The Human Apoptosis-inducing Protein AMID Is an Oxidoreductase with a Modified Flavin Cofactor and DNA Binding Activity
2005; Elsevier BV; Volume: 280; Issue: 35 Linguagem: Inglês
10.1074/jbc.m414018200
ISSN1083-351X
AutoresKer R. Marshall, Min Gong, Leigh Wodke, John H. Lamb, Donald J. L. Jones, Peter B. Farmer, Nigel S. Scrutton, Andrew W. Munro,
Tópico(s)Metabolism and Genetic Disorders
ResumoAMID (apoptosis-inducing factor-homologous mitochondrion-associated inducer of death; also known as PRG3 (p53-responsive gene 3)) is a human caspase-independent pro-apoptotic protein with some similarity to apoptosis-inducing factor. AMID was purified from a recombinant bacterial host, enabling biochemical analysis of the protein. AMID is a flavoprotein; possesses NAD(P)H oxidase activity; and catalyzes NAD(P)H-dependent reduction of cytochrome c and other electron acceptors, including molecular oxygen. NADPH binds ∼10-fold tighter than NADH. AMID binds 6-hydroxy-FAD (a cofactor that accumulates only adventitiously and at low abundance in other flavoprotein enzymes) to form a stoichiometric cofactor·protein complex. AMID has a distinctive electronic spectrum due to the modified flavin. NAD(P)+ binding perturbed the spectrum, enabling determination of Kd values for these coenzymes. 6-Hydroxy-FAD could be removed from AMID and the apoprotein reconstituted with FAD. FAD was converted to 6-hydroxy-FAD in reconstituted AMID during aerobic turnover with NADPH. AMID is a DNA-binding protein that lacks apparent DNA sequence specificity. Formation of the protein·DNA complex (i) effected a major protein conformational change and (ii) was prevented in the presence of nicotinamide coenzyme. Apo-AMID retains DNA binding activity. Our studies establish a link between coenzyme and DNA binding that likely impacts on the physiological role of AMID in cellular apoptosis. AMID (apoptosis-inducing factor-homologous mitochondrion-associated inducer of death; also known as PRG3 (p53-responsive gene 3)) is a human caspase-independent pro-apoptotic protein with some similarity to apoptosis-inducing factor. AMID was purified from a recombinant bacterial host, enabling biochemical analysis of the protein. AMID is a flavoprotein; possesses NAD(P)H oxidase activity; and catalyzes NAD(P)H-dependent reduction of cytochrome c and other electron acceptors, including molecular oxygen. NADPH binds ∼10-fold tighter than NADH. AMID binds 6-hydroxy-FAD (a cofactor that accumulates only adventitiously and at low abundance in other flavoprotein enzymes) to form a stoichiometric cofactor·protein complex. AMID has a distinctive electronic spectrum due to the modified flavin. NAD(P)+ binding perturbed the spectrum, enabling determination of Kd values for these coenzymes. 6-Hydroxy-FAD could be removed from AMID and the apoprotein reconstituted with FAD. FAD was converted to 6-hydroxy-FAD in reconstituted AMID during aerobic turnover with NADPH. AMID is a DNA-binding protein that lacks apparent DNA sequence specificity. Formation of the protein·DNA complex (i) effected a major protein conformational change and (ii) was prevented in the presence of nicotinamide coenzyme. Apo-AMID retains DNA binding activity. Our studies establish a link between coenzyme and DNA binding that likely impacts on the physiological role of AMID in cellular apoptosis. Apoptosis (or programmed cell death) is an autoregulatory process by which defective cells within tissues of multicellular organisms self-destruct. Apoptosis occurs via either a caspase-dependent or caspase-independent mechanism (1Strasser A. O'Connor L. Dixit V.M. Annu. Rev. Biochem. 2000; 69: 217-245Crossref PubMed Scopus (1381) Google Scholar, 2Saelens X. Festjens N. Vande Walle L. van Gurp M. van Loo G. Vandenabeele P. Oncogene. 2004; 23: 2861-2874Crossref PubMed Scopus (729) Google Scholar, 3Lorenzo H.K. Susin S.A. FEBS Lett. 2004; 557: 14-20Crossref PubMed Scopus (155) Google Scholar, 4Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3249) Google Scholar, 5Cande C. Vahsen N. Garrido C. Kroemer G. Cell Death Differ. 2004; 11: 591-595Crossref PubMed Scopus (188) Google Scholar, 6Jaattela M. Cande C. Kroemer G. Cell Death Differ. 2004; 11: 135-136Crossref PubMed Scopus (70) Google Scholar, 7Lipton S.A. Bossy-Wetzel E. Cell. 2002; 111: 147-150Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Apoptosis-inducing factor (AIF) 1The abbreviations used are: AIF, apoptosis-inducing factor; Ni-NTA, nickel-nitrilotriacetic acid; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MOPS, 4-morpholinepropanesulfonic acid. 1The abbreviations used are: AIF, apoptosis-inducing factor; Ni-NTA, nickel-nitrilotriacetic acid; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MOPS, 4-morpholinepropanesulfonic acid. is a flavin-containing oxidoreductase released from mitochondria during caspase-independent apoptosis (2Saelens X. Festjens N. Vande Walle L. van Gurp M. van Loo G. Vandenabeele P. Oncogene. 2004; 23: 2861-2874Crossref PubMed Scopus (729) Google Scholar, 5Cande C. Vahsen N. Garrido C. Kroemer G. Cell Death Differ. 2004; 11: 591-595Crossref PubMed Scopus (188) Google Scholar, 8Joza N. Susin S.A. Daugas E. Stanford W.L. Cho S.K. Li C.Y. Sasaki T. Elia A.J. Cheng H.Y. Ravagnan L. Ferri K.F. Zamzami N. Wakeham A. Hakem R. Yoshida H. Kong Y.Y. Mak T.W. Zuniga-Pflucker J.C. Kroemer G. Penninger J.M. Nature. 2001; 410: 549-554Crossref PubMed Scopus (1152) Google Scholar, 9Cande C. Cecconi F. Dessen P. Kroemer G. J. Cell Sci. 2002; 115: 4727-4734Crossref PubMed Scopus (428) Google Scholar, 10Daugas E. Nochy D. Ravagnan L. Loeffler M. Susin S.A. Zamzami N. Kroemer G. FEBS Lett. 2000; 476: 118-123Crossref PubMed Scopus (398) Google Scholar, 11Cregan S.P. Dawson V.L. Slack R.S. Oncogene. 2004; 23: 2785-2796Crossref PubMed Scopus (454) Google Scholar). Caspase-independent apoptosis ensues following the release of AIF from the mitochondrion and after its translocation to the nucleus, where AIF, possibly together with cyclophilin A (12Cande C. Vahsen N. Kouranti I. Schmitt E. Daugas E. Spahr C. Luban J. Kroemer R.T. Giordanetto F. Garrido C. Penninger J.M. Kroemer G. Oncogene. 2004; 23: 1514-1521Crossref PubMed Scopus (226) Google Scholar), initiates chromosomal condensation, margination, and degradation. The human pro-apoptotic protein AMID (apoptosis-inducing factor-homologous mitochondrion-associated inducer of death; also designated PRG3 (p53-responsive gene 3) was identified recently (13Ohiro Y. Garkavtsev I. Kobayashi S. Sreekumar K.R. Nantz R. Higashikubo B.T. Duffy S.L. Higashikubo R. Usheva A. Gius D. Kley N. Horikoshi N. FEBS Lett. 2002; 524: 163-171Crossref PubMed Scopus (78) Google Scholar, 14Wu M. Xu L.G. Li X. Zhai Z. Shu H.B. J. Biol. Chem. 2002; 277: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) on the basis of its amino acid sequence similarity (22% identity) to AIF. AMID is also thought to be a caspase-independent effector of apoptosis (14Wu M. Xu L.G. Li X. Zhai Z. Shu H.B. J. Biol. Chem. 2002; 277: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), but, unlike AIF, lacks a mitochondrial localization signal and either is located in the cytoplasm (13Ohiro Y. Garkavtsev I. Kobayashi S. Sreekumar K.R. Nantz R. Higashikubo B.T. Duffy S.L. Higashikubo R. Usheva A. Gius D. Kley N. Horikoshi N. FEBS Lett. 2002; 524: 163-171Crossref PubMed Scopus (78) Google Scholar, 14Wu M. Xu L.G. Li X. Zhai Z. Shu H.B. J. Biol. Chem. 2002; 277: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) or is associated with the outer mitochondrial membrane (14Wu M. Xu L.G. Li X. Zhai Z. Shu H.B. J. Biol. Chem. 2002; 277: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The N-terminal domains of both AMID and AIF, which contain putative FAD-binding motifs, can be mutated or deleted without apparent effects on the apoptogenic activity when overexpressed in human cell lines (13Ohiro Y. Garkavtsev I. Kobayashi S. Sreekumar K.R. Nantz R. Higashikubo B.T. Duffy S.L. Higashikubo R. Usheva A. Gius D. Kley N. Horikoshi N. FEBS Lett. 2002; 524: 163-171Crossref PubMed Scopus (78) Google Scholar, 14Wu M. Xu L.G. Li X. Zhai Z. Shu H.B. J. Biol. Chem. 2002; 277: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Loeffler M. Daugas E. Susin S.A. Zamzami N. Metivier D. Nieminen A.L. Brothers G. Penninger J.M. Kroemer G. FASEB J. 2001; 15: 758-767Crossref PubMed Scopus (211) Google Scholar, 16Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). AMID appears to be confined to eukaryotes, with orthologs identified in mammals (Mus musculus; 90% identity), birds (Gallus gallus; 68% identity), amphibians (Xenopus laevis; 61% identity), and fish (Fugu rubripes; 56% identity) and more primitive organisms such as sea squirt (Ciona intestinalis; 37% identity). AMID contains a putative Rossmann fold associated with dinucleotide binding and shares some sequence identity with bacterial oxidoreductases, including Escherichia coli NAD+ reductase and Pseudomonas putida putidaredoxin reductase. AMID expression is regulated by p53, which binds to p53-responsive elements in the AMID promoter region (13Ohiro Y. Garkavtsev I. Kobayashi S. Sreekumar K.R. Nantz R. Higashikubo B.T. Duffy S.L. Higashikubo R. Usheva A. Gius D. Kley N. Horikoshi N. FEBS Lett. 2002; 524: 163-171Crossref PubMed Scopus (78) Google Scholar, 17Wu M. Xu L.G. Su T. Tian Y. Zhai Z. Shu H.B. Oncogene. 2004; Google Scholar). However, AMID-induced apoptosis is not p53-dependent, as demonstrated by overexpression of AMID in p53-deficient colon cancer cells (14Wu M. Xu L.G. Li X. Zhai Z. Shu H.B. J. Biol. Chem. 2002; 277: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). AMID mRNA levels were recently reported to be down-regulated in tumor tissues compared with matched human normal tissues, suggesting a potential role for AMID in tumor suppression (17Wu M. Xu L.G. Su T. Tian Y. Zhai Z. Shu H.B. Oncogene. 2004; Google Scholar). However, an earlier report suggested that AMID mRNA levels are up-regulated in colon cancer cell lines, with levels undetectable in various normal tissue samples (10Daugas E. Nochy D. Ravagnan L. Loeffler M. Susin S.A. Zamzami N. Kroemer G. FEBS Lett. 2000; 476: 118-123Crossref PubMed Scopus (398) Google Scholar). During apoptosis, AIF binds to nuclear DNA and is associated with chromatin condensation and DNA fragmentation (18Daugas E. Susin S.A. Zamzami N. Ferri K.F. Irinopoulou T. Larochette N. Prevost M.C. Leber B. Andrews D. Penninger J. Kroemer G. FASEB J. 2000; 14: 729-739Crossref PubMed Scopus (709) Google Scholar). Mutants of AIF that do not bind DNA following targeted mutagenesis of positively charged surface residues also fail to induce apoptosis (19Ye H. Cande C. Stephanou N.C. Jiang S. Gurbuxani S. Larochette N. Daugas E. Garrido C. Kroemer G. Wu H. Nat. Struct. Biol. 2002; 9: 680-684Crossref PubMed Scopus (300) Google Scholar). Electron microscopy of AMID-expressing 293T cells indicates margination and condensation of chromatin (14Wu M. Xu L.G. Li X. Zhai Z. Shu H.B. J. Biol. Chem. 2002; 277: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and points to the possibility that AMID might also interact directly or indirectly with DNA. Herein, we report the biochemical and enzymological properties of purified human AMID. We show that AMID is an NAD(P)H-dependent oxidoreductase and that unusual spectral features of the protein are attributed to the presence of bound 6-hydroxy-FAD, which is associated noncovalently and stoichiometrically with AMID. We demonstrate that AMID is a DNA-binding protein with an apparently sequence-independent mode of interaction, consistent with a proposed role in chromatin condensation. We also establish a link between nicotinamide coenzyme binding, protein conformational change, and the ability to bind DNA that we suggest is of physiological significance in the apoptotic function of AMID. Construction of an AMID Expression Vector—A human cDNA clone containing the AMID open reading frame (IMAGE 3506309; GenBank™/EBI accession number NM_032797) was obtained from Medical Research Council Geneservice (Babraham, UK). The AMID coding sequence was PCR-amplified using Pfu Turbo DNA polymerase (Stratagene) from the cDNA using forward primer 5′-cat atg ggg tcc cag gtc tcg gtg gaa tcg g and reverse primer 5′-gga tcc atg cgc caa gca gtc cgt acg ccc acc, incorporating NdeI and BamHI restriction sites (underlined), respectively. The PCR (50 μl) contained 50 ng of cDNA clone, 20 pmol of each primer, 5 μl of 10× Pfu Turbo PCR buffer, 0.2 mm dNTPs, and 3 units of Pfu Turbo DNA polymerase. Cycling parameters were 30 cycles at 95 °C for 30 s, 63 °C for 1 min, and 72 °C for 2 min. The resultant fragment was then cloned into NdeI- and BamHI-digested pET15b (Novagen) to yield an N-terminally hexahistidine-tagged expression clone of AMID. The N-terminal sequence of the His-tagged version of AMID is His6 followed by a short peptide (SSGLVPRGSH) prior to the AMID start codon. Clones were found to be free of mutations following automated DNA sequencing. Purification of His-tagged AMID—AMID was expressed in E. coli strain HMS174 (DE3) in 2-liter culture flasks containing LB medium and 100 μg/ml ampicillin. Cultures were grown at 37 °C to A600 = 0.6, and the temperature was then reduced to 25 °C before induction at A600 = 0.8 with 1 mm isopropyl β-d -thiogalactopyranoside. Cells were grown overnight with shaking at 240 rpm and harvested by centrifugation at 4000 rpm for 15 min in a Beckman JLA 8.1000 rotor. Cell pellets were resuspended in 50 mm potassium phosphate (pH 8.0) containing 300 mm KCl, 10 mm imidazole, 1 mm benzamidine, and 60 μg/ml phenylmethylsulfonyl fluoride (buffer A) and subjected to six rounds of ultrasonication on ice at maximum power for 20 s with a 2-min rest between pulses. The cell lysate was then passed through a French pressure cell twice at 1000 p.s.i. and clarified by centrifugation at 18,000 rpm for 30 min in a Sorvall SS34 rotor. The cell lysate was loaded onto a nickel-nitrilotriacetic acid (Ni-NTA) column (Novagen) equilibrated with buffer A and washed with 5 column volumes of buffer A and then with 5 column volumes of wash buffer (buffer A with 37.5 mm imidazole and 1 m KCl). The column was washed with 2 volumes of buffer A before eluting the protein with elution buffer (buffer A containing 250 mm imidazole). Protein was concentrated by ultrafiltration using a 30-kDa molecular mass cutoff filter (Centriprep, Millipore Corp.) and then resolved on a Sephacryl S-200 gel filtration column (1 m × 1 cm) pre-equilibrated with buffer A to remove imidazole. After the final gel filtration, purity was assessed by SDS-PAGE. Steady-state Analysis of AMID—The kinetics of AMID-dependent oxidation of NAD(P)H were determined in 50 mm potassium phosphate (pH 8.0) containing 300 mm KCl (assay buffer) using AMID at a final concentration of 9 μm. Rates were determined using ϵ340 = 6210 m–1 cm–1. Steady-state NAD(P)H-dependent reduction of potassium ferricyanide (ϵ420 = 1020 m–1 cm–1), cytochrome c (ϵ550 = 22640 m–1 cm–1), and ferricenium hexafluorophosphate (ϵ300 = 4900 m–1 cm–1) was determined under similar conditions with AMID at a constant final concentration of 35 nm and either with the electron acceptors maintained at saturating levels (≥10 times the Km value) and the NAD(P)H concentration varied (to determine Km values for the pyridine nucleotides) or with the NAD(P)H concentration saturating and the concentrations of the electron acceptors varied (to determine Km values for the electron acceptors). Assays were performed using NADH at 0–2 mm and NADPH at 0–500 μm. Data were collected using a Cary UV50 Bio spectrophotometer (Varian, Inc.) at 30 °C. Rate versus substrate concentration data were analyzed using Origin software (MicroCal). Spectral Binding of Pyridine Nucleotides to AMID—To analyze binding of oxidized pyridine nucleotide coenzymes (NAD+ and NADP+) to AMID, a binding assay based on spectral perturbation was developed. UV-visible spectra for ligand-free AMID (22–28 μm) were recorded in assay buffer (1 ml) at 30 °C on a Cary UV50 Bio spectrophotometer. Thereafter, pyridine nucleotides were titrated into the AMID solutions by progressive additions of small volumes (typically 0.5–2 μl) of the ligand from concentrated nucleotide stocks prepared in assay buffer. Titrations were done in the range of 0–1 mm for both NAD+ and NADP+. Spectra were recorded after each addition of ligand. Following completion of the titrations (i.e. when no further spectral changes were induced), spectral data were base-lined and corrected for dilution with ligand. Difference spectra were generated by subtraction of the spectra for ligand-free AMID from those collected at each point in the titrations. Wavelengths reflecting the maximum overall changes induced (peaks and troughs in the difference spectra) were identified from each titration set. The maximum absorption changes induced (A456 minus A431 for NAD+ and A448 minus A430 for NADP+) were plotted versus the relevant ligand concentration, and data were fitted to a rectangular hyperbola to define the binding constant (Kd) for the interactions between NAD(P)+ and oxidized AMID. Isolation of the AMID Cofactor—The flavin cofactor was extracted from AMID by protein precipitation with 5% trichloroacetic acid, followed by centrifugation at 13,000 rpm in a microcentrifuge at 4 °C for 10 min in the dark. The cofactor was then lyophilized and resuspended in 82% (NH4)2CO3 and 18% methanol (pH 8.5) prior to reverse-phase high performance liquid chromatography (HPLC) purification using a Luma C18 column (1 mm × 15 cm) attached to an Agilent Series 1100 HPLC purifier equipped with a diode array UV spectrophotometer. The sample was eluted isocratically using the resuspension buffer. The flow rate was 0.25 ml/min. The instrument was set to collect fractions with absorbance peaks at 260 and 427 nm. The concentration of the protein-free cofactor (subsequently identified as 6-hydroxy-FAD) was determined using an extinction coefficient of ϵ427 = 22.6 mm–1 cm–1 at 427 nm at pH 9 (20Negri A. Massey V. Williams Jr., C.H. J. Biol. Chem. 1987; 262: 10026-10034Abstract Full Text PDF PubMed Google Scholar). Mass Spectrometry and Analysis of Purified 6-Hydroxy-FAD—The molecular mass of the AMID cofactor was determined using a Q-Tof Ultima global mass spectrometer (Waters Ltd). MALDI mode was used with positive ions analyzed. The matrix used was a mixture of anthranilic acid, nicotinic acid, and ammonium citrate (27.9 mg of anthranilic acid, 12.3 mg of nicotinic acid, 500 μl of acetonitrile, 300 μl of 100 mm ammonium citrate, and 300 μl of distilled water). Ions were selected in the quadrupole; the collision gas used was argon; collision energies were in the range of 15–30 eV; and product ions were analyzed in the time-of-flight analyzer in V-mode. The cofactor sample was dissolved in methanol and "ZipTipped" (using a Millipore C18 ZipTip) prior to mixing in a 1:1 (v/v) ratio with the matrix. Removal of 6-Hydroxy-FAD from AMID and Reconstitution with FAD—Exchange of 6-hydroxy-FAD with FAD was performed essentially as described (21Hefti M.H. Vervoort J. van Berkel W.J. Eur. J. Biochem. 2003; 270: 4227-4242Crossref PubMed Scopus (107) Google Scholar). AMID (∼15 mg of protein) was bound to Ni-NTA resin in buffer A, and 6-hydroxy-FAD was removed by washing with 2 m potassium bromate and 2 m urea until cofactor elution was complete as judged by complete loss of green color and by UV-visible spectrophotometry. FAD was then incorporated into AMID by addition to the column of buffer A containing 10 mm FAD until a prominent yellow-colored band was visible at the top of the column. The resin was then washed extensively with buffer A to remove all unbound FAD, and the protein was eluted from the column in elution buffer as described above. DNA Gel Retention Assay—To examine DNA binding by AMID by gel retention assay, 1 μg of a 100-bp DNA ladder ranging from 100 to 1500 bp (New England Biolabs Inc.) was added to 50 μg of purified AMID or YcgT (a flavoprotein from Bacillus subtilis) and incubated at room temperature for 30 min. Both His-tagged and non-tagged versions of AMID (with the tag removed from AMID by digestion overnight with 5 units of thrombin/mg AMID at 16 °C prior to incubation with DNA) were used in the assay. Protein/DNA mixtures were loaded onto 3% agarose gels containing 0.5 μg/ml ethidium bromide and electrophoresed as described (19Ye H. Cande C. Stephanou N.C. Jiang S. Gurbuxani S. Larochette N. Daugas E. Garrido C. Kroemer G. Wu H. Nat. Struct. Biol. 2002; 9: 680-684Crossref PubMed Scopus (300) Google Scholar). To examine the influence of binding of pyridine nucleotides upon binding of DNA by the gel retention assay, AMID (13 μg) was preincubated with NADP+ (0–17 mm) and DNA (1.5 μg) for 30 min at 25 °C. The amount of AMID used was 10% in excess of the minimum required to retain all DNA in the well in the NADP+-free sample. Thereafter, samples were electrophoresed and analyzed as described above. To examine the ability of 6-hydroxy-FAD-free AMID to bind DNA, apo-AMID was generated on Ni-NTA resin as described above. Apo-AMID was eluted from the column using imidazole as described above. Protein-containing fractions were identified spectrophotometrically (by the absorption maximum at 280 nm), and fractions containing the major proportion of apo-AMID were pooled, concentrated by ultrafiltration, and gel-filtered to remove imidazole as described above. Apoprotein concentration was estimated using an extinction coefficient of ϵ280 = 18,610 m–1 cm–1 (using us.expasy.org/tools/protparam.html). The gel retention assay (as described above for flavin-bound AMID) was repeated with apo-AMID. Assessment of Protein Conformation and AMID Binding to DNA by Circular Dichroism—The interaction of AMID with DNA was analyzed by measuring the far-UV CD spectrum (190–260 nm) of AMID (5 μm) in the presence and absence of DNA (2 mg of a DNA ladder). The CD spectra of AMID and DNA were measured individually, and then a spectrum of an equal volume mixture of AMID and DNA was measured. Differences in CD were computed by comparison of the spectra derived by arithmetical (0.5 times the sum of the individual spectra) and physical (AMID/DNA mixture) addition of the components. Spectra were collected on a Jasco J-715 spectropolarimeter at 25 °C using a 0.02-cm path length quartz cell. Expression, Purification, and Spectral Analysis of AMID— AMID was expressed in E. coli HMS174 (DE3) predominantly as a soluble protein to levels of 0.5–2 mg/g of cell paste. The His-tagged protein was purified to homogeneity by a single column chromatography step on Ni-NTA resin as determined by SDS-PAGE analysis (Fig. 1, inset). The protein had electrophoretic mobility consistent with its predicted mass of 42.7 kDa (including the His tag and intervening amino acids). Preliminary studies of the protein revealed a tendency of AMID to aggregate and precipitate in the presence of organic buffers (e.g. Tris-HCl and MOPS) or at low ionic strength. AMID was found to remain stable in solution at slightly alkaline pH (pH 8.0) in phosphate buffer. For assays and spectroscopic analysis, AMID was maintained in 50 mm potassium phosphate containing 300 mm potassium chloride. As a predicted flavin-binding protein, AMID was expected to have a yellow/orange color. However, AMID was instead found to have a green color, likely indicating a modification of the bound cofactor. The cofactor was released readily from the enzyme by acid precipitation or by heat treatment at 80 °C, indicating noncovalent association with AMID. The UV-visible spectrum of the purified oxidized protein revealed an atypical spectral signature with respect to regular flavoproteins. The absorption spectrum of oxidized AMID (Fig. 1, solid line) has a sharp peak at ∼430 nm and a broad long wavelength feature with a peak at ∼600 nm. Absorption shoulders are present at ∼412 and ∼475 nm. Extended incubation in air produced no noticeable change in the electronic absorption spectrum, indicating that the long wavelength transition did not derive from a neutral semiquinone flavin form. The spectrum is completely different from that of a typical flavoprotein. Fig. 1 shows the spectrum of AMID overlaid with that of the FAD-binding oxidoreductase YcgT from B. subtilis, which contains an FAD cofactor with absorbance maxima at 378 and 459 nm (Fig. 1, dashed line). A review of the literature showed that the oxidized spectrum of AMID resembles that of 6-hydroxy-FAD or 6-mercapto-FAD (20Negri A. Massey V. Williams Jr., C.H. J. Biol. Chem. 1987; 262: 10026-10034Abstract Full Text PDF PubMed Google Scholar, 22Sato K. Nishina Y. Shiga K. J. Biochem. (Tokyo). 2003; 134: 719-729Crossref PubMed Scopus (32) Google Scholar, 23Ghisla S. Massey V. Biochem. J. 1986; 239: 1-12Crossref PubMed Scopus (167) Google Scholar). Only the former occurs naturally in proteins. The complete lack of spectral features typical of a non-modified flavin indicates that AMID binds the modified cofactor stoichiometrically. Subsequent HPLC purification of the modified cofactor confirmed that only one cofactor species is associated with AMID. Characterization of the Modified FAD Cofactor in AMID— The cofactor was released from AMID by heat inactivation of the protein, which led to precipitation of AMID with the cofactor remaining in solution. The cofactor was purified by reverse-phase HPLC as described under "Experimental Procedures." Spectral analysis of the unbound cofactor showed that the peak at 430 nm observed in the protein-bound form was blue-shifted to 427 nm. In acidic solutions (e.g. following precipitation of AMID with trichloroacetic acid), the color of the cofactor changed from green to yellow, and the broad absorption band at 600 nm was lost. This is consistent with protonation of the anionic form of either 6-hydroxy-FAD or 6-mercapto-FAD, resulting in the yellow neutral form (20Negri A. Massey V. Williams Jr., C.H. J. Biol. Chem. 1987; 262: 10026-10034Abstract Full Text PDF PubMed Google Scholar, 24Mayhew S.G. Whitfield C.D. Ghisla S. Schuman-Jorns M. Eur. J. Biochem. 1974; 44: 579-591Crossref PubMed Scopus (63) Google Scholar). In AMID, the cofactor was present in the mesomeric, anionic green form across the pH range of 5.5–8.75 (data not shown). The purified cofactor was subjected to tandem mass spectrometry to determine whether the cofactor was the anionic form of 6-hydroxy-FAD or 6-mercapto-FAD. A sample containing purified FAD was also analyzed as a reference. Following MALDI-TOF mass spectrometry, an ion at m/z 802 was obtained for the modified cofactor relative to one at m/z 786 for FAD (Fig. 2A), indicating the presence of an additional oxygen atom in the AMID cofactor and consistent with its being anionic 6-hydroxy-FAD. To confirm that the oxygenation occurred on the isoalloxazine ring, the precursor ions were selected, fragmented, and analyzed in a second stage of mass spectrometry. The cofactor mass spectrum shown in Fig. 2B indicates the presence of two ions at m/z 259 and 456, which differ by 16 units from the ions at m/z 243 (isoalloxazine ring) and 440 (riboflavin monophosphate) obtained for FAD. This provides further evidence that the isoalloxazine ring is the site of substitution and that the cofactor bound by AMID is 6-hydroxy-FAD. On the basis of the previously determined extinction coefficient of ϵ427 = 22,600 m–1 cm–1 for free 6-hydroxy-FAD at pH 9.0 (19Ye H. Cande C. Stephanou N.C. Jiang S. Gurbuxani S. Larochette N. Daugas E. Garrido C. Kroemer G. Wu H. Nat. Struct. Biol. 2002; 9: 680-684Crossref PubMed Scopus (300) Google Scholar), we calculated an extinction coefficient of ϵ430 = 25,500 m–1 cm–1 for AMID-bound 6-hydroxy-FAD under our experimental conditions at pH 8.0. A number of enzymes have now been purified in which the FAD cofactor exists partially in a form that is hydroxylated at C-6 of the isoalloxazine ring (20Negri A. Massey V. Williams Jr., C.H. J. Biol. Chem. 1987; 262: 10026-10034Abstract Full Text PDF PubMed Google Scholar, 22Sato K. Nishina Y. Shiga K. J. Biochem. (Tokyo). 2003; 134: 719-729Crossref PubMed Scopus (32) Google Scholar, 25Pace C.P. Stankovich M.T. Biochim. Biophys. Acta. 1987; 911: 267-276Crossref PubMed Scopus (20) Google Scholar, 26Tedeschi G. Negri A. Ceciliani F. Ronchi S. Vetere A. D'Aniello G. D'Aniello A. Biochim. Biophys. Acta. 1994; 1207: 217-222Crossref PubMed Scopus (45) Google Scholar, 27Hallberg B.M. Henriksson G. Pettersson G. Divne C. J. Mol. Biol. 2002; 315: 421-434Crossref PubMed Scopus (130) Google Scholar, 28Igarashi K. Verhagen M.F. Samejima M. Schulein M. Eriksson K.E. Nishino T. J. Biol. Chem. 1999; 274: 3338-3344Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In many cases, this yields a catalytically inactive enzyme or one with reduced activity compared with the FAD-containing protein and represents only a small percentage of the total flavin content. However, to our knowledge, AMID is the first protein characterized in which 6-hydroxy-FAD is bound stoichiometrically. Through heat treatment, we were able to remove completely 6-hydroxy-FAD bound to AMID and subsequently to reconstitute the immobilized apoprotein with natural FAD. The spectra of the FAD- and 6-hydroxy-FAD-containing forms are clearly distinct, although both were reduced to their hydroquinone forms upon addition of NADH/NADPH, without obvious formation of any semiquinone species (Fig. 3A). However, for FAD-reconstituted AMID, reduction with both NADH and NADPH leads to the development of long wavelength absorption possibly consistent with development of a charge transfer species between the hydroquinone flavin and the oxidized nicotinamide cofactor (29R
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