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• W2058134930 abstract "Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6Å resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12Å deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates. Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6Å resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12Å deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates. Fructosamines are formed by condensation of glucose with the amino group of amino acids or proteins. Fructosamines are formed spontaneously, i.e. non-enzymatically, at a rate that depends on temperature, sugar anomerization rate, concentration, and turnover rate of the target proteins. In medicine, protein-bound fructosamines (also named glycated proteins) have attracted much attention since their formation is increased in diabetes and taken to be in part responsible for diabetic complications. Fructosamines are relatively unstable compounds and are precursors for advanced glycation end products (AGEs), 5The abbreviations used are: AGE, advanced glycation end product; FAOX, fructosamine oxidase; SeMet, selenomethionine; FSA, fructosyl thio-acetate; r.m.s.d., root mean square deviation; MSOX, monomeric sarcosine oxidase. some of which cause proteins cross-linking, extracellular matrix stiffening, and activation of the receptor for AGEs (RAGEs) (1.Monnier V.M. Mustata G.T. Biemel K.L. Reihl O. Lederer M.O. Zhenyu D. Sell D.R. Ann. N. Y. Acad. Sci. 2005; 1043: 533-544Crossref PubMed Scopus (185) Google Scholar, 2.Wendt T. Tanji N. Guo J. Hudson B.I. Bierhaus A. Ramasamy R. Arnold B. Nawroth P.P. Yan S.F. D'Agati V. Schmidt A.M. J. Am. Soc. Nephrol. 2003; 14: 1383-1395Crossref PubMed Scopus (217) Google Scholar). As an example, the fructosamine-derived lysine-arginine cross-link glucosepane is to date the single major cross-link known to accumulate in human collagen in diabetes and aging (3.Sell D.R. Biemel K.M. Reihl O. Lederer M.O. Strauch C.M. Monnier V.M. J. Biol. Chem. 2005; 280: 12310-12315Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Several years ago our laboratory initiated a search for deglycating enzymes in soil organisms with the goal of finding enzymes that could deglycate proteins. In doing so we found enzymes with “amadoriase” activity toward low molecular weight substrates in soil samples, first in Pseudomonas sp. (4.Saxena A.K. Saxena P. Monnier V.M. J. Biol. Chem. 1996; 271: 32803-32809Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and later in Aspergillus fumigatus (5.Takahashi M. Pischetsrieder M. Monnier V.M. J. Biol. Chem. 1997; 272: 3437-3443Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 6.Takahashi M. Pischetsrieder M. Monnier V.M. J. Biol. Chem. 1997; 272: 12505-125057Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 7.Wu X. Takahashi M. Chen S.G. Monnier V.M. Biochemistry. 2000; 39: 1515-1521Crossref PubMed Scopus (43) Google Scholar). The latter turned out to have similar properties with the enzyme first published by Horiuchi et al. (8.Horiuchi T. Kurokawa T. Sati N. Agric. Biol. Chem. 1989; 53: 103-110Crossref Scopus (2) Google Scholar) under the name fructose amino acid oxidase (EC 1.5.3). Considerable work has since been published on these enzymes, which we are referring to in this work under the generic name fructosamine oxidases (FAOX). In addition to FAOX enzymes, two different families of enzymes acting on fructosamines have been identified: fructosamine 3-kinases (found in mammals and birds) and fructoselysine-6-phosphate deglycase, which acts in functional association with fructoselysine-6-kinase (found in bacteria) (9.Delpierre G. Rider M.H. Collard F. Stroobant V. Vanstapel F. Santos H. Van Schaftingen E. Diabetes. 2000; 49: 1627-1634Crossref PubMed Scopus (137) Google Scholar, 10.Wiame E. Delpierre G. Collard F. Van Schaftingen E. J. Biol. Chem. 2002; 277: 42523-42529Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). All these enzymes ultimately catalyze the detachment of the sugar moiety from the amine of the fructosamine, leading to its “deglycation.” Those three families of enzymes use different catalytic mechanisms and have different physiological roles; fructosamine 3-kinase is an ATP-dependent protein-repair enzyme that clears fructosamines as they form on proteins in the cell, whereas the members of the last two families, fructoselysine-6-phosphate deglycase and FAOX, are microbial enzymes enabling the utilization of glycated amino acids as energy source. Fungal FAOXs are peroxisomal enzymes containing covalently bound FAD and catalyze the oxidation of the C-N bond between the nitrogen of the amino portion of the fructosamines and C1 of the fructosyl moiety. The resulting Schiff base is readily hydrolyzed to glucosone and an amino acid. After reduction, the reduced FAD is oxidized by molecular oxygen with concomitant release of hydrogen peroxide (Fig. 1). Many fungal species contain more than one FAOX in their genomes, suggesting complementary roles. A. fumigatus contains two FAOXs; FAOX-I, which acts best on fructosyl-ϵ-lysine and fructosyl aminocaproate, and FAOX-II, which has more affinity for fructose-α-glycine (5.Takahashi M. Pischetsrieder M. Monnier V.M. J. Biol. Chem. 1997; 272: 3437-3443Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 6.Takahashi M. Pischetsrieder M. Monnier V.M. J. Biol. Chem. 1997; 272: 12505-125057Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 7.Wu X. Takahashi M. Chen S.G. Monnier V.M. Biochemistry. 2000; 39: 1515-1521Crossref PubMed Scopus (43) Google Scholar). All FAOXs act best on glycated amino acids, and their activity decreases as the size of the fructosamine increases and is almost undetectable on glycated proteins. The lack of enzymatic activity of FAOX enzymes toward glycated proteins frustratingly prevented them from being utilized as tools to revert protein glycation in in vitro or animal models to study the pathological consequences of protein glycation in aging and diabetes. To understand and possibly overcome by protein engineering, the lack of activity of FAOX toward glycated protein, we have solved the high resolution crystal structures of FAOX-II from A. fumigatus in free and inhibitor-bound forms. Expression and Purification of FAOX-II—An overnight 200-ml pre-culture (37 °C, LB containing 50 mg/liter ampicillin and 25 mg/liter chloramphenicol) of Escherichia coli BL21(DE3)pLysS carrying the expression plasmid of FAOX-II (6.Takahashi M. Pischetsrieder M. Monnier V.M. J. Biol. Chem. 1997; 272: 12505-125057Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) was performed starting from a single freshly plated colony. 100 ml of this culture was used to inoculate 2 liters of LB medium containing 50 mg/liter ampicillin and 25 mg/liter chloramphenicol. Cells were grown at 37 °C under vigorous shaking until A600 reached 0.5, and isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 0.4 mm; the culture was grown for an additional 4 h at 37°C. Cells were pelleted by centrifugation at 6000 rpm for 10 min at 4 °C. The bacterial pellet was resuspended in 100 ml of 25 mm Tris, pH 8.0, 200 mm NaCl, 1 mg/ml hen egg lysozyme and submitted to three cycles of freezing and thawing. MgSO4 was added to a final concentration of 1 mm together with 20,000 units of DNase, and the preparation was incubated at 4 °C for 15 min before centrifugation at 16,000 rpm for 40 min at 4 °C. The supernatant was gently mixed with polyethylene glycol 6000 at a final concentration of 15% (w/v), and the preparation was centrifuged at 12,000 rpm for 20 min at 4 °C to give a pellet that contained >95% of FAOX-II. Resuspension in 25 mm Tris, pH 8.0, 200 mm NaCl was followed by dilution in buffer A (25 mm Tris, pH 8.0, 75 mm NaCl) to a final volume of 1100 ml before loading onto a 60-ml DEAE at 1.2 ml/min. The column was washed with 100 ml of buffer A, and the flow-through and washes, which contained FAOX-II, were collected. After adjusting the pH to 9.5 with 5 m NaOH, the volume was brought to 2000 ml by the addition of 800 ml of 25 mm Tris pH 9.5. The preparation was loaded on a 30-ml Q-Sepharose column at 5 ml/min, and the column was washed with 100 ml of buffer (25 mm Tris 8.0) before elution of proteins using a 300-ml gradient (0–100% B: 25 mm Tris, pH 8.0, 0.5 m NaCl) at 2 ml/min. Fractions were collected, and A450 nm was monitored to identify the fractions with the most intense yellow color (eluted at 15–30% B). The pooled fractions (∼16 ml) were concentrated to 40 mg/ml, and buffer was exchanged into 10 mm Tris, pH 8.0, and 10 mm NaCl, during which some protein precipitate formed and was removed by centrifugation. The final yield was 35 mg of pure protein with a specific activity of 3.3 units/mg using fructosyl-glycine as a substrate. Analysis of the Purity of FAOX-II—The final purified FAOX-II preparation showed a single band with an apparent mass of about 50 kDa by silver-stained SDS-PAGE. Dynamic light scattering analysis indicated that the sample was monodisperse and had an apparent mass of 47 kDa. Mass spectrometry analysis revealed one peak of mass 49,703 kDa, in excellent agreement with the calculated mass of a FAD-bound enzyme; no mass was observed at 48,931 kDa, expected for apoenzyme lacking FAD (the latter represented about 80% of FAOX-II in the bacterial crude extract and was removed during the purification). The A280/A450 ratio was 0.125, in good agreement with the calculated theoretical ratio of 0.124. Mutagenesis—Mutagenesis was performed using a Stratagene QuikChange kit according to the manufacturer's instructions. The mutant proteins were expressed under the same conditions as the wild-type enzyme. The purification procedure was simplified and involved only the DEAE-Sepharose and the Q-Sepharose purifications steps. Purity of the mutant proteins was assayed by SDS-PAGE analysis using Coomassie Blue staining and was >70%. Expression and Purification of Selenomethionine FAOX-II— An overnight 200-ml pre-culture (37 °C, LB ampi-chloramphenicol) of bacteria E. coli BL21(DE3)pLysS carrying the expression plasmid of FAOX-II was performed starting from a single freshly plated colony. The culture was centrifuged at 4000 rpm for 20 min at 4 °C, and the pellet was resuspended in 20 ml of a minimal medium (27 g/liter Na2HPO4, 13.5 g/liter KH2PO4, 4.5 g/liter NH4Cl, 2.25 g/liter NaCl, 4.5 mm MgSO4, 18 g/liter glucose, 2.25 mg/liter thiamine, 125 mg Fe(II)SO4·7H2O) and the antibiotics ampicillin (to 50 mg/liter) and chloramphenicol (25 mg/liter). Two liters of minimal media were inoculated with 12 ml of this preparation, and the culture was grown at 37 °C under vigorous shaking until A600 reached 0.5; at this stage, 200 mg of lysine, 200 mg of phenylalanine, 200 mg of threonine, 100 mg of isoleucine, 100 mg of leucine, 100 mg of valine, and 100 mg of selenomethionine (SeMet) were added to the culture. After 20 min isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 0.4 mm and CoCl2 and ZnCl2 to 0.1 mm, and the culture was pursued for another 6 h. The preparation of the bacterial extract and the purification procedure were performed identically as for wild-type FAOX-II with the exception that 1 mm dithiothreitol was added to all buffers to prevent selenomethionine oxidation. This procedure allowed the preparation of 10 mg of pure SeMet-FAOX-II. Mass spectrometric analysis indicated that all eight methionine residues were replaced by selenomethionine (data not shown). Expression and Purification of FAOX-I—His-tagged FAOX-I from A. fumigatus was expressed and purified on a His-Trap column as described previously (7.Wu X. Takahashi M. Chen S.G. Monnier V.M. Biochemistry. 2000; 39: 1515-1521Crossref PubMed Scopus (43) Google Scholar). Crystallization and Cryoprotection—Crystals of wild-type and selenomethionine FAOX-II were obtained by the sitting drop vapor diffusion method at 20 °C by mixing 1 μl of the protein solution (15 mg/ml in 10 mm Tris buffer, pH 8.0) in the presence or absence of a 3 mm concentration of the inhibitor fructosyl thioacetate (FSA) with 1 μl of the reservoir containing 0.1 m Hepes, pH 7.4, 10% isopropanol, and 18% polyethylene glycol 4000. Crystals took 2 weeks to grow and are monoclinic, with space group P21, and contain 2 FAOX-II molecules in the asymmetric unit to give a calculated Matthews coefficient of 2.1 Å3/Da. SeMet crystals grew under the same conditions as the wild type and were isomorphous. Crystals with and without inhibitor grew under similar conditions but had different unit cell dimensions (Table 1); the smaller SeMet crystals had more regular morphology and were used for data collection. The crystals were transferred to a cryoprotectant solution containing 0.1 m Hepes, pH 7.4, 20% isopropanol, 18% PEG 4000 and dunked in liquid nitrogen for data collection.TABLE 1Data collection, phasing, and refinement statistics Numbers in parentheses refer to the highest resolution shell. EPE, Hepes.CrystalFAOX-II (SeMet)FAOX-II-FSA (SeMet)Data collection Cell dimensions a (Å)74.387.1 b (Å)54.353.4 c (Å)106.3103.3 β (°)95.92113.33 Wavelength (Å)0.979291.08100 Resolution (Å)20-1.75 (1.81-1.75)30-1.6 (1.66-1.6) Rsym or Rmerge (%)6.9 (59.5)10.4 (24.0) I/σI26.8 (2.9)14.6 (3.3) Completeness (%)99.7 (98.4)91.3 (52.0) Redundancy6.8 (6.2)6.8 (3.4)Refinement Resolution (Å)20-1.830-1.6 No. of reflections74,880105,596 Rwork/Rfree (%)16.7/20.417.0/19.8 No. of atoms Protein6,6787,018 Ligand:FAD106106 Ligand:FSA32 Ligand:EPE15 Water842925 Average B-factors (Å2) Protein17.516.6 Ligands21.819.7 Water29.330.0 r.m.s.d. Bond lengths (Å)0.0130.010 Bond angles (°)1.41.3 Ramachandran Most favored (%)88.488.7 Additional allowed (%)11.611.3 Disallowed (%)00 Open table in a new tab Data Collection and Structure Determination—Single wave-length SeMet anomalous dispersion data from a crystal containing the free form of FAOX-II were measured at beamline 19-ID at the Advanced Photon Source and processed using HKL2000 (11.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-325Crossref PubMed Scopus (38617) Google Scholar) (Table 1). The sites of 16 selenium atoms were determined and used for initial phasing to 1.8 Å resolution in SOLVE (12.Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar); phases were improved by solvent-flattening and non-crystallographic symmetry averaging in RESOLVE (13.Terwilliger T.C. Acta Crystallogr. Sect. D. 2000; 56: 965-972Crossref PubMed Scopus (1636) Google Scholar, 14.Terwilliger T.C. Acta Crystallogr. Sect. D. 2002; 59: 34-44PubMed Google Scholar) such that the mean figure of merit increased from 0.38 to 0.66. An initial FAOX-II model automatically built by RESOLVE provided the starting point for iterative cycles of interactive model building carried out in COOT (15.Emsley P. Cowtan K. Acta Crystallogr. Sect. D. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar) and refinement calculations in REFMAC (16.Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar). The quality of all models was assessed by PROCHECK (17.Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and MolProbity (18.Davis I.W. Leaver-Fay A. Chen V.B. Block J.N. Kapral G.J. Wang X. Murray L.W. Arendall W.B. Snoeyink J. Richardson J.S. Richardson D.C. Nucleic Acids Res. 2007; 34: 375-383Crossref Scopus (3051) Google Scholar). The final refined model contained residues 3–57, 68–109, and 117–437 in molecule A, residues 3–57, 67–109, and 120–438 in molecule B, FAD cofactors in both molecules, and 842 waters. Diffraction data for FAOX-II bound to FSA were measured from a SeMet crystal. Data were measured at beamline X29 at the National Synchrotron Light Source and processed using HKL2000 (11.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-325Crossref PubMed Scopus (38617) Google Scholar) (Table 1). This structure was initially solved using the coordinates of the unbound FAOX-II. The final refined model of the FAOX-II includes residues 2–437 in molecule A, 2–438 in molecule B, FAD and FSA bound to both molecules, 926 waters, and a molecule of Hepes presumably from the crystallization mixture, bound to the surface of FAOX-II molecule B distant from the active site. Data and refinement statistics are provided in Table 1. Molecular figures were generated using PyMol (19.DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). Synthesis of Substrates and Inhibitor—Nα-(1-Deoxy-d-fructos-1-yl)-glycine, Nα-(1-deoxy-d-fructos-1-yl)-l-valine, Nα-(1-deoxy-d-fructos-1-yl)-l-glutamic acid, Nα-t-Boc-Nϵ-(1-deoxy-d-fructos-1-yl)-l-lysine (where t-Boc represents tert-butoxycarbonyl), Nϵ-(1-deoxy-d-fructos-1-yl)-l-lysine, Nϵ-(1-deoxy-d-fructos-1-yl)-amino caproic acid, Nα-t-Boc-Nϵ-(1-deoxy-d-ribulos-1-yl)-l-lysine, and Nϵ-(1-deoxy-d-ribulos-1-yl)-l-lysine were synthesized as previously described (20.Röper H. Röper S. Heyns K. Carbohydr. Res. 1983; 116: 183-195Crossref Scopus (94) Google Scholar, 21.Beksan E. Schieberle P. Robert F. Blank I. Fay L.B. Schlichtherle-Cerny H. Hofmann T. J. Agric. Food Chem. 2003; 51: 5428-5436Crossref PubMed Scopus (84) Google Scholar, 22.Thornalley P.J. Langborg A. Minhas H.S. Biochem. J. 1999; 344: 109-116Crossref PubMed Scopus (1026) Google Scholar, 23.Grandhee S.K. Monnier V.M. J. Biol. Chem. 1991; 266: 11649-11653Abstract Full Text PDF PubMed Google Scholar). For synthesis of the inhibitor fructosyl-thioacetate (FSA), trifluoromethane sulfonic anhydride (300 mg, 1.1 mmol) was added into a solution of 2,3:4,5-di-O-isopropylidene-β-d-fructopyranose (260 mg, 1 mmol) in dichloromethane (10 ml). The resulting solution was stirred at room temperature for 1 h and evaporated under reduced pressure to give triflate 2 (see Scheme), which was dissolved in N,N-dimethylformamide (5 ml). Methyl-thioglycate (106 mg, 1 mmol) and cesium carbonate (325 mg, 1 mmol) were added, and the resulting mixture was stirred for 0.5 h and evaporated. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate 5/1) to give the ester 3 (260 mg, 74.7% yield), which was hydrolyzed by lithium hydroxide (9.5 mg, 4 mmol) in isopropanol/water (1:1, 5 ml) to give 4. The final product 1 was achieved by deprotection of 4 in TFA/water (4:1) at room temperature. Glycated bovine serum albumin and glycated histones were prepared as described previously (9.Delpierre G. Rider M.H. Collard F. Stroobant V. Vanstapel F. Santos H. Van Schaftingen E. Diabetes. 2000; 49: 1627-1634Crossref PubMed Scopus (137) Google Scholar). Enzymatic Assays—FAOX activity was measured as previously described by following the formation of hydrogen peroxide using the Amplex Red peroxidase assay (Molecular Probes) according to the manufacturer's instructions (7.Wu X. Takahashi M. Chen S.G. Monnier V.M. Biochemistry. 2000; 39: 1515-1521Crossref PubMed Scopus (43) Google Scholar). Briefly, FAOX was incubated with the appropriate concentration of fructosamine in the presence of horseradish peroxidase (0.02 milliunits) and 10-acetyl-3,7-dihydroxyphenoxazine (50 μm) at 30 °C in a final volume of 200 μl, and fluorescence was detected at 590 nm using excitation at 540 nm in a microplate reader (Tecan Instrument). One unit of enzyme is defined as producing 1 mmol of hydrogen peroxide/min/mg of enzyme at 30 °C at a saturating concentration of the substrate. Previous attempts to obtain crystals from His-tagged FAOX-I (Amadoriase I) were fruitless, leading us to isolate and purify the non-tagged recombinant enzyme FAOX-II (Amadoriase II) instead. Crystals of FAOX-II in free form and bound to its FSA inhibitor grew under similar conditions, and their structures were determined to 1.75 and 1.6 Å resolution, respectively. The free FAOX-II and inhibitor-bound FAOX-II-FSA structures each have two independent molecules in the asymmetric unit. Superposition of the Cα atoms of the two molecules in FAOX-II gives an r.m.s.d. of 0.3 Å; a similar superposition for the FAOX-II-FSA complex crystal gives a 0.2-Å r.m.s.d., indicating that the two molecules in each crystal are in nearly identical conformations. In the free FAOX-II structure, no continuous electron density was seen for residues 58–66 and 110–116, revealing that in the absence of ligand bound in the active site, these two surface regions are flexible. Aside from the two flexible segments near the active site, the conformation of the rest of the enzyme is very similar in the presence and absence of inhibitor, as evidenced by a low r.m.s.d. value of 0.5 for superposition of the Cα atoms of the free and complexed FAOX-II-FSA (Figs. 2, A and B). Unless otherwise indicated, structural details in the following sections are provided for molecule A in the FAOX-II-FSA crystal and are similar in both FAOX-II and FAOX-II-FSA structures. FAOX-II is a two-domain protein belonging to the d-amino acid oxidase FAD-binding protein family. One domain contains a classic FAD binding motif that includes two β-sheets that are flanked by four α-helices on one side and consists of residues Ala-2—Gly-48, Ala-163—Ala-221, and Cys-335—Glu-382 (Figs. 2, A and B, in dark gray). The second, “catalytic” domain contains an 8-stranded mixed β-sheet that provides a curved wall of the active site substrate binding pocket, a long helix in a flap that covers the active site, and a second long helix at the periphery of the structure. An SSM structural homolog search (24.Krissinel E. Henrick K. Acta Crystallogr. Sect. D. 2004; 60: 2256-2268Crossref PubMed Scopus (3189) Google Scholar) against the PDB data base identified monomeric sarcosine oxidase (MSOX, PDB code 1el9) from Bacillus subtilis, a protein sharing 18% sequence identity with FAOX-II (25.Trickey P. Wagner M.A. Jorns M.S. Mathews F.S. Structure. 1999; 7: 331-345Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), as the closest match, with an r.m.s.d. of 2.8 Å for 357 equivalent Cα atoms. The C-terminal 36 residues in FAOX-II form a loop over the substrate binding site which is missing in MSOX (Figs. 2, A and B, pink). l-Proline dehydrogenase (PDB code 1y56), glycine oxidase (PDB code 1ng3), the β chain of heterotetrameric sarcosine oxidase (PDB code 2gag), and N-methyl-l-tryptophan oxidase (PDB code 2apg) were also identified as structural homologs but share no significant sequence similarity with FAOX-II. Flavin Binding—The FAD is bound to FAOX-II similarly in the presence and absence of the FSA inhibitor and in a manner that is also similar to that seen in MSOX (Fig. 2C). The cofactor is in an extended conformation and is nearly completely encased in the enzyme. In the two independent molecules in the free and inhibitor-bound structures, the isoaloxazine ring varied from planar to slightly bent (by up to 10°). The covalent, hydrogen-bonding and electrostatic interactions between FAD and FAOX-II are summarized in Fig. 3A. These include one covalent bond to Cys-335, 21 hydrogen bonds of which 3 are to solvent molecules, and 2 helix dipoles. The positive end of one of the helices (residues 367–383) is directed toward the FAD N1 and O2 atoms, helping to stabilize the electron-rich character of the flavin ring; the positive end of the other dipole points toward the negatively charged pyrophosphate group of FAD and serves to stabilize/neutralize its charge. Arginine residues at positions 112, 343, and 411 and lysine residues at positions 53 and 368 contribute to a basic environment surrounding the FAD. Of particular interest is Lys-53, which approaches the si-side of the flavin aromatic ring; in MSOX, an Arg is found in the equivalent position. Lys-276 forms a water-mediated hydrogen bond with the flavin ring N5 atom. This interaction is also seen in MSOX and maize polyamine oxidase (PDB code 1b37), the latter of which has an unrelated protein fold. Glu-280 is the only acidic residue found in the flavin environment and is involved in hydrogen-bonding with the hydroxyl group of the fructosyl moiety of the inhibitor (see below). FSA Inhibitor Binding—Studies on MSOX successfully took advantage of the replacement of the nitrogen of the C-N bond of the substrate by sulfur, tellurium, or selenium in the design of inhibitors (25.Trickey P. Wagner M.A. Jorns M.S. Mathews F.S. Structure. 1999; 7: 331-345Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 26.Wagner M.A. Trickey P. Chen Z.W. Mathews F.S. Jorns M.S. Biochemistry. 2000; 39: 8813-8824Crossref PubMed Scopus (91) Google Scholar). We, therefore, synthesized an analog of fructosyl-glycine, the fructosamine for which FAOX-II displays the highest affinity, in which the α-nitrogen was replaced by a sulfur atom. This compound, FSA, was found to be a good competitive inhibitor with a Ki of 5 μm, i.e. ∼50-fold lower than the Km for fructosyl-glycine, without being a substrate of FAOX-II. Spectroscopic studies indicated that FSA formed a charge transfer species with FAOX, presumably due to the close proximity of the electron-rich sulfur of FSA and the flavin. The soaking of FAOX-II crystals in the presence of FSA caused their dissolution; co-crystallization was successfully used to obtain complex crystals. The FSA ligand could be located unambiguously on the re-side of the flavin ring (Figs. 2, B–D), with its fructosyl portion displaying a pyranose conformation, the most abundant form of fructosamines in solution (27.Tagami U. Akashi S. Mizukoshi T. Suzuki E. Hirayama K. J. Mass Spectrom. 2000; 35: 131-138Crossref PubMed Scopus (43) Google Scholar). This finding confirms previous studies with stereospecific inhibitors adopting both furanose and pyranose conformations (28.Wu X. Chen S.G. Petrash J.M. Monnier V.M. Biochemistry. 2002; 41: 4453-4458Crossref PubMed Scopus (26) Google Scholar). The sulfur atom of the inhibitor, which would correspond to the nitrogen atom of a substrate, is 3.4 Å from C4α of FAD, whereas the adjacent carbon of the inhibitor, corresponding to the nascent imine carbon of a substrate, is 3.4 Å from N5 of FAD. Thus, the inhibitor appears to be bound in a position and orientation corresponding to a productive substrate binding mode. The interactions made by the ligand with the protein are summarized in Figs. 2B, 3B, and 4A and fall into three groups, those involving the inhibitor carboxylate, those involving the fructosyl group, and those forming a hydrophobic interface along one side of the inhibitor. The FSA carboxylate oxygens are hydrogen-bound to the side chains of Tyr-60, Arg-112, and Lys-368 and to two water molecules (Fig. 4A). Interestingly, the MSOX active site contains Tyr-55, Arg-52, and Lys-348 in similar positions (Fig. 4B). To address their possible importance for enzyme function, these three residues were mutated to impair their interactions with the carboxylic portion of the substrate. Mutation of Tyr-60 to Phe, Arg-112 to Glu, and Lys-368 to Met resulted in decreases of Km for fructosyl-glycine by 3-, 10-, and 5-fold, respectively, without changing the Vmax significantly (Table 2).TABLE 2Effect of amino acids substitution on kinetic properties of FAOX-II ND, not detecte" @default.
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• W2058134930 title "Crystal Structure of the Deglycating Enzyme Fructosamine Oxidase (Amadoriase II)" @default.
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• W2058134930 cites W1539796472 @default.
• W2058134930 cites W1965277349 @default.
• W2058134930 cites W1967945049 @default.
• W2058134930 cites W1968332280 @default.
• W2058134930 cites W1974794664 @default.
• W2058134930 cites W1984711870 @default.
• W2058134930 cites W1986191025 @default.
• W2058134930 cites W2006954456 @default.
• W2058134930 cites W2007664069 @default.
• W2058134930 cites W2021458914 @default.
• W2058134930 cites W2022011077 @default.
• W2058134930 cites W2026948867 @default.
• W2058134930 cites W2033596810 @default.
• W2058134930 cites W2037816461 @default.
• W2058134930 cites W2038840577 @default.
• W2058134930 cites W2042499399 @default.
• W2058134930 cites W2045746015 @default.
• W2058134930 cites W2053302543 @default.
• W2058134930 cites W2058451618 @default.
• W2058134930 cites W2063366649 @default.
• W2058134930 cites W2071540582 @default.
• W2058134930 cites W2079226100 @default.
• W2058134930 cites W2083873311 @default.
• W2058134930 cites W2086255061 @default.
• W2058134930 cites W2088565094 @default.
• W2058134930 cites W2089034761 @default.
• W2058134930 cites W2121472843 @default.
• W2058134930 cites W2122339645 @default.
• W2058134930 cites W2125593296 @default.
• W2058134930 cites W2130892097 @default.
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• W2058134930 cites W2143694829 @default.
• W2058134930 cites W2144081223 @default.
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