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• W1995076640 abstract "Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and p-nitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (α/β)8-barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the β-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg336) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH2-terminal Tyr28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys26, Tyr28, Lys109, and Arg336. Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs. Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and p-nitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (α/β)8-barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the β-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg336) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH2-terminal Tyr28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys26, Tyr28, Lys109, and Arg336. Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs. YqjM from the soil bacterium Bacillus subtilis is a member of the Old Yellow Enzyme (OYE) 1The abbreviations used are: OYE, Old Yellow Enzyme; FMN, flavin mononucleotide; pHBA, p-hydroxybenzaldehyde; pNP, p-nitrophenol; SeMet, selenomethionine; SAD, single-wavelength anomalous diffraction; PETN, pentaerythritol tetranitrate; r.m.s.d., root mean square deviation; MR, morphinone reductase; OPR, 12-oxophytodienoate reductase. family of flavin oxidoreductases as inferred by sequence comparison and detailed biochemical investigations (1Fitzpatrick T.B. Amrhein N. Macheroux P. J. Biol. Chem. 2003; 278: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). OYE itself is an enzyme of significant historical importance, since studies performed with OYE revolutionized the whole field of enzymatic catalysis, thereby highlighting the importance of small vitamin-derived molecules that act as enzymatic cofactors or prosthetic groups. OYE was first isolated in 1932 from brewers' bottom yeast by Warburg and Christian (2Warburg O. Christian W. Naturwissenschaften. 1932; 20: 688Crossref Scopus (64) Google Scholar), and was found to contain a non-covalently bound flavin mononucleotide (FMN). Hugo Theorell (3Theorell H. Biochem. Z. 1935; 275: 344-346Google Scholar, 4Theorell H. Nygaard A.P. Arkiv. Kemi. 1954; 7: 205-209Google Scholar, 5Theorell H. Nygaard A.P. Acta Chem. Scand. 1954; 8: 1104-1105Crossref Google Scholar) then went on to demonstrate that the reaction catalyzed by OYE is strictly dependent on this vitamin B2 (riboflavin) derivative. Consequently he postulated one of the main paradigms in biochemistry: neither apoprotein nor cofactor alone can catalyze the corresponding redox reaction, and only both together yield the active holoenzyme. Intensive studies on OYE spanning more than 70 years contributed greatly to enhancing the general understanding of flavoenzyme catalysis. More specifically, in the so-called reductive half-reaction, NADPH was established to serve as reductant of FMN (6Abramovitz A.S. Massey V. J. Biol. Chem. 1976; 251: 5327-5336Abstract Full Text PDF PubMed Google Scholar, 7Massey V. Schopfer L.M. J. Biol. Chem. 1986; 261: 1215-1222Abstract Full Text PDF PubMed Google Scholar). The cofactor is converted back to its original form in the oxidative half-reaction where flavin then acts as an electron donor reducing the substrate during this step. Despite detailed investigations of OYE, the natural substrates have remained elusive for a long time. Several studies, however, showed that quinones as well as a large variety of α,β-unsaturated aldehydes and ketones function as efficient oxidants of flavin, leading to the reduction of the olefinic bond (8Stott K. Saito K. Thiele D.J. Massey V. J. Biol. Chem. 1993; 268: 6097-6106Abstract Full Text PDF PubMed Google Scholar, 9Vaz A.D.N. Chakraborty S. Massey V. Biochemistry. 1995; 34: 4246-4256Crossref PubMed Scopus (133) Google Scholar). Recently, two-hybrid screen studies have suggested a possible role of one OYE isozyme, OYE2 from Saccharomyces cerevisiae, in controlling the redox state of actin and thereby maintaining the proper plasticity of the actin cytoskeleton (10Haarer B.K. Amberg D.C. Mol. Biol. Cell. 2004; 15: 4522-4531Crossref PubMed Scopus (49) Google Scholar). In fact, reactive oxygen species generated after oxidative stress have been shown to react with the actin sulfhydryl groups of Cys285 and Cys374. A disulfide bond is then formed, resulting in cross-linking of actin filaments (10Haarer B.K. Amberg D.C. Mol. Biol. Cell. 2004; 15: 4522-4531Crossref PubMed Scopus (49) Google Scholar). This actin oxidation might be prevented by the action of OYE2 that seems to function as a flavin disulfide reductase. However, OYE3, another OYE isozyme from S. cerevisiae, failed to interact with actin, and therefore general functional conclusions for the whole OYE family cannot be drawn. The findings from Haarer and Amberg (10Haarer B.K. Amberg D.C. Mol. Biol. Cell. 2004; 15: 4522-4531Crossref PubMed Scopus (49) Google Scholar) do, however, confirm former observations that OYE serves as a detoxification enzyme in the antioxidant defense system and disarms a variety of reactive oxidative species. Among others, this has been also inferred by Fitzpatrick et al. (1Fitzpatrick T.B. Amrhein N. Macheroux P. J. Biol. Chem. 2003; 278: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) who demonstrated that treatment with hydrogen peroxide or nitro compounds such as TNT induces the expression of YqjM, an OYE homolog, from B. subtilis. Data base searches have indicated that YqjM displays a high degree of sequence similarity to OYE1 from Saccharomyces carlsbergensis and its homologs (1Fitzpatrick T.B. Amrhein N. Macheroux P. J. Biol. Chem. 2003; 278: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Pseudomonas putida XenA, a xenobiotic reductase, appears to be the closest homolog exhibiting 40% identity. Biochemical analysis of YqjM has shown that the enzyme shares some important common features with members of the OYE family (1Fitzpatrick T.B. Amrhein N. Macheroux P. J. Biol. Chem. 2003; 278: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). For example, YqjM binds the FMN cofactor non-covalently and reduces the flavin in the reductive half-reaction at the expense of NADPH. Like other members of the family, YqjM transfers electrons from the reduced flavin to the double bond of a variety of α,β-unsaturated carbonyl compounds and accepts also electrophilic xenobiotics such as nitroglycerin, N-ethylmaleimide, and trinitrotoluene as substrates for the oxidative half-reaction. Finally, phenolic compounds such as p-hydroxybenzaldehyde (pHBA) bind tightly to this enzyme and form charge transfer complexes due to the overlapping π-electrons of the electron-rich phenolate ligand and the electron-deficient flavin isoalloxazine ring (1Fitzpatrick T.B. Amrhein N. Macheroux P. J. Biol. Chem. 2003; 278: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). However, on comparing YqjM with OYE, several important differences are apparent. For example, photoreduction of YqjM leads directly to the fully reduced state; whereas in the yeast and plant homologs the red flavin semiquinone is kinetically stabilized (1Fitzpatrick T.B. Amrhein N. Macheroux P. J. Biol. Chem. 2003; 278: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). A further unique feature of YqjM is attributed to its oligomerization state. While all other OYE homologs are either monomeric or dimeric enzymes, YqjM is the only known family member that functions as a homotetramer (11Fitzpatrick T.B. Auweter S. Kitzing K. Clausen T. Amrhein N. Macheroux P. Protein Expression Purif. 2004; 36: 280-291Crossref PubMed Scopus (25) Google Scholar) suggesting the evolution of particular residues involved in the interface formation between the protomers. This implies that the different modes of self-assembly within the OYE family may be correlated with a functional diversity. Finally, sequence comparisons have suggested significant changes in the active site of the enzyme. For example, Thr37 of OYE, which regulates the redox potential of the FMN cofactor (12Xu D. Kohli R.M. Massey V. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3556-3561Crossref PubMed Scopus (61) Google Scholar), is replaced by Cys26 in YqjM, whereas the OYE-Tyr375, which is essential for substrate and inhibitor binding, has no conserved counterpart in YqjM. The three-dimensional structures from OYE (13Fox K.M. Karplus P.A. Structure. 1994; 2: 1089-1105Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) and several of its homologs (14Barna T. Messiha H.L. Petosa C. Bruce N.C. Scrutton N.S. Moody P.C.E. J. Biol. Chem. 2002; 277: 30976-30983Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 15Barna T.M. Khan H. Bruce N.C. Barsukov I. Scrutton N.S. Moody P.C.E. J. Mol. Biol. 2001; 310: 433-447Crossref PubMed Scopus (87) Google Scholar, 16Breithaupt C. Strassner J. Breitinger U. Huber R. Macheroux P. Schaller A. Clausen T. Structure. 2001; 9: 419-429Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 17Malone T.E. Madson S.E. Wrobel R.L. Jeon W.B. Rosenberg N.S. Johnson K.A. Bingman C.A. Smith D.W. Phillips Jr., G.N. Markley J.L. Fox B.G. Proteins. 2005; 58: 243-245Crossref PubMed Scopus (18) Google Scholar) have been elucidated and point to the fundamental fold of YqjM. However, to determine the molecular basis of the unique properties of YqjM, we solved the structures of the oxidized and reduced holoenzyme and of complexes with the inhibitors pHBA and p-nitrophenol (pNP), respectively. The data presented here confirm that YqjM shares the overall fold of the OYE family. Also it demonstrates unequivocally that YqjM is in fact the first characterized representative of a new class of OYE homologs showing fundamental differences to the classical OYE enzymes. Materials—All materials used were of analytical grade. All chemicals were purchased from Sigma. Chromatographic columns were from Amersham Biosciences. Expression and Purification of the Native YqjM—The construct of pET21a (Novagen) with the open reading frame of yqjM was expressed and purified as described in Ref. 1Fitzpatrick T.B. Amrhein N. Macheroux P. J. Biol. Chem. 2003; 278: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar with the following modifications: purification was performed with an AEKTA-FPLC system from Amersham Biosciences using a HiPrep 16/10 DEAE FF column in the anion exchange step and a HiPrep 16/10 Phenyl FF column for the hydrophobic interaction chromatography. Additionally, a final gel filtration step was introduced using a HiLoad Superdex 200 gel filtration column equilibrated with 10 mm HEPES, pH 7.5, 150 mm potassium chloride, and 4 mm dithiothreitol. Fractions containing YqjM were pooled and concentrated to 10 mg/ml by ultrafiltration using Centriprep-30 (Amicon). Selenomethionine-labeled YqjM (SeMet-YqjM) was expressed from the met-Escherichia coli strain B834(DE3). The purification of SeMet-YqjM followed the same protocol as for the native enzyme. Protein Crystallization—Crystallization trials were performed at 19 °C using the sitting drop vapor diffusion method. After intensive screening, well diffracting crystals were obtained by mixing 3 μl of protein with 1.5 μl of a crystallization solution containing 0.1 m Tris/HCl, pH 8.5, 25% polyethylene glycol 3500, and 0.2 m sodium chloride. Crystal trials were set up in cryschem plates with a reservoir volume of 400 μl. After 5-7 days, crystal plates appeared that had the characteristic yellow color of the oxidized, enzyme-bound FMN cofactor. They belonged to the orthorhombic space group C2221 with unit cell dimensions of a = 51.5 Å, b = 185.2 Å, and c = 169.8 Å and contained two molecules per asymmetric unit corresponding to a solvent content of 54%. However the above-mentioned condition did not yield crystals of the SeMet-YqjM derivative. After a new screening round, a similar crystallization condition was found containing 0.1 m Tris/HCl, pH 8.5, 25% polyethylene glycol 3500, 0.2 m lithium sulfate, and 0.01 m strontium chloride. YqjM-SeMet crystals complexed with pHBA and pNP were obtained by cocrystallization using a ligand concentration of 3 mm. The presence of either pHBA or pNP resulted in a color change of the yellow YqjM crystals toward red. Reduction of YqjM crystals was achieved by soaking crystals of the oxidized enzyme in 100 mm NADPH until they turned colorless (approximately after 10 min). For cryo-measurements, crystals were transferred from the crystallization drop to the mother liquor supplied with 15% 2-methyl-2,4-pentanediol as cryo-protectant and rapidly frozen in a 100 K stream of nitrogen gas. Data Collection and Structure Determination—Initial attempts to overcome the crystallographic phase problem by molecular replacement using several search models derived from other members of the OYE family failed. Therefore experimental phase information was required to determine the structure of YqjM. Phase information could be obtained by collecting a single-wavelength anomalous diffraction (SAD) data set from a single SeMet-YqjM crystal. SAD data of SeMet-YqjM and diffraction data of the complexes with pHBA and pNP were collected at the BW6 beamline at the Deutsches Elektronen Synchroton (Hamburg, Germany) using a MarCCD detector. Crystals of the oxidized and reduced YqjM were measured in house using a MarResearch imaging plate. All diffraction data were processed and scaled with the programs DENZO and SCALEPACK from the CCP4 package (18Bailey S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (42) Google Scholar). For the SAD experiment, an x-ray absorption spectrum was recorded in the vicinity of the selenium edge. Diffraction data up to 1.8 Å resolution were then collected at a wavelength corresponding to the peak of this spectrum (f ″ maximum, 0.9792 Å). Subsequently, difference Fourier analyses performed with SHELX (19Sheldrick G.M. Schneider T.R. Macromolecular Crystallography, Part B. 1997; 277: 319-343Crossref Scopus (1886) Google Scholar, 20Sheldrick G.M. Z. Kristallogr. 2002; 217: 644-650Crossref Scopus (360) Google Scholar) enabled us to identify all 10 of the theoretical selenium sites. Refinement of heavy atom parameters and phase calculation were conducted with SHARP (21de La Fortelle E. Bricogne G. Carter Jr, C.W. Sweet R.M. Macromolecular Crystallography, Part A. 276. Academic Press, New York1997: 472-494Google Scholar) leading to a figure of merit of 0.68 for data between 20 and 1.82 Å resolution. Solvent flattening was performed with SOLOMON (22Abrahams J.P. Leslie A.G.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1142) Google Scholar) and resulted in an electron density map of excellent quality, in which the entire backbone model of YqjM was built in with ARP/wARP (23Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2564) Google Scholar). Energy-restrained crystallographic refinement against the 1.3 Å resolution data set was carried out with maximum likelihood algorithms implemented in CNS (24Brünger A.T. Adams P.D. Clore G.M.L.D.W. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar) using the protein parameters of Engh and Huber (25Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2545) Google Scholar). Refinement, model rebuilding with the program O (26Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar), and water incorporation proceeded smoothly via rigid body, positional, and later B-factor optimization. The entire structure was checked using simulated annealing composite omit maps. Finally, after the addition of the FMN cofactor molecules, the refinement converged at a R-factor of 18.9% (Rfree = 20.8%). All residues of YqjM could be traced in the electron density map and exhibited good stereochemistry (Table I). Also the FMN cofactor molecules were well defined by electron density as well as four sulfate ions, two of which were bound within the active sites and the other two stabilizing crystal lattice contacts. In the Ramachandran plot, 91% of the 674 residues were found in the most favorable, 8.6% in the favorable, 0.3% in the generously allowed, and no residue was found in the disallowed region. The data collection, phasing, and refinement statistics are summarized in Table I. All parameter and topology files were created with the program XPLOR2D (27Kleywegt G. Jones T. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 1119-1131Crossref PubMed Scopus (496) Google Scholar).Table ICrystallographic data collection and refinement statisticsOxidized YqjMReduced YqjMYqjM/pHBAYqjM/pNPData collectionUnit cellC2221C2221C2221C2221Cell constants (Å)a = 51.5a = 51.2a = 51.2a = 51.4b = 185.2b = 184.6b = 184.7b = 184.8c = 169.8c = 168.9c = 169.2c = 172.0Resolution (Å)aValues for the highest resolution shells are given in parentheses8-1.30 (1.32-1.30)10-1.70 (1.73-1.70)10-1.85 (1.88-1.85)8-1.40 (1.42-1.40)No. of unique reflections183,799 (8068)86,971 (4275)66,570 (3211)144,550 (7312)Redundancy2.35 (2.09)2.96 (2.93)2.71 (2.65)2.82 (2.85)Completeness (%)92.7 (82.2)99.0 (98.7)97.3 (94.8)96.3 (97.5)Rsym (%)bRsym = ΣhklΣj|Ij - 〈I〉|/ΣhklΣjI j(hkl), where 〈I〉 is the mean intensity of reflection hkl4.3 (34.2)3.3 (14.6)8.5 (22.1)7.5 (14.1)I/σ(I)20.6 (2.0)26.5 (7.0)16.6 (4.6)18.2 (4.9)RefinementRcryst/Rfree (%)cRcryst = Σhkl‖Fobs| - |Fcalc‖/Σhkl|Fobs|, where Fobs and Fcalc are the observed and calculated structure factor amplitude for reflections hkl included in the refinement, respectively. Rfree is the R-value for 5% of randomly selected reflections excluded from refinement18.9/20.817.9/19.918.2/20.419.6/21.2No. of atoms (mean B (Å2))Protein5274 (18.3)5274 (20.4)5274 (15.0)5274 (13.6)FMN62 (12.8)62 (17.7)62 (10.0)62 (9.6)Sulfate20 (47.7)10 (57.6)10 (58.7)Ligand18 (15.5)30 (23.8)Water842 (32.7)669 (33.8)622 (26.7)642 (25.6)r.m.s.d. bond length (Å)0.0140.0060.0050.006r.m.s.d. bond angles (°)1.551.281.281.29r.m.s.d. B-factors (Å2)2.132.282.222.19a Values for the highest resolution shells are given in parenthesesb Rsym = ΣhklΣj|Ij - 〈I〉|/ΣhklΣjI j(hkl), where 〈I〉 is the mean intensity of reflection hklc Rcryst = Σhkl‖Fobs| - |Fcalc‖/Σhkl|Fobs|, where Fobs and Fcalc are the observed and calculated structure factor amplitude for reflections hkl included in the refinement, respectively. Rfree is the R-value for 5% of randomly selected reflections excluded from refinement Open table in a new tab Graphical presentations were made using the programs MOLSCRIPT (28Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), RASTER3D (29Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2857) Google Scholar), DINO (www.dino3d.org) and PYMOL (www.pymol.org). Sequence Alignment and Phylogenetic Reconstruction—Amino acid sequences were retrieved by BLAST searches using the Swiss-Prot data base and aligned using the web-based program MULTALIN (available at prodes.toulouse.inra.fr/multalin/multalin.htmlI). To perform a phylogenetic analysis, the amino acid sequences were aligned with ClustalW and the result saved in the NEXUS format. The program PAUP* 4.0b10 was used to analyze the phylogenetic relationships of the OYE/YqjM homologs. Enzyme Assays—The apparent steady state kinetic constants were determined as described in Ref. 1Fitzpatrick T.B. Amrhein N. Macheroux P. J. Biol. Chem. 2003; 278: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar. Briefly, the assays were performed in 0.1 m Tris/HCl, pH 7.5, at 25 °C in the presence of an oxygen-consuming system and followed by the oxidation of β-NADPH (100 μm) at 340 nm in a Uvikon spectrophotometer while systematically varying the substrate concentration. Overall Structure—The x-ray structure of native oxidized YqjM was solved by SAD and refined at 1.3 Å resolution to an R-factor of 18.9% (Table I). The monomer of YqjM is comprised of one compact domain representing the frequently observed (α/β)8-barrel or TIM barrel fold, where a cylindrical core of eight twisted β-strands is surrounded by eight helices (Fig. 1). Similar to other (α/β)8-structures, all of the turns at the NH2-terminal end of the barrel are composed of only three or four residues, while the loops at the COOH-terminal end are much longer and build up the active site. The numbering of these loops refers to the preceding β-strands, e.g. loop L6 follows strand 6 (see also Fig. 1B for nomenclature of secondary structure). The COOH-terminal loops range in length from 5 to 31 residues and harbor additional secondary structural elements, namely helices αB, αC, αD, and αE and strands βC and βD. In addition, YqjM has a 310-helix αA and two short antiparallel β-strands prior to β-strand 1 that close the barrel on its NH2-terminal side. A short 310-helix αF is present after the COOH-terminal helix 8. The overall structure of YqjM strongly resembles the structures of other OYE homologs (13Fox K.M. Karplus P.A. Structure. 1994; 2: 1089-1105Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 14Barna T. Messiha H.L. Petosa C. Bruce N.C. Scrutton N.S. Moody P.C.E. J. Biol. Chem. 2002; 277: 30976-30983Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 15Barna T.M. Khan H. Bruce N.C. Barsukov I. Scrutton N.S. Moody P.C.E. J. Mol. Biol. 2001; 310: 433-447Crossref PubMed Scopus (87) Google Scholar, 16Breithaupt C. Strassner J. Breitinger U. Huber R. Macheroux P. Schaller A. Clausen T. Structure. 2001; 9: 419-429Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 17Malone T.E. Madson S.E. Wrobel R.L. Jeon W.B. Rosenberg N.S. Johnson K.A. Bingman C.A. Smith D.W. Phillips Jr., G.N. Markley J.L. Fox B.G. Proteins. 2005; 58: 243-245Crossref PubMed Scopus (18) Google Scholar). The closest structural relative was found to be pentaerythritol tetranitrate (PETN) reductase from Enterobacter cloacae where 278 residues could be aligned with a root mean square deviation (r.m.s.d.) value of 1.29 Å, followed by morphinone reductase (MR) from P. putida (286 aligned Cα with an r.m.s.d. of 1.41 Å), 12-oxophytodienoate reductase (OPR) 1 from Lycopersicon esculentum (286 Cα, 1.46 Å) and OYE (282 Cα, 1.48 Å). A common feature among these oxidoreductases is the NH2-terminal β-hairpin structure that lids the β-barrel. Except for the loops L4 and L8, all COOH-terminal loops of the β-barrel adopt conformations that are distinct in the OYE flavoproteins. In general, these loops form the active site and provide substrate as well as reaction specificity. While other members of the OYE family show some similarities in the respective loop structures, YqjM exhibits unique structural features in the corresponding active site region. Especially the loops L3, L5, and L6 adopt different conformations and vary considerably in length. For example, the crystal structures of OPR1 (16Breithaupt C. Strassner J. Breitinger U. Huber R. Macheroux P. Schaller A. Clausen T. Structure. 2001; 9: 419-429Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), PETN reductase (15Barna T.M. Khan H. Bruce N.C. Barsukov I. Scrutton N.S. Moody P.C.E. J. Mol. Biol. 2001; 310: 433-447Crossref PubMed Scopus (87) Google Scholar), and MR (14Barna T. Messiha H.L. Petosa C. Bruce N.C. Scrutton N.S. Moody P.C.E. J. Biol. Chem. 2002; 277: 30976-30983Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) have shown that the loop L3 forms a part of the hydrophobic active site tunnel and seems to play a role in substrate discrimination. In these enzymes loop L3 is extended and folds as an additional two stranded β-sheet, which covers half of the active site. In contrast, the corresponding loop of YqjM is folded as a short 310-helix (αC), which is packed on the wall of the β-barrel. As a result, the YqjM active site is hardly protected by any loop structure and thus is remarkably wide and accessible for potential substrate molecules. Oligomerization State and Oligomer Interfaces—The oligomeric state of YqjM observed in the crystal is the same as in solution (11Fitzpatrick T.B. Auweter S. Kitzing K. Clausen T. Amrhein N. Macheroux P. Protein Expression Purif. 2004; 36: 280-291Crossref PubMed Scopus (25) Google Scholar). It is a tetrameric enzyme, in which the subunits are organized with 222 (D2) point group symmetry (Fig. 2). The tetramer has overall dimensions of ∼84 × 92 × 47 Å3 and resembles a four-petaled cloverleaf with a central hole. The subunits are arranged in such a way that their active sites, defined by the bound FMN molecule, open up in different directions to solvent but are connected with each other by the central hole. In the C2221 crystal form, two of the 2-fold rotation axis coincide with the crystallographic a and b axis, respectively. The two monomers, which are present in the asymmetric unit (named A and B, their crystallographic symmetry mates C and D), are essentially identical and are related by a non-crystallographic 2-fold axis. As discussed below, the YqjM tetramer is organized as a dimer of active dimers, i.e. AB and CD, respectively. The (α/β)8-domains of the corresponding monomers interact extensively around the dimer 2-fold axis such that their barrel axis are approximately parallel to each other but open up into opposite directions. The dimer axis passes between helix 1 of the two monomers, where both elements come close together. Additionally, interaction of helix αE and the entire COOH terminus including helix αF with the neighboring counterpart contribute considerably to the binding energy of both subunits. The interaction sites α1/α1* and the αF/αF* are arranged in a perpendicular fashion and are stabilized by the formation of a large hydrophobic cluster involving residues Met27, Pro39, Phe40, Met42, Ala43, Ile46, Ile50, Pro314, and Phe315. This cluster is enclosed by a pronounced hydrogen bonding network formed by Ser29, His44, Arg48, Leu311, Arg312, Gln333, Tyr334, Arg336, Gly337, and Trp338. Most notably, the COOH-terminal end is directed by the latter interactions toward the active site of the neighboring subunit. One of the COOH-terminal residues Arg336* (the asterisk denotes the neighboring subunit) is even protruding as an arginine finger into the adjacent active site, where it forms a part of the substrate binding pocket (near the flavin dimethyl benzene ring). Thus the shared active site strongly suggests that the dimer is the active unit of YqjM. Its formation shields 1241 Å2 per monomer (9% of the entire monomer surface), while the other monomer-monomer contacts are considerably smaller. The contact area between A and C comprises 5% of the accessible monomer surface and the monomers A and D do not interact at all with each other. The contacts between the dimers AB and CD are mediated by the helix-loop-helix motifs of helices 6 and 7 and comprise mainly hydrophobic interactions involving Val260, Phe261, Pro262, Tyr264, Val266, Met285, and Met291. Th" @default.
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• W1995076640 date "2005-07-01" @default.
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• W1995076640 title "The 1.3 Å Crystal Structure of the Flavoprotein YqjM Reveals a Novel Class of Old Yellow Enzymes" @default.
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