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

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

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Characterization of the flavin association in hexose oxidase from Chondrus crispus Thomas Rand 1 , Karsten B. Qvist 1 , Clive P. Walter 1 and Charlotte H. Poulsen 1,2 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 + O 2 fi d-d-gluconolactone + H 2 O 2 d-d-gluconolactone + H 2 O fi 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. FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO 2693 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 having 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 3 310 360 410 460 Wavelength (nm) ε (m M -1 cm -1 ) B 310 360 410 460 ε (m M -1 cm -1 ) 0 5 10 15 20 Wavelength (nm) C 310 360 410 460 -10 -5 0 5 10 Wavelength (nm) HOX ox -HOX red 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. Characterization of flavin in hexose oxidase T. Rand et al. 2694 FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO This stoichiometry in HOX is further substantiated by reports of extinction coefficients (e 445 –e 448 ) for 8a- and 6-substituted flavins in the range 11.3–12.3 mm )1 Æcm )1 [12]. Assuming a 1 : 2 enzyme ⁄ FAD ratio, the extinc- tion coefficients, with respect to the covalently bound flavins in HOX, are e 364 ¼ 11.9 mm )1 Æcm )1 , e 437 ¼ 12.3 mm )1 Æcm )1 and e 450 ¼ 12.2 mm )1 Æcm )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 (HOX ox –HOX red ), 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: K m ¼ 4.2 mm, V max 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 2468 pH Relative Fluorescence (%) 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 pK a of 5.8. Flavin released from WT HOX by acid hydro- lysis (o) shows the same pH dependence (pK a 5.8) as COX. At pH 8, each sample showed a similar, low, but distinct fluorescence emission maximum at 525 nm, when excited at 445 nm. T. Rand et al. Characterization of flavin in hexose oxidase FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO 2695 apparent extinction coefficient of the flavin in the mutant enzyme (e 437 ¼ 5.5 mm )1 Æcm )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 H 2 O 2 Æmin )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 M 1 K 5 46 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 7α 6 4 N H 3 2 N 1 O C H 3 O H 2 C N N O C H 2 C H C C H C H 2 O P O O OH P O OH O C H 2 N N N H 2 OH OH OH OH OH I74 VSGG H C*YE DFVF DEC*V K91 V132 LPG GS C YSV GLG GHI VGG GDGI LA R157 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. Characterization of flavin in hexose oxidase T. Rand et al. 2696 FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO 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 10 20 30 40 50 60 70 Time [min] 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 815.8005- 3005.1735 840.4535- N N O OP O OH O C H 2 N N NH 2 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. T. Rand et al. Characterization of flavin in hexose oxidase FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO 2697 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. Characterization of flavin in hexose oxidase T. Rand et al. 2698 FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO 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 )1 Æcm )1 and 12.3 mm )1 Æcm )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-binding 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 pK a 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 pK a 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 T. Rand et al. Characterization of flavin in hexose oxidase FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO 2699 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 K m of 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 K m and k cat 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 K m 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 Bis ⁄ Tris, and the pH was adjusted to 6.3. A Q-Sepharose FF (XK50) column (200 mL) was prepared as described by the manufacturer (Amersham Biosciences, Hillerød, Denmark). The column was equilibrated with 20 mm Bis ⁄ Tris buffer (pH 6.3), and elution was performed using a linear gradient of 0–0.5 m NaCl in 20 mm Bis ⁄ Tris buffer (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 enzyme was approximately 150 UÆmg )1 . The final purity of the enzyme was more than 98% as judged by SDS ⁄ PAGE 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 (11.3–12.3 mm -1 Æcm )1 ) [12], absorbance at 445 nm of a 0.005 mm solution of the recombinant WT enzyme (0.115) supports a 1 : 2 enzyme ⁄ FAD stoichiometry. The H79K mutant enzyme was purified using a slightly different procedure. The ionic strength of 1.7 L of the crude sample was reduced to 11 mS with 20 mm Bis ⁄ Tris, and the 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 20 mm Bis ⁄ Tris buffer (pH 6.3) and elution was performed at ambient temperature using a linear gradient of 0–1 m NaCl in 20 mm Bis ⁄ Tris buffer (pH 6.3). SDS ⁄ PAGE with 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 enzyme was more than 98% as judged by SDS ⁄ PAGE ana- lysis and western blotting (data not shown). A 0.005 mm solution of the H79K mutant enzyme was found to exhibit an A 437 nm of 0.055. Judging by the extinction coefficient of a 6-substituted flavin (12.3 mm )1 Æcm )1 ) [14], the H79K 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 phos- phate buffer, pH 6.3, using a SPECTRAMAX TM 190 spectrophotometer with a 96-well ELISA reader (Molecular Characterization of flavin in hexose oxidase T. Rand et al. 2700 FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO 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 according to the 1 : 2 enzyme ⁄ FAD stoichiometry in the holoenzyme. Substrate sugar (glucose) was added in excess at 2.5 mm to a total volume of 300 lL(d ¼ 1 cm). The 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 N 2 gas. Enzyme (0.005 mm) was added while flushing and bubbling through with N 2 gas for 30 min. The cuvettes were 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 cit- rate ⁄ phosphate buffers adjusted to pH values between 3 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 16 h at 95 °C and lyophilized. Samples were resuspended in 1 mL of deionized water and diluted to a final concentra- tion of 1.8 lm in phosphate ⁄ citrate buffers, pH 3–8, 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 each run. The flow (0.1 mLÆmin )1 ) from each RP-HPLC run 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 a guard column (Zorbax StableBond Guard 1.0 · 17 mm; 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, 2.1 · 150 mm). This was done primarily because 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 simplification (m ⁄ z > 3000). 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. modeller was then used through the web interface at 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/ cgi-bin/homology_model/build_model_data.py.) Forty-five T. Rand et al. Characterization of flavin in hexose oxidase FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO 2701 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 sequence alignment. (http://phylogenomics.berkeley.edu/ 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. References 1 Hansen OC & Stougaard P (1997) Hexose oxidase from the red alga Chondrus crispus. Purification, molecular cloning, and expression in Pichia pastoris. 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Arch Biochem Biophys 208, 388–394. 22 Wagner MA, Khanna P & Jorns MS (1999) Structure of the flavocoenzyme of two homologous amine oxi- dases: monomeric sarcosine oxidase and N-methyltryp- tophan oxidase. Biochemistry 38, 5588–5595. 23 Edmondson DE, Kenney WC & Singer TP (1976) Struc- tural elucidation and properties of 8alpha-(N1-histidyl) riboflavin: the flavin component of thiamine dehydro- genase and beta-cyclopiazonate oxidocyclase. Biochemis- try 15, 2937–2945. Characterization of flavin in hexose oxidase T. Rand et al. 2702 FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO [...]... Tcoffee@igs: a web server for computing, evaluating and FEBS Journal 273 (2006) 2693–2703 ª 2006 DANISCO Characterization of flavin in hexose oxidase combining multiple sequence alignments Nucleic Acids Res 31, 3503–3506 28 Berezin C, Glaser F, Rosenberg J, Paz I, Pupko T, Fariselli P, Casadio R & Ben Tal N (2004) ConSeq: the identification of functionally and structurally important residues in protein...T Rand et al 24 Bean RC & Hassid WZ (1956) Carbohydrate oxidase from a red alga, Iridophycus flaccidum J Biol Chem 218, 425–436 25 Savary BJ, Hicks KB & O’Connor JV (2001) Hexose oxidase from Chondrus crispus: improved purification using perfusion chromatography Enzyme Microb Technol 29, 42–51 26 Notredame C, Higgins DG & Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence... 3503–3506 28 Berezin C, Glaser F, Rosenberg J, Paz I, Pupko T, Fariselli P, Casadio R & Ben Tal N (2004) ConSeq: the identification of functionally and structurally important residues in protein sequences Bioinformatics 20, 1322– 1324 29 Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucleic Acids Res 32, 1792–1797 2703 . to distinguish between this type of flavin- bind- ing motif and other covalent-binding motifs. The fol- lowing line of reasoning may be employed in this. 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

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