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J. Mol. Biol. (2006) 361, 140–152 Xenobiotic Reductase A in the Degradation of Quinolineby Pseudomonas putida 86: PhysiologicalFunction, Structure and Mechanism of8-Hydroxycoumarin Reduction Julia J. Griese1 †, Roman P. Jakob1 †, Stephan Schwarzinger2and Holger Dobbek1⁎ A continuous evolutionary pressure of the biotic and abiotic world has led to the development of a diversity of microbial pathways to degrade and Universität Bayreuth, biomineralize aromatic and heteroaromatic compounds. The heterogeneity Universitätsstrasse 30, of compounds metabolized by bacteria like Pseudomonas putida indicates the 95447 Bayreuth, Germany large variety of enzymes necessary to catalyse the required reactions.
Quinoline, a N-heterocyclic aromatic compound, can be degraded by Lehrstuhl Biopolymere, microbes along different pathways. For P. putida 86 quinoline degradation by Universität Bayreuth, Germany the 8-hydroxycoumarin pathway has been described and several inter-mediates were identified. To select enzymes catalysing the later stages of the8-hydroxycoumarin pathway P. putida 86 was grown with quinoline. TheFMN-containing enzyme xenobiotic reductase A (XenA) was isolated andanalysed for its reactivity with intermediates of the 8-hydroxycoumarinpathway. XenA catalyses the NADPH-dependent reduction of 8-hydro-xycoumarin and coumarin to produce 8-hydroxy-3,4-dihydrocoumarin and3,4-dihydrocoumarin, respectively. Crystallographic analysis of XenA aloneand in complex with the two substrates revealed insights into themechanism. XenA shows a dimeric arrangement of two (β/α)8 barreldomains each binding one FMN cofactor. High resolution crystal structuresof complexes with 8-hydroxycoumarin and coumarin show different modesof binding for these molecules in the active site. While coumarin is ideallypositioned for hydride transfer from N-5 of the isoalloxazine ring to C-4 ofcoumarin, 8-hydroxycoumarin forms a non-productive complex withoxidised XenA. Orientation of 8-hydroxycoumarin in the active site appearsto be dependent on the electronic state of the flavin.
We postulate that XenA catalyses the last step of the 8-hydroxycoumarin pathway before the heterocyclic ring is hydrolysed to yield 3-(2,3-dihydroxyphenyl)-propionic acid. As XenA is also found in P. putida strainsunable to degrade quinoline, it appears to have more than one physiologicalfunction and is an example of how enzymes with low substrate specificitycan help to explain the variety of degradation pathways possible.
2006 Published by Elsevier Ltd.
Keywords: xenobiotic reductase; flavin; quinoline; Pseudomonas putida 86; *Corresponding author Old Yellow Enzyme † J.J.G. and R.P.J. contributed equally to the work.
Present addresses: J.J. Griese, Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Germany; R.P. Jakob, Lehrstuhl Biochemie, Universität Bayreuth, Germany.
Abbreviations used: XenA, xenobiotic reductase A; OYE, Old Yellow Enzyme; NG, nitroglycerin; PETN, pentaerythritol tetranitrate.
E-mail address of the corresponding author: 0022-2836/$ - see front matter 2006 Published by Elsevier Ltd.

Structure and Function of XenA xycoumarin 4 and 3-(2,3-dihydroxyphenyl)-propionic acid 5 (The enzymes producing Approximately 15 × 106 tons of coal tar are and converting these two compounds are not produced worldwide every year and are the source known. Quinoline degrading P. putida strains do for condensed aromatics and N-heteroaromatics like not grow with coumarin as a sole source of carbon and energy. However, when bacteria that were have developed strategies to metabolize naturally grown with quinoline are incubated with coumarin occurring aromatic compounds and xenobiotics and 6, 3-(2-hydroxyphenyl)-propionic acid 7 accumu- as they are able to biomineralize potentially toxic latesCharacteristic reactions of coumarins are compounds, they can be exploited in the bioreme- additions to the C-3/C-4 double bond and the diation of polluted soils and Quinoline is a nucleophilic opening of the lactone group.Addi- ubiquitous, soluble, heteroaromatic pollutant with tion of a hydride and a proton to the C-3/C-4 double cancerogenic propertiSeveral bacterial species bond and hydrolysis of the lactone explains the are capable of degrading quinoline and can grow observed products 3-(2,3-dihydroxyphenyl)-propio- with quinoline or its derivatives as sole sources of nic acid 5 and 3-(2-hydroxyphenyl)-propionic acid 7 carbon, nitrogen and energy. A unique pathway and defines the activity of the enzymes able to exists in Pseudomonas putida 86 that was isolated convert 8-hydroxycoumarin 4 and coumarin 6 from soil near a coal tar factory (Rütgerswerke, Castrop-Rauxel, Germany) by quinoline enrichment acid 5 can be further degraded to intermediates of culture Within the 8-hydroxycoumarin the tricarboxylic acid 3-(2-hydroxyphenyl)- pathway of P. putida 86 the N-heterocyclic ring of propionic acid 7 cannot, explaining why P. putida 86 quinoline 1 is cleaved preferentially to form 8- does not grow with coumarin as carbon source hydroxycoumarin 4. Four intermediates (2−5) have Bacterial enzymes known to catalyse the reduc- been identified in the 8-hydroxycoumarin path- tion of the olefinic bond of α,β-unsaturated carbonyl way,and the enzymes that catalyse the first two compounds including ketones and esters are a group steps have been investigated (The of enzymes called xenobiotic reductaseThese molybdo-iron-sulfur flavoprotein quinoline oxido- enzymes constitute a bacterial subgroup of the Old reductase (QOR) hydroxylates quinoline 1 at C-2 to Yellow Enzyme (OYE) family and are monomeric, yield 1H-2-oxoquinoline 2The multicomponent homodi- or tetrameric, NAD(P)H-dependent, FMN- enzyme 1H-2-oxoquinoline 8-monooxygenase cata- containing oxidoreductases with a subunit size of lyses the NADH-dependent second hydroxylation ∼40 kDa. Like for OYE itself, the physiological reaction at C-8 to yield 1H-8-hydroxy-2-oxoquino- oxidant and with it the physiological function is line 3The next two intermediates are 8-hydro- unknown for almost all members of the OYE family.
Figure 1. Degradation of quinoline and coumarin by P. putida 86. Intermediates of the 8-hydroxycoumarin pathway are depicted.The first reaction of the pathway is labelled with A and is catalysed by quinoline 2-oxidoreductase.The secondreaction, labelled with B, is catalysed by 2-oxoquinoline Triple arrows indicate that the reactionsequence between the different intermediates is unclear and multiple steps could be involved. In the following the names ofall substances depicted are given: (1) quinoline, (2) 1H-2-oxoquinoline, (3) 1H-8-hydroxy-2-oxoquinoline, (4) 8-hydroxycoumarin, (5) 3-(2,3-dihydroxyphenyl)-propionic acid, (6) coumarin, and (7) 3-(2-hydroxyphenyl)-propionic acid.
Structure and Function of XenA Recently, some enzymes of the OYE family were substrate 2-cyclohexenone was used as positive implicated in antioxidant defense by detoxification controQuinoline, 2-quinolinol, 4-quinolinol and of reactive electrophilic substrates.,Xenobiotic 2,8-quinolinediol did not oxidise reduced XenA reductases catalyse three types of reactions whereby (data not shown). Coumarin and 8-hydroxycou- they accept a broad range of electrophilic, xenobiotic marin were reduced by XenA, albeit more than compounds: (i) reduction of the olefinic bond of α,β- tenfold slower than 2-cyclohexenone. XenA has an unsaturated carbonyl compounds like 2-cyclohex- apparent turnover number of 8 s−1 with 2-cyclohex- enone; (ii) reductive denitration of aliphatic nitro enone, 0.4 s−1 with coumarin and 0.2 s−1 with 8- esters like nitroglycerin (NG) or pentaerythritol hydroxycoumarin (). XenA shows an ∼four- tetranitrate (PETN); (iii) reduction of nitroaromatic fold preference for NADPH over NADH. It will also compounds like 2,4,6-trinitrotoluene.
reduce molecular oxygen with a specific activity of Xenobiotic reductase A (XenA) was originally 0.2 unit/mg at oxygen saturation of the buffer identified in a P. putida strain that had been isolated (263 μM at 25 °C, 1 bar). Therefore, assays were from NG-contaminated This strain, P. putida II- conducted under anaerobic conditions. Apparent B, grows with NG as sole nitrogen source because values for the Michaelis constant (KM) for all three XenA denitrates NG. XenA also reduces 2-cyclohex- substrates are in the same range with 33(±4) μM for 2- enone to cyclohexanone and the nitro groups of cyclohexenone, 23(±2) μM for coumarin and 34(±3) trinitrotoluene to amino groups.XenA is an μM for 8-hydroxycoumarin. The apparent KM value NADPH-dependent intracellular enzyme with a for NADPH, determined with 2-cyclohexenone as subunit size of 39.8 kDa (for the apoprotein). It oxidative substrate is 61(±7) μM ( contains one molecule of FMN per subunit. Based on Real-time NMR spectra were recorded to deter- gel filtration chromatography it was suggested to be mine the products of the reactions with 8-hydroxy- monomeric in solution.
coumarin and coumarin For comparison, To investigate the late steps of the 8-hydroxycou- reference spectra of educts (8-hydroxycoumarin, marin pathway, we were searching for enzymes able coumarin, and NADPH) and possible products to convert 8-hydroxycoumarin 4 and coumarin 6, explaining the reactive principles of the pathway nic acid and NADP+) were measured. The newly ). We found that XenA is expressed in P.
appearing resonances agreed well with the forma- putida 86 grown with quinoline as sole carbon, tion of 8-hydroxy-3,4-dihydrocoumarin and 3,4- nitrogen and energy source. Ligand titration and dihydrocoumarin, respectively, and NADP+, while crystallographic analysis of XenA showed its ability the resonances disappearing corresponded to 8- to bind 8-hydroxycoumarin 4 and coumarin 6 in hydroxycoumarin and coumarin, respectively, and solution and the crystalline state. Kinetic analysis NADPH. Approximate rate constants derived for the proved its ability to reduce the C-3/C-4 double time-dependent evolution of selected resonances bond of 8-hydroxycoumarin 4 and coumarin 6.
show similar rates for NADP+ and 8-hydroxy-3,4-dihydrocoumarin production and for NADPH and Results and Discussion 8-hydroxycoumarin decay (data not shown). Thenearly complete decay of resonances of the eductsshows that the equilibria of both reactions (8- Expression and purification of XenA hydroxycoumarin and coumarin) are on the side ofproducts No resonances indicating a Escherichia coli BL21(DE3) (Stratagene) was used hydrolysis of the lactone group were detectable for overexpression of recombinant XenA. Four during the 5 h of the real time NMR experiment.
column chromatography steps were used to purifymore than 30 mg XenA with a purity exceeding 95%per 10 g of cells (wet weight). The protein solutionwas reconstituted with FMN before the gel filtration Table 1. Steady-state kinetic properties of xenobiotic step because E. coli produces XenA to ∼70% in the apoform, as approximated by the ratio of absorbance at 280 nm to 464 nm of the protein before and after reconstitution. The reconstituted recombinant pro- tein displayed an absorbance ratio A280/A464 of 7.5.
The same ratio was determined for XenA purified using the same strategy from P. putida 86, indicating that reconstitution with FMN was complete.
Apparent kinetic constants were derived from steady-state kineticanalyses. The assays were performed at 25 °C in 1 ml of 50 mM Reactivity of XenA potassium phosphate buffer (pH 7.0). The compounds 7-hydro-xycoumarin, quinoline, 2-quinolinol, 4-quinolinol, 2,8-quinoline-diol, 2,4-dinitrophenol, picric acid and metronidazole were also To investigate whether XenA participates in the 8- tested, but no activity was detected.
hydroxycoumarin pathway, its ability to catalyse the a Reductive substrate: 150 μM NADPH.
reduction of quinoline, coumarin and several of their b Oxidative substrate: 300 μM 2-cyclohexenone.
derivatives with NADPH as a reducing substrate The reaction mixture contained 250 nM XenA.
d The reaction mixture contained 1 μM XenA and 1% DMSO.
was tested. The previously identified oxidative

Structure and Function of XenA Figure 2. Kinetic analysis of XenA substrate turnover by real-time NMR spectroscopy. Series of1D proton NMR spectra recorded at600 MHz. The time between eachspectrum shown is approximately18 min. The last spectrum shown wasrecorded 288 min after addition ofXenA. (a) A portion of the spectrashowing the decrease and increase ofthe signals corresponding to theadenosine protons A8 and A2 ofNADPH and NADP+, respectively.
A2 of NADP+ overlaps with theincreasing signal of the nicotinamideresonance N5 of NADP+ (labelomitted for clarity). The decayingdoublet at 7.88 ppm corresponds toH4 of the substrate 8-hydroxycou-marin (8OHC). (b) The increasingsignal of protons H3 (2.69 ppm) H4(2.88 ppm) of the enzymatic product,8-hydroxy-3,4-dihydrocoumarin(8OHDHC). H3 partially overlapswith the decaying signal of theprotons at position 4a of the nicotin-amide moiety of NADPH (4a: 2.66and 4b: 2.55 ppm, respectively). Thepositions of H3 and H4 are almostexactly identical to those of 3,4-dihydrocoumarin but different fromthe hydrolysed form, clearly suggest-ing that 8-hydroxy-3,4-dihydrocou-marin is the end product of theenzymatic reaction of XenA. Thereaction with coumarin yields essen-tially identical results, except thatenzymatic turnover is approxi-mately two to threefold higher (datanot shown).
a phenolic group.There is no hyperchromic effectaround 600 nm, and the hypochromic effect at The titration of oxidised XenA with coumarin and 464 nm is accompanied by a red shift of the entire 8-hydroxycoumarin resulted in perturbations of the flavin absorbance (c)). Plots of absorption electronic absorption spectrum of the enzyme- change at 464 nm versus ligand concentration and bound FMN and allowed determination of the fitting to equation (1) gave dissociation constants dissociation constant of the complexes ).
(Kd) of 2.5(±0.5) μM and 5.0(±1.8) μM for 8- Optical titration with the two substrates showed hydroxycoumarin and coumarin, respectively isosbestic points at 502 nm for 8-hydroxycoumarin (b) and (d)).
(and 496 nm for coumarin (The absorption changes recorded upon titration of Overall structure and oligomerisation state oxidised XenA with 8-hydroxycoumarin are characteristic of charge transfer complexes The structure of native oxidised XenA from P.
between ligands with a deprotonated phenolic hy- putida 86 was solved by single isomorphous droxyl group and the oxidised flavin.The spectra replacement combined with anomalous dispersion display a hypochromic effect at 464 nm, corre- of an iodide derivative ). The resulting sponding to the flavin absorption maximum, and a electron density was basically interpretable and hyperchromic effect at 600 nm probably elicited by displayed the overall fold of the (β/α)8-barrel. To the charge transfer interaction. Coumarin causes optimize automatic model building by using differ- only a perturbation of the electronic absorption ent phase sources, a Patterson search was carried out spectrum of FMN without additional charge with a search model constructed from the crystal transfer interactions (c)), as found for structure of morphinone reductase (PDB- titrations of OYE with ligands that do not contain ID:1GWJ)The combined phases from SIRAS

Structure and Function of XenA Figure 3. Titrations of XenA with 8-hydroxycoumarin and coumarin. Conditions: 30 μM XenA in 50 mM potassium phosphate buffer (pH 7.0), 20 °C. (a) Spectral changes recorded on titrating XenA with 8-hydroxycoumarin. For clarity,only selected spectra are shown, including the spectra of start and end points of the titration. (b) Plot of absorbance changeat 464 nm versus 8-hydroxycoumarin concentration. The continuous line results from the fit to equation (1). (c) and (d) Asfor (a) and (b), but for titration of XenA with coumarin. Arrows indicate the direction of spectral changes.
phasing and the molecular replacement solution helices surround eight twisted β-strands, forming a allowed automatic tracing and model building cylindrical structure (a)). On the N-terminal including the majority of the water structure. The side of the β-strands, the barrel is closed by a β- structure was refined to 1.5 Å resolution with a final hairpin, whereas on the C-terminal side it is wide R-factor of 18.2% (). Stereochemical para- open. The flavin is bound at the C-terminal end.
meters of the model are good except for one residue, The longer C-terminal loops contain additional Trp302, which is found in the disallowed region of secondary structure elements, namely several short the Ramachandran plot (). The XenA mono- 310-helices, α-helices and two short β-strands mer adopts a (β/α)8-barrel fold where eight α- (the C-terminal loops are numbered Table 2. Phasing statistics Phasing power.
Friedel mates were treated as independent reflections.
a Heavy atom derivative: 12 iodide sites per a.u.
b Rs=Σh Σi Ii(h)−<I(h)> /Σh Σi Ii(h); where i are the independent observations of reflection h.
c RCullis=Σh ( FPH (h)–FP(h) − FHcalc(h)) /Σh FPH(h)–FP(h) .

Structure and Function of XenA Table 3. Crystallographic data and refinement Total/unique refl.
20−1.50 (1.6−1.5) 20−1.42 (1.50−1.42) 20−1.42 (1.50−1.42) Model R/Rfree-factor (%)b rms deviation from ideal geometry Ramachandran statistics (%) generously allowed/disallowed regions Friedel mates were merged. XenA: XenA crystal with sulphate ion in the active site. XenA8-OH-Cum: crystal of XenA soaked with 8-hydroxycoumarin. XenACum: crystal of XenA soaked with coumarin. All three crystals belonged to space group I222. The values given inparantheses are for the highest resolution shell. The Ramachandran statistics were calculated with PROCHECK.50 a Rs=Σh Σi Ii(h)−<I(h)> /Σh Σi Ii(h); where i are the independent observations of reflection h.
b The free R-factor was calculated from 5% of the data, which were removed at random before the structure was refined.
according to the preceding β-strands). Loop L3 is the between helices α1 of the two subunits. Additional longest loop, consisting of 48 residues, and contains interactions between the monomers are built up by most of the barrel's extra secondary structure the C terminus from helix αF to αH. Trp358 in helix elements and active site residues.
αH protrudes into the active site of the neighbouring Like many other OYE family members, XenA monomer and forms part of its FMN and substrate forms a homodimer. Monomer A in the asymmetric binding pocket (see below). The dimer interface unit is related to monomer B by a crystallographic 2- shields 10% of the monomer surface (1380 Å2 per fold axis, resulting in approximately opposite monomer). It lies on the opposite side of the barrel in directions for the barrel openings. The crystal- comparison to the dimer interface of OYE that is lographic 2-fold axis forming the dimer runs formed by helices 4, 5, and 6.
Figure 4. Overall structure of XenA. (a) Ribbon diagram of theXenA dimer. Monomer A is colouredaccording to its secondary structure:red, helices; yellow, β-strands; green,coils. Monomer B is coloured blue.
The FMN cofactors of both mono-mers are shown in stick form withcarbon in cyan, nitrogen in blue,oxygen in red, and phosphorus inorange. (b) Topology of XenA. He-lices are displayed as rectangles,strands as arrows. Helices andstrands of the barrel are numberedaccording to their order in the barrel.
Extra barrel secondary structure ele-ments are designated by letters. Thenumbers at the beginning and end ofeach secondary structure elementaccount for the amino acid number.

Structure and Function of XenA The structure of XenA is highly similar to that of occupation of the active site: in the absence of a other OYE homologues. The closest homologue of ligand, three different conformations were discern- XenA is the flavoprotein YqjM from Bacillus ible in the electron density, whereas in the presence subtilis,The two proteins share an overall of a ligand, the γ-sulfhydryl group pointed exclu- amino acid sequence identity of 40% and similarity sively to O-4 of the flavin. It was suggested that this of 54% and are closely related in structure: 292 residue modulates the redox potential of the flavin residues can be superimposed with a root mean depending on the presence of substratHow- square deviation of 1.2 Å.The major structural ever, in XenA, Cys25 is in hydrogen-bonding difference in the monomers is in loop L3, which distance to O-4 of the cofactor in all three structures, consists of 48 residues in XenA, whereas loop L3 of indicating a role of Cys25 independent of the YqjM consists of only 30 residues. YqjM has been occupation of the active site.
described as a dimer of dimers, while XenA is a The dimethylbenzene ring of flavin is stabilised homodimer in the crystal. In place of the active site by hydrophobic interactions with Met24 on its re residue Trp358 of XenA, YqjM has an arginine finger side and Trp358 of the other monomer on the si protruding into the adjacent active site.Based on side. Trp358 extends into the active site of the sequence comparisons of the OYE homologue YqjM adjacent monomer and closes it on the side of the from B. subtilis with other OYE family members, a dimethylbenzene moiety of the cofactor through a new bacterial subgroup of the OYE family has been face-on-edge π−π interaction (The term- suggested.However, several C-terminal residues inal phosphate group is embedded into an electro- of YqjM suggested to be invariant in this bacterial positive groove of the protein formed by loops L7 subgroup are not found in XenA from P. putida 86, and L8 and the positive end of the macrodipole of the closest relative of YqjM.Specifically, Arg312, Gln333, and Arg336 of YqjM align with Ala330,Pro354 and His357 of XenA, respectively. Arg336 is Ligand complexes in the active site of XenA the residue that extends into the adjacent active siteof YqjM. In XenA, this residue is functionally The crystal structure of XenA has been analysed in replaced by Trp358, resulting in a comparable active complex with sulphate and the substrates coumarin site composition from two monomers. However, other distinctive features of the proposed new The active site structure of oxidised, uncomplexed subgroup of the OYE family are found in XenA as XenA is shown in An anion binds above well, such as Cys25 and Tyr27, which are conserved the oxidised flavin as found for other members of in this subgroup, but not in the OYE family the OYE family.,A sulphate molecule could bemodelled in the electron density. The sulphate ion is The FMN binding site in hydrogen-bonding distance to His178, His181,and Tyr183 ().
A pocket leading to the active site is wide open The coumarin complex is stabilized by π−π with an average diameter of around 18 Å measured stacking interactions between coumarin and the between the Cβ-atoms of the surrounding amino isoalloxazine ring (b)). The carbonyl acids. It is formed between the two monomers andhas at its bottom the FMN cofactor in an elongatedconformation (). The FMN cofactor is boundat the C-terminal end of the β-barrel, above β-strands 1 and 8, with its si face exposed to thesolvent. The protein matrix interacts with thepyrimidine ring and the ribityl side-chain of theflavin through hydrogen bonds. Most of the inter-actions between apoprotein and flavin are con-served within the OYE family, for example aglutamine residue (Gln99), a histidine pair (His178and His181), and an arginine residue (Arg231) bindN-3, O-2 and N-1 of the cofactor, respectively. O-4 ofthe FMN is bound by the amide proton of Ala57 andthe γ-sulfhydryl group of Cys25. The amide protonof Cys25 binds N-5. OYE and most of its char-acterised homologues contain a threonine residue ina position equivalent to Cys25 (Thr37 in OYE),reported to increase the redox potential of thecofactor by stabilising the negative charge of the Figure 5. Active site of oxidised XenA in complex with reduced flavin through hydrogen bonding with O- sulphate. All residues are displayed in stick mode with Based on structural comparisons, the same carbon atoms of the apoprotein in cyan, carbon atoms of function can be assigned to Cys25 of XenA. For the flavin in violet, nitrogen in blue, oxygen in red, and instance, the corresponding cysteine residue in YqjM phosphorus and sulphur in orange. The asterisk denotes a adopts different conformations depending on the residue from monomer B.

Structure and Function of XenA Figure 6. Stereo view of the active site of XenA in complex with (a) 8-hydroxycoumarin, (b) coumarin. All residues are displayed in stick mode with carbon atoms of the apoprotein in cyan, carbon atoms of the flavin in violet, carbon atoms ofthe ligand in grey, nitrogen in blue, oxygen in red, and phosphorus in orange. Initial Fo−Fc electron density maps for theligands are contoured at 3 σ.
oxygen of coumarin is in hydrogen-bonding dis- donor. This observation fits the catalytic mechan- tance to His178 and His181. His181 also binds O-1 ism that has been proposed for OYE with a hydride of the coumarin. There are no further interactions transfer from the flavin N-5 to the β-carbon, with the protein. Notably, Tyr27 does not con- followed by proton transfer from the active site tribute to substrate binding. The corresponding acid Tyr196 to the α-carbon of α,β-unsaturated residue in YqjM and the C-terminal tyrosine residue carbonyl substrates,,However, mutational Tyr375 that occupies the corresponding position in studies of PETN reductase have shown that the OYE have been shown to coordinate the carbonyl corresponding tyrosine residue of this OYE homo- oxygen atom of the inhibitor p-hydroxybenzalde- logue is not essential for catalysis,while morphi- hyde, leading to the assumption that this tyrosine none reductase contains a cysteine residue in place residue contributes to substrate specifity. In XenA, of Tyr196 of OYE, which is not the essential active the histidine pair His178 and His181 appears to be site For PETN reductase and morphinone exclusively responsible for substrate specificity and reductase, it was suggested that the proton is orientation by hydrogen-bonding in the active site.
transferred from the solvent to the substrate., Through coordination of the carbonyl oxygen of an The studies on PETN reductase and morphinone α,β-unsaturated carbonyl substrate like coumarin reductase show that, despite a conserved active site by the histidine pair, the reactive olefinic bond is architecture, the function of individual residues positioned optimally for proton and hydride may not be conserved and has to be investigated transfer and becomes polarised, activating the β- for each enzyme individually. Furthermore, a carbon for nucleophilic attack: C-4 (the β-carbon) of dependence on the type of substrate cannot be coumarin is in 3.7 Å distance to the hydride donor, ruled out. Reactivity analysis of XenA mutations N-5 of the FMN cofactor, whereas C-3 (the α- will have to reveal if Tyr183 is essential for carbon) is close to Tyr183, the presumptive proton protonation of the substrate.
Structure and Function of XenA In contrast to coumarin, 8-hydroxycoumarin hydroxycoumarin in reduced XenA, which is in binds in the active site in a fashion that does not agreement with the apparent kinetic constants and allow hydride transfer from flavin N-5 to C-4 of 8- the product detected by NMR experiments ( hydroxycoumarin, as the two atoms are 4.7 Å Non-productive binding modes in the oxidised apart 8-Hydroxycoumarin is flipped states have also been found for ligand complexes by 180° around the central C-1a – C-4a axis in of the related flavoproteins PETN reductasand comparison to coumarin, so that the phenolic nitroreductase.It has been suggested that hydroxyl group at C-8 is hydrogen bonded to changes in charge distribution upon reduction of the histidine pair. His181 also coordinates O-1. The the flavin could help to prevent some molecules ligand thus does not bind parallel to the long axis from binding in flavoproteins with broad substrate of the isoalloxazine ring like coumarin, but is specificity, like nitroreductases and xenobiotic rotated by ∼45° relative to this axis. Again, there are no further interactions with the protein except Single crystal absorption spectra of uncomplexed those mediated by the histidine pair and the flavin.
and complexed XenA are comparable to the solution It has been proposed that phenolic compounds spectra, and we can conclude that ligand binding in bind to oxidised OYE in the deprotonated state, solution and in the crystalline state are equivalent forming a charge transfer complex with the flavin (data not shown).
with characteristic These typicalabsorption changes have been seen upon titrationof oxidised XenA with 8-hydroxycoumarin and Physiological role of XenA make it likely that it binds in the deprotonatedphenolate state to oxidised XenA. As the phenolate Evidence has been obtained that XenA can group is able to form stronger hydrogen bonds participate in the 8-hydroxycoumarin pathway of with the histidine pair than the carbonyl oxygen P. putida 86. XenA is expressed in P. putida 86 the deprotonation would explain why the binding when grown with quinoline as a sole source of modes of 8-hydroxycoumarin and coumarin differ carbon, nitrogen and energy, and it catalyses the so remarkably. The binding mode of 8-hydroxy- NADPH-dependent reduction of 8-hydroxycou- coumarin appears to be non-productive, as the marin, a known intermediate in the degradation reactive olefinic bond of 8-hydroxycoumarin does of quinoline by P. putida Chemical reactivity not lie above the flavin N-5 or in close proximity of coumarin and its derivatives, our NMR analysis to Tyr183. Due to steric clashes, it is unlikely that of the products formed and the orientation of re-orientation of the substrate in the active site is coumarin in the active site complex argue that the possible. Thus, substrate bound in the unproduc- C-3/C-4 double bond of 8-hydroxycoumarin is tive mode would have to be released before reduced by XenA to yield 8-hydroxy-3,4-dihydro- binding in the productive mode would be possible.
coumarin. Additionally, the conversion of cou- The presence of a dominant non-productive binding marin by P. putida 86 is explained by our kinetic mode would be expected to result in substantially analysis of XenA with coumarin as an oxidising lower apparent KM and kcat values for 8-hydro- substrate yielding 3,4-dihydrocoumarin, which, xycoumarin compared to coumarin. However, when subsequently hydrolysed, produces 2-hydro- both ligands show comparable apparent KM and xyphenylpropionic acid, which was found to kcat values (), arguing for similar binding accumulate in P. putida 86. NMR clearly shows geometries in the reduced enzyme. A different that XenA only catalyses reduction of the C-3/C-4 binding geometry of 8-hydroxycoumarin in the double bond, but not the hydrolysis of the reduced enzyme could originate from an influence of the electron distribution and delocalization of XenA appears to have several distinct biological the isoalloxazine ring on the pKa value of the functions in addition to its participation in the phenol hydroxyl group of 8-hydroxycoumarin.
degradation of quinoline, as it is also found in the While oxidised FMN can stabilise the phenolate genome sequence of P. putida KT2440 that does not anion by charge transfer interactions, thereby contain the genes for other enzymes of the 8- effectively lowering the pKa of the phenol, hydroxycoumarin pathway. Furthermore, XenA is FMNH2 will do so to a much smaller extent. It also highly expressed in P. putida II-B grown in the appears likely that in oxidised XenA 8-hydroxy- absence of coumarins, constituting ∼14% of the total coumarin binds in the phenolate form while in the soluble protein.The broad substrate specificity of reduced state the phenol would be more stable. As XenA makes it an ideal candidate for participation the orientation of the ligands in the active site is in the degradation of several heteroaromatic and dominated by the strength of the hydrogen potentially toxic compounds. Enzymes with such a bonding interaction with the donating histidine broad substrate specificity can to some extent pair, the deprotonated form would bind preferen- explain the wide variety of aromatic, and often tially with the phenolate oxygen while the proto- xenobiotic, substances that are degraded by bacteria nated 8-hydroxycoumarin would bind with the like P. putida 86. Remarkably, XenA is to our carbonyl oxygen of the lactone group to the knowledge the first OYE homologue implicated in histidine pair. The consequence would be the the in vivo degradation of heteroaromatic com- prevalence of a productive binding mode of 8- Structure and Function of XenA Materials and Methods FMN at its absorption maximum (464 nm) was calculatedfrom the known extinction coefficient of 12.2 mM–1cm–1 at450 nm for free FMN.
Substrate specificity All chemicals used were of analytical grade. Coumarin and 2-cyclohexenone were obtained from Fluka. 8-Hydroxycoumarin was prepared essentially as described Apparent steady-state kinetic constants were by Cerqueira et All chromatography materials were recorded by following the oxidation of NADPH at from Amersham Biosciences.
340 nm in a Specord 30 spectrophotometer (AnalytikJena) while systematically varying the substrate con-centration. Reactions were initiated by addition of en- Growth of P. putida 86 zyme and monitored for 2 min. All experiments wereconducted three times and results were averaged. One P. putida 86 was cultured aerobically in 35 l fermentors at unit of enzyme activity was defined as the oxidation 30 °C in a quinoline minimal Whenever quinoline of 1 μmol NADPH per min at 25 °C in the assay and 2-oxoquinoline were undetectable in the fermentation broth, further portions of quinoline (0.5 ml/l) were added. At The reaction mixture was made anaerobic by flush- an optical density (600 nm) of approximately 3.5, cells were ing with nitrogen. Enzyme assays were measured at harvested by centrifugation, frozen in liquid nitrogen, and 25 °C in 1 ml of 50 mM potassium phosphate buffer stored at −30 °C until further use.
(pH 7.0), containing 150 μM NADPH, and 250 nMXenA, where 2-cyclohexenone was used as oxidativesubstrate, or 1 μM XenA, where coumarin or 8- PCR, cloning, sequencing and expression hydroxycoumarin were used as oxidative substrate.
Apparent steady-state kinetic constants for NADPH The nucleotide sequence of xenA from P. putida KT2440 were determined with 300 μM 2-cyclohexenone as was obtained from GenBank (accession no. AF154061).
oxidative substrate. 8-Hydroxycoumarin and coumarin The open reading frame of P. putida 86 xenA was amplified were dissolved at 100-fold concentration in dimethyl by polymerase chain reaction, using a P. putida 86 colony sulphoxide (DMSO), keeping the DMSO concentration as template, and cloned into pET-11a (Novagen). The in the reaction mixture constant at 1%. The software construct was verified by sequence analysis and was GraFit version 5.0.10 (Erithacus Software Limited) was transformed into E. coli BL21(DE3) (Stratagene) for used for evaluation of the data.
expression. E. coli strain BL21(DE3) harbouring plasmidpET-11a with the insertion of xenA was grown aerobically at 25 °C in 4 l of LB medium containing 100 μg/mlampicillin after 1% inoculation. Expression was inducedwith 0.1 mM IPTG at an optical density (600 nm) of ∼0.7.
1D-proton NMR spectra of the individual substances Cells were harvested by centrifugation 18 h after induc- tion, frozen in liquid nitrogen, and stored at −30 °C until 3-(2-hydroxyphenyl)-propionic acid, NADPH, and further use.
NADP+) in an aqueous buffer (pH 7.0), containing50 mM potassium phosphate and 5% 2H2O weremeasured on a Bruker Avance 400 spectrometer Purification of XenA equipped with a HCN-triple-resonance probe headwith z-axis gradients at 293 K. The residual water The enzyme heterologously expressed in E. coli BL21 signal was suppressed using double-pulsed field (DE3) was purified by four column chromatography steps: gradient echo The concentration of each anion exchange chromatography, hydrophobic interaction substance was 100 μM. 256 transients with 16.384 chromatography, gel filtration and a second anion complex data points were recorded and zero-filled to exchange chromatography. All chromatography steps 64,000 data points prior to Fourier transformation.
were carried out at 16 °C; in between the protein solution Signal assignments for NADPH, NADP+, and coumarin was kept on ice. After the hydrophobic interaction were taken from the literature.Assignments for chromatography step XenA-containing fractions were the other compounds were derived from incubated with 5 mM FMN on ice over night.
spectra and interpretation of 1D-spectra. 2D-NOESY From P. putida 86, XenA was purified in essentially the experiments with a mixing time of 800 ms and same way, except that the protein was not reconstituted with double-pulsed field gradient echos for solvent suppressionwere recorded at 600 MHz with4000 complex data points in the direct dimensionand 256 data points in the indirect dimension. The Determination of protein concentration assignments were used to identify compounds in thereaction mixture during kinetic experiments (data not The concentration of XenA holoprotein was determined using a millimolar extinction coefficient of 12.2 mM−1cm−1 Kinetic NMR experiments of the turnover of cou- at 464 nm. The extinction coefficient of XenA-bound FMN marin and 8-hydroxycoumarin by XenA were recorded was evaluated as follows: a spectrum of the native protein on a Bruker DRX 600 spectrometer equipped with a was recorded. The protein was then denatured by the HCN-triple resonance probe head with x,y,z-axis addition of 0.1 vol. 50% trichloroacetic acid, and the gradients at 288 K. The concentration of the substrate precipitated protein removed by centrifugation. The was 100 μM and 150 μM the cofactor NADPH in each supernatant was neutralised with solid sodium bicarbo- case. After the first data point enzyme was directly nate and the spectrum recorded against a reagent blank added into the NMR-sample tube to a final concen- without XenA. The extinction coefficient of XenA-bound tration of 0.8 μM. The dead time for mixing and Structure and Function of XenA equilibration was approximately 5 min. A series of 50 approximately 30 s in 400 mM potassium iodide dissolved experiments with 128 transients was recorded, each in the harvesting buffer. The crystals were immediately experiment requiring approximately 6 min. Spectra frozen after soaking with iodide. Iodide sites were localized were evaluated with MESTREC The time- using SHELXInitial phases were calculated using dependent intensities of selected signals were fit using SHAR(), and modified with SOLOMONTo Microcal™ Origin® version 6.0 (Microcal Software, Inc.) allow for automatic model building the modified experi- assuming exponential decay/build up to extract mental phases were combined with phases calculated from approximate rates. All chemical shifts were referenced a positioned model of morphinone reductase (PDB-ID.: relative to internal TSP.
1GWJ)using AMoRfor the Patterson search. Thecombined phases were further modified using the auto-matic building routine of wARP.The model was built Ligand binding titrations using MAIN 2000 , and atomic positions and B-factorswere refined with C).
30 μM oxidised XenA in 50 mM potassium phosphate buffer (pH 7.0), at 20 °C, was titrated by systematicallyvarying the ligand concentration. Spectra were recorded Protein Data Bank accession codes between 240 and 650 nm with a 8452 A diode arrayspectrophotometer (Hewlett Packard). Spectral changes The coordinates and structure factor amplitudes are evoked by the addition of ligand to XenA agree with a deposited in the RCSB Protein Data Bank with ID codes: 1:1 binding stoichiometry, and the isosbestic points 2H8X, 2H8Z, and 2H90.
during the titration are attestive of a single step process.
Absorption changes at two selected wavelengths withthe largest spectral perturbations were plotted againstligand concentration. The data were fitted with thesoftware Microcal™ Origin® version 6.0 (Microcal Software, Inc.) to the quadratic function (equation (1))to calculate the dissociation constant (Kd) for the The authors thank Susanne Fetzner for donation enzyme−ligand complex: of the P. putida 86 strain and helpful discussions, Juan Manuel Urbina-González and Ellen Wiede- mann for assistance in the preparation of 8- hydroxycoumarin, Ilme Schlichting for the mea- surement of single crystal absorption spectra and T þ ET þ KdÞ ðLT þ ET þ KdÞ2ð4LT ET Þ Paul Roesch for access to the NMR facilities. R.P.J. is supported by a PhD fellowship of the Fonds derChemischen Industrie. H.D. acknowledges the where ΔAmax is the maximum absorption change at the Deutsche Forschungsgemeinschaft (DO-785/2) for selected wavelength, LT is the total ligand concentration,and E financial support.
T is the total enzyme concentration.
Protein crystallisation and structure determination XenA was crystallised by vapour diffusion using the 1. Fetzner, S. (1998). Bacterial degradation of pyridine, hanging-drop setup at 16 °C. The reservoir solution (500 μl) indole, quinoline, and their derivatives under different contained 1.4 – 1.7 M ammonium sulphate in 0.1 M Hepes redox conditions. Appl. Microbiol. Biotecholn. 49, buffer (pH 7.5). Two drops were set up per reservoir by mixing 2 μl of protein solution (10 and 15 mg/ml in 10 mM 2. Fetzner, S., Tshisuaka, B., Lingens, F., Kappl, R. & Tris-HCl (pH 8.0), respectively) with 2 μl of reservoir Huttermann, J. (1998). Bacterial degradation of quino- line and derivatives - Pathways and their biocatalysts.
For determination of the uncomplexed structure, single Ang. Chem.-Int. Ed. 37, 577–597.
crystals were transferred to a harvesting buffer containing 3. Schwarz, G., Senghas, E., Erben, A., Schafer, B., ammonium sulphate at 1.5-fold the concentration of the Lingens, F. & Hoke, H. (1988). Microbial-metabolism reservoir in 0.1 M Hepes buffer (pH 7.5). To soak XenA of quinoline and related-compounds .1. Isolation and crystals with coumarin, 8-hydroxycoumarin and 7-hydro- characterization of quinoline-degrading bacteria. Syst.
xycoumarin, crystals were cross-linked with 0.025% glu- Appl. Microbiol. 10, 185–190.
taraldehyde in harvesting buffer for 1 h, and then 4. Shukla, O. P. (1986). Microbial transformation of transferred to harvesting buffer containing 5 mM of the quinoline by a Pseudomonas sp. Appl. Environ. Micro- respective ligand and 5% DMSO for at least 30 min. The biol. 51, 1332–1342.
crystals belong to space group I222 with cell dimensions 5. Blase, M., Bruntner, C., Tshisuaka, B., Fetzner, S. & a = 57.9 Å, b = 83.4 Å, c = 156.8 Å, α = β = γ= 90° and contain Lingens, F. (1996). Cloning, expression, and sequence one molecule per asymmetric unit. Visible absorption analysis of the three genes encoding quinoline 2- spectra of single XenA crystals were recorded using a oxidoreductase, a molybdenum-containing hydroxy- micro spectrophotometer (4DXray Systems AB). The XenA lase from Pseudomonas putida 86. J. Biol. Chem. 271, crystals were mounted in a loop and kept at 100 K during 6. Kappl, R., Huttermann, J. & Fetzner, S. (2002). The The structure of XenA was determined by SIRAS phasing molybdenum-containing hydroxylases of quinoline, with a potassium iodide derivative, which is routinely being isoquinoline, and quinaldine. Met. Ions Biol. Syst. 39, carried out with protein crystals of unknown structure. The derivative has been prepared by incubating the crystals for 7. Bonin, I., Martins, B. M., Purvanov, V., Fetzner, S., Structure and Function of XenA Huber, R. & Dobbek, H. (2004). Active site geometry Bourenkov, G. P., Macheroux, P. & Clausen, T. (2005).
and substrate recognition of the molybdenum hydro- The 1.3 A crystal structure of the flavoprotein YqjM xylase quinoline 2-oxidoreductase. Structure, 12, reveals a novel class of Old Yellow Enzymes. J. Biol.
Chem. 280, 27904–27913.
8. Rosche, B., Tshisuaka, B., Fetzner, S. & Lingens, F.
25. Holm, L. & Sander, C. (1993). Protein structure (1995). 2-Oxo-1,2-dihydroquinoline 8-monooxygen- comparison by alignment of distance matrices.
ase, a two-component enzyme system from Pseudo- J. Mol. Biol. 233, 123–138.
monas putida 86. J. Biol. Chem. 270, 17836–17842.
26. Xu, D., Kohli, R. M. & Massey, V. (1999). The role of 9. Martins, B. M., Svetlitchnaia, T. & Dobbek, H.
threonine 37 in flavin reactivity of the old yellow (2005). 2-Oxoquinoline 8-monooxygenase oxygenase enzyme. Proc. Natl Acad. Sci. USA, 96, 3556–3561.
component: active site modulation by Rieske-[2Fe- 27. Barna, T. M., Khan, H., Bruce, N. C., Barsukov, I., 2S] center oxidation/reduction. Structure, 13, Scrutton, N. S. & Moody, P. C. (2001). Crystal structure of pentaerythritol tetranitrate reductase: "flipped" 10. Eicher, T. & Hauptmann, S. (2005). The Chemistry of binding geometries for steroid substrates in different Heterocycles. Wiley-VCH GmbH, Weinheim.
redox states of the enzyme. J. Mol. Biol. 310, 433–447.
11. Fetzner, S. (2000). Enzymes involved in the aerobic 28. Vaz, A. D., Chakraborty, S. & Massey, V. (1995). Old bacterial degradation of N-heteroaromatic com- Yellow enzyme: aromatization of cyclic enones and the pounds: molybdenum hydroxylases and ring-opening mechanism of a novel dismutation reaction. Biochem- 2,4-dioxygenases. Naturwissenschaften, 87, 59–69.
istry, 34, 4246–4256.
12. Binks, P. R., French, C. E., Nicklin, S. & Bruce, N. C.
29. Brown, B. J., Deng, Z., Karplus, P. A. & Massey, V.
(1996). Degradation of pentaerythritol tetranitrate by (1998). On the active site of Old Yellow Enzyme. Role Enterobacter cloacae PB2. Appl. Environ. Microbiol. 62, of histidine 191 and asparagine 194. J. Biol. Chem. 273, 13. Fitzpatrick, T. B., Amrhein, N. & Macheroux, P. (2003).
30. Khan, H., Barna, T., Bruce, N. C., Munro, A. W., Leys, Characterization of YqjM, an Old Yellow Enzyme D. & Scrutton, N. S. (2005). Proton transfer in the homolog from Bacillus subtilis involved in the oxida- oxidative half-reaction of pentaerythritol tetranitrate tive stress response. J. Biol. Chem. 278, 19891–19897.
reductase. Structure of the reduced enzyme-proges- 14. Blehert, D. S., Knoke, K. L., Fox, B. G. & Chambliss, terone complex and the roles of residues Tyr186, G. H. (1997). Regioselectivity of nitroglycerin denitra- His181, His184. FEBS J. 272, 4660–4671.
tion by flavoprotein nitroester reductases purified from 31. Messiha, H. L., Bruce, N. C., Sattelle, B. M., Sutcliffe, two Pseudomonas species. J. Bacteriol. 179, 6912–6920.
M. J., Munro, A. W. & Scrutton, N. S. (2005). Role of 15. Snape, J. R., Walkley, N. A., Morby, A. P., Nicklin, S. & active site residues and solvent in proton transfer and White, G. F. (1997). Purification, properties, and the modulation of flavin reduction potential in bacterial sequence of glycerol trinitrate reductase from Agro- morphinone reductase. J. Biol. Chem. 280, 27103–27110.
bacterium radiobacter. J. Bacteriol. 179, 7796–7802.
32. Race, P. R., Lovering, A. L., Green, R. M., Ossor, A., 16. Miura, K., Tomioka, Y., Suzuki, H., Yonezawa, M., White, S. A., Searle, P. F. et al. (2005). Structural and Hishinuma, T. & Mizugaki, M. (1997). Molecular mechanistic studies of Escherichia coli nitroreductase cloning of the nemA gene encoding N-ethylmaleimide with the antibiotic nitrofurazone. Reversed binding reductase from Escherichia coli. Biol. Pharm. Bull. 20, orientations in different redox states of the enzyme. J.
Biol. Chem. 280, 13256–13264.
17. French, C. E. & Bruce, N. C. (1994). Purification and 33. Cerqueira, N. M. F. S. A., Oliveira-Campos, A. M. F., characterization of morphinone reductase from Pseu- Coelho, P. J., de Carvalho, L. H. M., Samat, A. & domonas putida M10. Biochem. J. 301, 97–103.
Guglielmetti, R. (2002). Synthesis of photochromic 18. Rohde, B. H., Schmid, R. & Ullrich, M. S. (1999).
dyes based on annulated coumarin systems. Helv.
Thermoregulated expression and characterization of Chim. Acta, 85, 442–450.
an NAD(P)H-dependent 2-cyclohexen-1-one reduc- 34. Tshisuaka, B., Kappl, R., Huttermann, J. & Lingens, F.
tase in the plant pathogenic bacterium Pseudomonas (1993). Quinoline oxidoreductase from Pseudomonas syringae pv. glycinea. J. Bacteriol. 181, 814–822.
putida 86: an improved purification procedure and 19. Kohli, R. M. & Massey, V. (1998). The oxidative half- electron paramagnetic resonance spectroscopy. Bio- reaction of Old Yellow Enzyme. The role of tyrosine chemistry, 32, 12928–12934.
196. J. Biol. Chem. 273, 32763–32770.
35. Whitby, L. G. (1953). A new method for preparing 20. Blehert, D. S., Fox, B. G. & Chambliss, G. H. (1999).
flavin-adenine dinucleotide. Biochem. J. 54, 437–442.
Cloning and sequence analysis of two Pseudomonas 36. Hwang, T. L. & Shaka, A. J. (1995). Water suppression flavoprotein xenobiotic reductases. J. Bacteriol. 181, that works - excitation sculpting using arbitrary wave- forms and pulsed-field gradients. J. Magn. Reson. Ser.
21. Abramovitz, A. S. & Massey, V. (1976). Interaction of A, 112, 275–279.
phenols with old yellow enzyme. Physical evidence 37. Auchere, F., Bertho, G., Artaud, I., Girault, J. P. & for charge-transfer complexes. J. Biol. Chem. 251, Capeillere-Blandin, C. (2001). Purification and struc- ture of the major product obtained by reaction of 22. Barna, T., Messiha, H. L., Petosa, C., Bruce, N. C., NADPH and NMNH with the myeloperoxidase/ Scrutton, N. S. & Moody, P. C. (2002). Crystal structure hydrogen peroxide/chloride system. Eur. J. Biochem.
of bacterial morphinone reductase and properties of 268, 2889–2895.
the C191A mutant enzyme. J. Biol. Chem. 277, 38. Miura, S. & Ichikawa, Y. (1994). Interaction of NADPH-adrenoferredoxin reductase with NADP+ 23. Fox, K. M. & Karplus, P. A. (1994). Old yellow enzyme and adrenoferredoxin. Equilibrium and dynamic at 2 A resolution: overall structure, ligand binding, and properties investigated by proton nuclear magnetic comparison with related flavoproteins. Structure, 2, resonance. J. Biol. Chem. 269, 8001–8006.
39. Martinez-Martinez, F. J., Padilla-Martinez, I. I. & 24. Kitzing, K., Fitzpatrick, T. B., Wilken, C., Sawa, J., Trujillo-Ferrara, J. (2001). H-1 and C-13 NMR Structure and Function of XenA assignments of 2-oxo-2H-1-benzopyran-3-acyl and-3- The CCP4 Suite: programs for protein crystallogra- amide derivatives. Magn. Reson. Chem. 39, 765–767.
phy. Acta Crystallog. sect. D, 50, 760–763.
40. Jeener, J., Meier, B. H., Bachmann, P. & Ernst, R. R.
46. Navaza, J. (1994). AMoRe: an automated package for (1979). Investigation of exchange processes by 2-di- molecular replacement. Acta Crystallog. sect. A, 50, mensional NMR-spectroscopy. J. Chem. Phys. 71, 47. Perrakis, A., Sixma, T. K., Wilson, K. S. & Lamzin, V. S.
41. Cobas, J. C. & Sardina, F. J. (2003). Nuclear magnetic (1997). wARP: Improvement and extension of crystal- resonance data processing. MestRe-C: a software lographic phases by weighted averaging of multiple- package for desktop computers. Concepts Magnetic refined dummy atomic models. Acta Crystallog. sect.
Reson. 19A, 80–96.
D, 53, 448–455.
42. Schneider, T. R. & Sheldrick, G. M. (2002). Substruc- 48. Turk, D. (1992). Weiterentwicklung eines Programms ture solution with SHELXD. Acta Crystallog. sect. D, für Molekülgraphik und Elektronendichte-Manipula- 58, 1772–1779.
tion und seine Anwendung auf verschiedene Protein- 43. Uson, I., Schmidt, B., von Bulow, R., Grimme, S., von Strukturaufklärungen, TU München.
Figura, K., Dauter, M. et al. (2003). Locating the 49. Brünger, A. T., Adams, P. D., Clore, G. M., Delano, anomalous scatterer substructures in halide and sulfur W. L., Gros, P., Grosse Kunstleve, R. W. et al. (1998).
phasing. Acta Crystallog. sect. D, 59, 57–66.
Crystallography and NMR system: a new software 44. La Fortelle, E. D., Irwin, J. J. & Bricogne, G.
suite for macromolecular structure determination.
(1997). SHARP: A maximum-likelihood heavy- Acta Crystallog. sect. D, 54, 905–921.
atom parameter refinement and phasing program 50. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & for the MIR and MAD methods. Crystallog.
Thornton, J. M. (1993). PROCHECK: a program to Comput. 7, 1–9.
check the stereochemical quality of protein structures.
45. Collaborative Computational Project No. 4. (1994).
J. Appl. Crystallog. 26, 283–291.
Edited by R. Huber (Received 4 April 2006; received in revised form 1 June 2006; accepted 7 June 2006) Available online 21 June 2006


Université de Poitiers Faculté de Médecine et Pharmacie ANNEE 2013 Thèse n° POUR LE DIPLOME D'ETAT DE DOCTEUR EN MEDECINE (décret du 16 janvier 2004) présentée et soutenue publiquement le 2 juillet 2013 à Poitiers par Monsieur Paul LOUMAIGNE

Keeping the Auckland Airport community informed Issue 73 April 2008 ISSN1176-9432 for airport emergency teamInside this issue: • Golf day benefits charity • Airport wins bronze award • Greening the airport • Auckland Cup race day • Plus much more… Cover: Brian Chase (left) and Tony Beattie (right) of the Airport