The Mo-Se of nicotinate dehydrogenase

Nadine Wagenera, Antonio J. Pierikb, Abdellatif Ibdahc, Russ Hillec, and Holger Dobbeka,1

aBioanorganische Chemie, Universita¨t Bayreuth, 95440 Bayreuth, Germany; bInstitut fu¨r Zytobiologie, Philipps-Universita¨t Marburg, 35037 Marburg, Germany; and cDepartment of Biochemistry, University of California, Riverside, CA 92521

Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved May 19, 2009 (received for review February 27, 2009) Nicotinate dehydrogenase (NDH) from Eubacterium barkeri is a with substrates and inhibitors are known (19–27). These struc- molybdoenzyme catalyzing the hydroxylation of nicotinate to tures demonstrate that the molybdenum coordination sphere 6-hydroxynicotinate. Reactivity of NDH critically depends on the typically consists of 1 oxo, 1 hydroxo, and 1 labile sulfido ligand presence of labile (nonselenocysteine) selenium with an as-yet- in a square-pyramidal coordination sphere, with the MoAO unidentified form and function. We have determined the crystal occupying the apical position. The sulfido ligand is prone to structure of NDH and analyzed its active site by multiple wave- cyanolysis, and its replacement with a second oxo ligand in the lengths anomalous dispersion methods. We show that selenium is process leads to inactivation of the (10). An extension bound as a terminal MoASe ligand to molybdenum and that it of the active site is found in aerobic carbon monoxide dehydro- occupies the position of the terminal sulfido ligand in other genases. While in an earlier report on the structure the presence molybdenum hydroxylases. The role of selenium in has of selenium in the active site was suggested (25), a latter study been assessed by model calculations, which indicate an accelera- using analytical multiple wavelength anomalous dispersion tion of the critical hydride transfer from the to the methods showed that the active site does not contain selenium selenido ligand in the course of substrate hydroxylation when but a linearly-coordinated Cu(I) ion bridged by a ␮-sulfido ligand compared with an active site containing a sulfido ligand. The to the molybdenum (28). This ␮-sulfido bridge occupies the position MoO(OH)Se active site of NDH shows a novel type of utilization of the MoAS seen in other molybdenum hydroxylases. and reactivity of selenium in nature. The anaerobic soil bacterium E. barkeri is able to ferment nicotinate to propionate, acetate, carbon dioxide, and ammonia Eubacterium barkeri ͉ hydroxylase ͉ molybdoprotein ͉ with the gain of 1 mol of ATP per mol of nicotinate. The ͉ molybdopterin selenium fermentation of nicotinate is initiated by its hydroxylation to BIOCHEMISTRY 6-hydroxynicotinate catalyzed by NDH (12). NDH has a (␣␤␥␦)2 elenium is an essential component of several enzymes and subunit structure and contains [2Fe-2S] clusters, FAD and a Shas a key role in various biological redox processes. Usually molybdenum center with a pyranopterin that has been selenium occurs in proteins as selenocysteine, which is cotrans- elaborated as the dinucleotide of cytosine and termed molyb- lationally inserted as the 21st amino acid (1) and is found in a dopterin cytosine dinucleotide (MCD) (18, 29). The genes variety of proteins in all 3 kingdoms of life (2). Selenium also encoding NDH occur in the transcriptional order ndhFSLM and finds a natural use as 5-methylaminomethyl-2-selenouridine in are part of a 23.3-kb gene cluster dedicated to the fermentation the ‘‘wobble’’ position of some tRNAs (3). The ionic radii and of nicotinate (30). The NdhF subunit (33 kDa) carries 1 FAD electronegativities of selenium and sulfur are similar, but se- molecule and the NdhS subunit (23 kDa) contains 2 [2Fe-2S] lenide is a stronger reducing agent than sulfide. Because of the clusters. Contrary to all structurally characterized hydroxylases lower pKa value of selenols compared with thiols selenocysteine the molybdenum cofactor appeared to be contained not in 1 but is deprotonated under physiological conditions, whereas cysteine 2 subunits: the NdhL subunit (50 kDa) and NdhM subunit (37 is mostly protonated (4). The ionization state together with the kDa). The most remarkable feature of NDHs is the presence of better polarizability of selenium makes selenocysteine a good labile (nonselenocysteine) selenium (14), which is essential to nucleophile. Several molybdenum- and tungsten-containing en- catalyze the hydroxylation reaction (18). Here, we report the zymes have been shown to contain selenium (5), and a recent X-ray crystal structure of NDH and its Se-containing active site, comprehensive genomic analysis has revealed a clear relation- together with computational studies demonstrating the catalytic ship between selenium and molybdenum utilization across all 3 profit of the natural selection of Se over its congeners S and O. domains of life (6). Selenium is found as selenocysteine in some prokaryotic molybdenum-containing oxotransferases like for- Results mate dehydrogenase H from Escherichia coli, where it coordi- Overall Structure. NDH was crystallized by vapor diffusion meth- nates molybdenum in the oxidized state of the (7–9). In ods under anoxic conditions in an atmosphere of 95% N2/5% H2. other enzymes, notably members of the molybdenum hydroxy- Crystals belonged to the space group P21 and contained the lase family (10, 11), like nicotinate dehydrogenase (NDH) from complete dimer of heterotetramers in 1 asymmetric unit. The Eubacterium barkeri (12–14) and the xanthine structure of NDH was determined with Patterson search tech- (XORs) of Clostridium purinolyticum (15), Clostridium acidiurici niques by using a substructure of 4-hydroxybenzoyl-CoA reduc- (16) and Eubacterium barkeri (17) and the purine hydroxylase tase (23) as search model. The final model contains all residues from C. purinolyticum (15) contain a labile (nonselenocysteine) and was refined to 2.2-Å resolution (Table 1). selenium in an unidentified form essential for their reactivity (18). Molybdenum hydroxylases catalyze the hydroxylation of var- ious organic molecules following the general scheme: Author contributions: N.W., R.H., and H.D. designed research; N.W., A.J.P., and A.I. per- formed research; N.W., A.J.P., A.I., R.H., and H.D. analyzed data; and A.J.P., R.H. and H.D. ϩ 3 ϩ Ϫ ϩ ϩ wrote the paper. RH H2O ROH 2e 2H . The authors declare no conflict of interest. This hydroxylation reaction is unique in biology as it uses water This article is a PNAS Direct Submission. as the source for the hydroxyl oxygen and not dioxygen (10). The Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, active site of molybdenum hydroxylases contains molybdenum www.pdb.org (PDB ID codes 3HRD). coordinated by the enedithiolate group of a pyranopterin co- 1To whom correspondence should be addressed. E-mail: [email protected]. factor, commonly referred to as molybdopterin. The crystal This article contains supporting information online at www.pnas.org/cgi/content/full/ structures of different molybdenum hydroxylases in complex 0902210106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902210106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 29, 2021 Table 1. Statistics on diffraction data and structure refinement Dataset

Statistic Above Se edge Below Se edge Native

Data collection Wavelength, Å 0.9780 0.9803 1.5418

Space group P21 P21 Cell dimensions (a,b,c in Å, b in °) 100.00, 72.15, 217.10, 90.56 97.08, 71.70, 214.49, 90.23 Total/unique reflections 396,539/208,277 360,921/196,213 519,267/148,165

Rs,% 7.8 (31.0) 8.7 (44.8) 9.7 (53.6) Resolution, Å 30–2.5 (2.6–2.5) 30–2.5 (2.6–2.5) 30–2.2 (2.3–2.2) Completeness, % 99.2 (97.6) 93.5 (74.9) 98.9 (96.8) (I)/(␴I) 6.8 (2.4) 6.5 (1.5) 10.8 (2.3) Refinement

Model Rwork/Rfree factor, % 21.2/25.0 No. atoms Protein 17,180 Ligand 249 Water 954 B factors Protein 36.3 Ligand 26.1 Water 29.3 rmsd Bond lengths, Å 0.006 Bond angles, ° 1.3

In the datasets at the Se edge the values given are for unmerged Friedel mates. The values in parentheses indicate the highest resolution.

ϩ ϩ NDH is a dimer of heterotetramers with (FSLM)2 subunit NAD /NADP access to the N5 position is usually blocked by composition in solution (12) and all subunits are present in the a tyrosine (carbon monoxide dehydrogenase from Oligotropha crystal structure (Fig. 1). The dimer has overall dimensions of carboxydovorans, quinoline 2-) or tryptophan 148 ϫ 100 ϫ 70 Å3. The F subunit (296 residues) harbors the side chain (carbon monoxide dehydrogenase from Hydrog- FAD cofactor, which interacts with the N terminal (residues enophaga pseudoflava). In contrast, the less bulky side chain of 1–57) and middle domain (residues 58–178) through its adenosin isoleucine is found in the NADϩ/NADPϩ-reducing NDH and and ribityl moiety. The C-terminal domain (residues 179–291) bovine and bacterial XOR (21, 22). All 3 domains of the F only interacts with FAD through K187F, whose amino group is subunit show a mixed ␣/␤-fold. The S subunit (157 residues) in hydrogen-bonding distance to O4 of the isoalloxazine ring. As comprises 2 domains, each coordinating 1 [2Fe-2S] cluster. The in other molybdenum hydroxylases, the C-terminal domain N-terminal domain (residues 1–79) is similar to ‘‘plant-type contributes to shield the N5 position of the isoalloxazine ring ferredoxins’’ and binds the [2Fe-2S] cluster close to the FAD, from the solvent. In molybdenum hydroxylases unable to reduce which is designated as Fe/S II or type II on the basis of its EPR characteristics in homologous molybdenum hydroxylases (31– 33). The C-terminal domain (residues 80–157) displays a 4-helix bundle with 2-fold symmetry unique to molybdenum hydroxy- lases (19) and coordinates the [2Fe-2S] cluster (type I) closest to the molybdopterin. The L subunit (425 residues) and M subunit (330 residues) harbor the Mo-bound pyranopterin cofactor. Both subunits have an extended structure and lie approximately perpendicular on top of each other. The L subunit interacts with the M and S subunits and has an N-terminal extension (residues 1–28L) that wraps around the C-terminal domain of the S subunit. A middle domain (residues 29–129L and 181–277L) with 2 antiparallel ␤-sheets of 2 and 7 strands and 3 ␣-helices follows the N-terminal extension. The C-terminal domain of the L subunit is dominated by a mixed 5-stranded ␤-sheet flanked on 1 side by 2 ␣-helices that continue into a 2-stranded antiparallel ␤-sheet and a C-terminal ␣-helix. The M subunit can be divided into 2 domains, both containing mixed 4-stranded ␤-sheets and Fig. 1. Overall structure of NDH. Ribbon plot representation of the NDH 3 ␣-helices (Fig. 1). dimer. In the left monomer each subunit has its own color with green and red Both monomers of NDH show the same arrangement of for the MCD coordinating L and M subunits, blue for the [2Fe-2S] clusters subunits and cofactors building 2 independently working elec- containing S subunit, and yellow for the FAD containing F subunit. The right tron transfer chains (Fig. 1), similar to other structurally char- monomer is colored in different shades of gray. Cofactors are labeled, and FeSI acterized molybdenum hydroxylases. However, NDH is unusual and FeS indicate the position of the type I and type II [2Fe-2S] clusters of the II in containing 4 subunits per monomer, whereas other molybde- S subunit. The shortest distances between the cofactors are 5.4 Å (MCD-FeSI), 11.5 Å (FeSI-FeSII), and 6.4 Å (FeSII-FAD), with the shortest metal-to-metal num hydroxylases of known structure, contain 1, 2, or 3 subunits. distances of 14.7 Å (MCD-FeSI) and 12.4 Å (FeSI-FeSII). All pictures were pre- Compared with all structurally characterized molybdenum hy- pared by using PyMol (48). droxylases, the Mo-pyranopterin binding subunit/domain has

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902210106 Wagener et al. Downloaded by guest on September 29, 2021 anomalous scattering from only 1 of the equatorial ligands of the Mo ion (Fig. 2B). That this position is indeed occupied by selenium is supported by the stronger electron density observed at this position and from a Mo ligand bond length of 2.3 Å (Fig. 2C), which is longer than the typical MoOS bond (Table S1). Se is a direct ligand and coordinates Mo in the equatorial plane. The lack of additional density around Se indicates that it is a terminal ligand. We estimated the occupancy of the Se ligand to be Ϸ80% by adjusting the Se occupancy of the model such that the Se atom refines to a similar B factor as the neighboring atoms including the other Mo ligands. The second coordination sphere around the Mo-ion is formed by E289M in trans to the apical oxo-ligand, Q208L, in hydrogen- bonding distance to the apical oxo-ligand and residues that may contribute to the binding and stabilization of the substrate, like Y312L, R319L, F353L, and Y13M (Fig. 2A). An additional small ligand bound near the active site has been modeled as nitrate, which is present in the crystallization buffer. Nitrate interacts with the main chain of residues forming the substrate-binding pocket and is 8 Å away from the Mo ion and 6 Å away from the Se. Nitrate is found at the same position where an acetate molecule has been modeled in the crystal structure of bXOR (21) and both show the same pattern of interactions with the surrounding amino acids. A physiological role for this conserved anion- near the active site has not been demonstrated.

Structure-Based Reaction Mechanism of NDH. Increasing evidence

on the chemistry of XORs supports a mechanism of substrate BIOCHEMISTRY hydroxylation involving a base-assisted nucleophilic attack of the equatorial Mo-OH group on the substrate, with a concomitant Fig. 2. The Mo-Se active site of NDH. (A) Stereoview of the active-site ϩ A environment. Transparent ribbons are colored as in Fig. 1 (left monomer). (B) hydride transfer from the substrate to the Mo( VI) S group to Selenium identification by anomalous scattering detected at energies higher give Mo(ϩIV)-SH. Two residues in the direct environment of (␭ ϭ 0.9780 Å in red) and smaller (␭ ϭ 0.9803 Å in green) than the energy of molybdenum, Q208L in NDH (Q767 in bXOR) and E289M the Se K edge. Both Bijvoet difference maps are contoured at the ϩ5.0-␴ level. (E1261 in bXOR), are conserved among the molybdenum Ϫ (C) Fobs Fcalc map for the Mo and pyranopterin cofactor at a contour level of hydroxylases. E1261 serves as general base catalyst in accepting ϩ5.0 ␴. For the calculation of the map all atoms of the Mo ion and the the proton from Mo-OH upon reaction in bXOR (11) (Fig. S3) pyranopterin cofactor were omitted. and, accordingly, replacement of this residue in bacterial XOR by alanine profoundly compromises the reactivity of the enzyme (35). Additionally, NDH and bXOR have common residues in been split into 2 polypeptides in NDH: the L subunit corresponds the active site like R319 (R880 in bXOR) and F353 (F914 in to the N-terminal domain and the M subunit is homologous to L L bXOR) (Fig. S3). R880 of bXOR was suggested to stabilize the the C-terminal domain. Bioinformatic analysis has revealed split developing negative charge on the substrate during the hydroxy- molybdopterin subunits in 1 of 3 other bacterial NDHs (SI Text lation step, and its mutation results in an increase of the and Fig. S1). Moorella thermoacetica, Carboxydothermus hydrog- dissociation constant, K , for substrate binding and a decrease enoformans, Petrotoga mobilis, Clostridium asparagiforme,5 D ␣ in the rate constant of enzyme reduction, kred (36). The conser- -proteobacterial species, Alkaliphilus oremlandii, and Alkaliphi- vation of amino acids essential for catalysis indicates common lus metalliredigens have split subunits in NDH homologs of ways of substrate binding and transition state stabilization in hitherto unknown catalytic activity (SI Text and Fig. S2). NDH and XORs. Based on these similarities (Fig. S3), we constructed a structural model for nicotinate binding in the Active-Site Structure and Selenium Detection. The substrate channel active site of NDH in analogy to the structure of bXOR in is formed by residues from the M and L subunits, which create complex with 2-hydroxy-6-methylpurine (27). It has recently a funnel leading to the active site. The active site contains the been shown that 2-hydroxy-6-methylpurine binds between phe- molybdenum ion with a distorted square pyramidal coordination nylalanine residues in the active site of bXOR, with the carbon (Fig. 2A). Two enedithiolate sulfurs and 1 hydroxo-ligand occupy atom to be hydroxylated in close proximity to the equatorial the equatorial positions at the Mo-ion and an oxo-ligand is found hydroxyl ligand. To allow binding of nicotinate in the active site in the apical position. The activity of NDH is known to depend of NDH the side chain of F353L has to rotate to assume a similar on the presence of selenium in the active site, and different Se conformation as found for substrate/ligand-bound bXOR (21, occupancies depending on preparation and specific activity have 27) (Fig. S3). Modeling of substrate binding in analogy to bXOR been observed (14, 18, 34). Therefore, detection of selenium produces a mechanistically reasonable complex. The nitrogen could not solely rely on the strength of the observed electron atom and carboxylate of nicotinate are at hydrogen-bonding densities, which depend on the occupancy of the ligands. To distance to Y13M and R319M, respectively. C6, the carbon atom locate Se in the structure of NDH we collected complete to be hydroxylated, is in a distance of Ϸ2 Å from the hydroxyl crystallographic datasets at 2 different X-ray wavelengths, one at ligand and 2.5 Å from the Se ligand. This binding mode would the low-energy side (␭ ϭ 0.9803 Å) and one at the high-energy facilitate the nucleophilic attack of the hydroxyl ligand on C6 and side (␭ ϭ 0.9780 Å) of the X-ray absorption K-edge of selenium the concomitant hydride transfer from C6 to the Se ligand (Fig. S4). (Table 1). Whereas Bijvoet difference maps of the low-energy dataset show only a signal at the Mo ion, Bijvoet difference maps Computational Study Comparing MoASe and MoAS. The functional calculated from the high-energy dataset reveal strong additional advantage of selenium over sulfur as a ligand is not immediately

Wagener et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 29, 2021 with an equatorial MoASe rather than MoAS group, with ethylaldehyde and formamide as simple substrates (Fig. 4 A and B, respectively). Ethylaldehyde and formamide were used as simple analogs for xanthine and nicotinate to reduce the nec- essary computational resources. Additionally, it is not necessary to include alternative protonation states with these 2 substrates. The calculations indicate that the transition state for the first step of the reaction is stabilized by 3.1–3.4 kcal/mol upon substitution of selenium for sulfur in the reaction with both ethylaldehyde (⌬H‡ is 3.47 kcal/mol for MoAS and 0.13 kcal/mol for MoASe) and formamide (⌬H‡ is 14.81 kcal/mol for MoAS and 11.67 kcal/mol for MoASe). This amounts to a factor of 200–300 in rate acceleration for this step. The geometry of the optimized MoASe-containing structure Fig. 3. Molecular orbital view and the relative energy of MoAE ␲ and ␲*(Eϭ is square pyramidal, with the MoAE(Eϭ S, Se), the hydroxyl O, S, Se). Depicted are the bonding interactions between the dxy orbital of the group, and the enedithiolate ligand of the pyranopterin cofactor molybdenum and the p orbital of the coordinated chalcogen in bonding x in the equatorial plane (Fig. S5). Table S1 gives the metric (lower) and antibonding configurations, with the relative energies for A A MoAO, MoAS, and MoASe species indicated. parameters for both Mo S and Mo Se structures. It can be seen that substitution of selenium for sulfur does not have a significant influence on the bond angles made by the ligands, evident. The molybdenum sulfido ligand found in the active site although the MoASe is longer than the MoAS (2.31 vs. 2.18 Å), of molybdenum hydroxylases like XOR (MoAS) plays an im- as expected given the larger selenium and weaker MoASe bond. portant role in the catalytic activity of these enzymes. Its The calculated geometry of the selenium-containing model is replacement by a (second) MoAO group to give the so-called very similar to what has been found in the structure of NDH and desulfo form of the enzyme leads to its complete inactivation. both show a Mo-Se distance of Ϸ2.3 Å. The hydride transfer taking place in the initial step of the reaction can be considered a nucleophilic attack of the hydride Discussion on the MoAE antibonding orbital (␲*) (E ϭ O, S). The stronger The nature of the selenium moiety of NDH has been investigated ␲ interaction leads to higher ␲* anti-bonding energy, which over the last decade by using various approaches. After the raises the barrier of the reaction. The strength of the MoAO ␲ discovery that selenium is essential for hydroxylase activity (13), bond relative to that of MoAS leads to a very high ␲* anti- Dilworth (14) showed that selenium is a component of NDH, bonding orbital and is presumably the basis for the lack of which could be released by heat treatment or the addition of activity seen in the desulfo form of the enzymes (Fig. 3). chaotrophic agents like urea. That selenium in NDH is not part AMoASe rather than MoAS is expected to yield an even of a stable organic molecule but that is instead present as a weaker ␲ bond with molybdenum than sulfur and oxygen (O Ͼ neutral selenol or an inorganic selenide has been indicated by the S Ͼ Se) (Fig. 3) and is thus expected to increase the reactivity liberation of selenium from NDH by incubation with alkylating of the enzyme in any mechanism involving hydride transfer. In agents, resulting in the formation of dialkylselenides (14). The the case of less reactive carbon centers, the increased reactivity approximate ratio of 1 mol of selenium per mol of NDH was may be important in catalyzing the hydroxylation. Computa- established by EPR studies of NDH with the 77Se isotope that tional studies have been carried out after the reaction of a showed the nuclear spin of 77Se couples to the Mo(V) electron molybdenum center such as that found in bXOR and NDH, but spin, suggesting that the selenium could be a ligand to molyb-

Fig. 4. Calculated reaction coordinates. (A) Reaction of ethylaldehyde with the MoO(OH)S (Left) and the MoO(OH)Se analogue (Right). (B) Reaction of formamide with the MoO(OH)S (Left) and the MoO(OH)Se analogue (Right).

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902210106 Wagener et al. Downloaded by guest on September 29, 2021 denum (18). This left the possibilities that selenium replaced the used to purify the protein until freezing the protein crystals used for structure sulfido ligand at the molybdenum or that it could be weakly determination. For all steps, including crystallization of NDH, buffer condi- bound to a heteroatom of a separate cofactor adjacent to tions were used for which NDH was reported to be most stable and active (34). ⅐ molybdenum. The crystal structure of NDH presented here Frozen E. barkeri cells were resuspended in 50 mM Tris HCl (pH 7.8), 10 mM shows that selenium indeed replaces sulfur as a molybdenum NaCl, 1 mM EDTA, and 2 mM DTT (buffer A) and broken by 5 cycles of A sonication. After centrifugation for 30 min the cell-free extract was loaded on ligand, present as Mo Se. The refined bond length of 2.3 Å a Source 30Q column. NDH was eluted from the anionic exchange column by agrees well with the expected bond length for such a terminal a linear gradient of buffer A containing 0.5 M NaCl. Pooled fractions were selenido ligand and specifically is too short for a selenol ligand loaded onto a hydroxyapatite column without preceding buffer exchange. (37). No further stabilization of the selenium by other interac- Active NDH eluted with 120 mM KPO4, pH 7.3. Active fractions were collected, tions is observed, nor do we find residues such as cysteines in the rebuffered in 50 mM Tris⅐HCl (pH 7.8), 0.2 M KCl, 1 mM EDTA, and 2 mM DTT vicinity of the Mo ion, which might form a bond to the selenido and concentrated to 20 mg/mL. The protein was stored at ϩ4 °C and used ligand. Access to the selenido ligand is partly blocked by F353L within 5 days. Approximately 1.5 mg NDH could be obtained from 10 g of cells. (Fig. 2A), which could explain why incubation of active NDH To obtain protein with higher purity, an additional gelfiltration step (Super- ⅐ with potassium cyanide does not lead to a rapid inactivation of dex 200) was performed in 50 mM Tris HCl (pH 7.8), 0.2 M KCl, 1 mM EDTA, and the enzyme and inactive enzyme could not be reactivated by the 2 mM DTT. All purification steps were carried out under anoxic conditions in a glove box containing 95% N and 5% H . addition of sodium selenide or selenophosphate (18, 34). 2 2 Whether selenium is also a ligand to molybdenum in other Activity Measurement. Enzyme assays were conducted according to Gladyshev selenium-dependent molybdenum hydroxylases remains to be et al. (34) with some modifications. Hydroxylase activity was measured under established in future structural studies. For the selenium- anoxic conditions in 100 mM KPO4 (pH 7.0), 50 mM nicotinate (pH 7.5), 1 mM containing XOR from E. barkeri it has been shown that the NADPϩ, and 5 mM DTT. The reaction was started by addition of NADPϩ and enzyme can be inactivated with potassium cyanide and enzyme followed by absorption increase at 340 nm. thus inactivated can be reactivated by incubation with selenide under reducing conditions (17), observations that could be Crystallographic Methods. NDH was crystallized by the vapor diffusion method explained by the presence of a selenido ligand bound to molyb- in a hanging drop. The enzyme preparations used for crystallization had Ϫ denum such as is seen here with NDH. However, with the purine specific activities of 11–20 units⅐mg 1. The drop contained a 1:1 mixture of Ϫ1 hydroxylase from C. purinolyticum no magnetic interaction be- protein solution (10 mg/mL ) with reservoir solution containing 18–20% PEG ⅐ tween the nuclear spin of 77Se and the Mo(V) electron spin could 3350, 0.1 M Tris HCl (pH 7.5), 75 mM NaNO3, 5% glycerol, or 1% 2,4- methylpentandiol. Crystals were harvested in the corresponding soaking be detected, raising the possibility that the labile selenium is not solutions containing 15% (vol/vol) (2R,3R)-butanediol (Sigma), shock-frozen, BIOCHEMISTRY coordinated to Mo in all forms of this enzyme (38). The catalytic and stored in liquid nitrogen. Diffraction data were collected at Ϫ180 °C on a advantage of the incorporation of selenium in a molybdenum rotating anode X-ray generator (Nonius FR591; Bruker) equipped with an hydroxylase is evident from comparing XORs of different image plate detector (mar345dtb; Marresearch) (native, Table 1) and at the organisms. While for bXORs with a sulfido ligand at the BM-14, European Synchrotron Radiation Facility, Grenoble (above and below molybdenum turnover number of 2–25 sϪ1 has been reported Se-edge, Table 1). The protein structure model was build with Coot (41) and (39), the selenium containing XOR from E. barkeri achieves MAIN 2000 (42). Positional and temperature refinement was carried out with turnover rates Ͼ400 sϪ1 (17). If the XOR from E. barkeri has a CNS (43). Anomalous difference Fourier maps were calculated by using CNS selenido ligand like NDH, the higher rates are likely caused by (43). The final refinement statistics and stereochemical analyses using PRO- an acceleration of the hydride transfer step, as suggested by our CHECK (44) are shown in Table 1. Ramachandran statistics revealed 86% in the most favored, 13% in the additionally allowed, 1% in the generously allowed, model calculations. and 0% in the disallowed regions. Simulated annealing omit maps were used For the molybdenum hydroxylase family we now see that at to validate the active-site structure reported. least 3 variations for the common theme MoO(OH)X coordi- nation sphere exist, where X is: (i) S for XORs (40), quinoline Model Calculations. Calculations were carried out by using hybrid density oxidoreductase (24), and 4-hydroxybenzoyl-CoA reductase (23); functional theory (B3LYP) as implemented in Gaussian 03. The structures were (ii) SCu for carbon monoxide dehydrogenase (28); and, as fully optimized and confirmed as minima or transition state by calculating the detailed above (iii) Se for NDH. All active sites can be converted vibrational frequency. The structures were optimized with B3LYP using to an inactive state in which X is O, to give a second MoAO LANL2DZ ECP (45, 46) augmented with f polarization functions (47) for Mo and ligand. These variations demonstrate how nature adapts a pro- 6–31G(d) for the rest of atoms. For the hydrogen that performs the hydride ϩϩ tein bound ligand-metal motif for different substrates and transfer 6–31 G(d,p) was used. The zero point energies were calculated reactivities by the exchange of 1 ligand and indicate a flexible with same basis set and level of theory. All quoted energies are corrected to zero point energy and are without temperature correction. biological chemistry of molybdenum enzymes by ligand tuning. ACKNOWLEDGMENTS. H.D. was supported by Deutsche Forschungsgemein- Materials and Methods schaft Grant DO 785/2–2 and the Fonds der Chemischen Industrie. R.H. and Purification of NDH. As NDH is instable and looses activity with time (34) our H.D. received travel funds from the Bavaria California Technology Center to experimental strategy was to take Ͻ1 week from breaking the E. barkeri cells initiate the collaboration.

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