.5 lL) from the purified flavopeptide (collected from run 3) was mixed with 0.5 lL of 2,5-dihydroxybenzoic acid in 20% MeCN MALDI target plates (as detailed above) were used for spotting A mass gate was inserted for spectrum

Edman sequencing

Edman sequencing was performed on a sample of purified flavopeptide eluted in run 3 The sequencer used was a PROCISE, sequencer (Applied Biosystems, Naerum, Den-mark) A total of 20 cycles were performed

Homology modelling

WU-BLAST at EBI (European Bioinformatics Institute) established GOOX (1zr6.pdb without and 2axr.pdb with a product analogue) as the most suitable templates for mod-elling of HOX An alignment of HOX and GOOX was determined using Tcoffee [26,27], and some manual editing

Berkley University to generate a three-dimensional model for HOX, based on the GOOX structure, and its alignment with the HOX sequence (http://phylogenomics.berkeley.edu/

models were generated, and the one with the best value of

the modeller objective function was chosen Molecular

graphics were obtained using the pymol Molecular

Graph-ics System (DeLano Scientific, San Carlos, CA)

Multiple alignment of selected GOOX

homologues

The 50 closest homologues to GOOX were found using the

CONSEQ-server (http://conseq.bioinfo.tau.ac.il/) [28], with

three rounds of psi-blast The sequence of HOX was

added, and muscle [29] was used to generate a multiple

cgi-bin/muscle/input_muscle.py)

Acknowledgements

We thank M R Zargahi for kindly providing

assist-ance in the fermentation, extraction and purification of

wild-type hexose oxidase and mutant H79K hexose

oxidase, both from H polymorpha

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