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The Critical Role of Tryptophan-116 in the Catalytic Cycle Of FEBS 28256 FEBS Letters 563 (2004) 197^202 View metadata, citation and similar papers at core.ac.uk brought to you by CORE The critical role of tryptophan-116 in the catalytic cycleprovided of by Elsevier - Publisher Connector dimethylsulfoxide reductase from Rhodobacter capsulatus Justin P. Ridgea, Kondo-Francois Aguey-Zinsoub, Paul V. Bernhardtb, Graeme R. Hansonc, Alastair G. McEwana;Ã aCentre for Metals in Biology, School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Qld. 4072, Australia bDepartment of Chemistry, School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Qld. 4072, Australia cCentre for Magnetic Resonance, University of Queensland, Brisbane, Qld. 4072, Australia Received 18 December 2003; revised 10 March 2004; accepted 10 March 2004 First published online 23 March 2004 Edited by Hans Eklund periplasmic DMSO reductase [4^8], periplasmic nitrate reduc- Abstract In dimethylsulfoxide reductase of Rhodobacter cap- sulatus tryptophan-116 forms a hydrogen bond with a single oxo tase (NAP) [9], formate dehydrogenases (FDH-H and FDH- ligand bound to the molybdenum ion. Mutation of this residue to N) [10,11], trimethylamine-N-oxide (TMAO) reductase [12] phenylalanine a¡ected the UV/visible spectrum of the puri¢ed and arsenite oxidase (ASO) [13]. The common protein struc- MoVI form of dimethylsulfoxide reductase resulting in the loss tural features of these enzymes give no indication as to how of the characteristic transition at 720 nm. Results of steady- their speci¢c catalytic properties are determined. However, an state kinetic analysis and electrochemical studies suggest that examination of the Mo active site does reveal some signi¢cant tryptophan 116 plays a critical role in stabilizing the hexacoor- di¡erences between enzymes of the DMSO reductase family. VI dinate monooxo Mo form of the enzyme and prevents the In DMSO reductase and TMAO reductase a ¢fth ligand to VI formation of a dioxo pentacoordinate Mo species, generated the Mo is provided by a serine side chain [4,12], while in NAP as a consequence of the dissociation of one of the dithiolene it is a cysteine residue [9] and in FDH a selenocysteine residue ligands of the molybdopterin cofactor from the Mo ion. ß 2004 Published by Elsevier B.V. on behalf of the Federation [10]. In ASO, there is no amino acid ligand to the Mo atom of European Biochemical Societies. [13]. In addition, a terminal oxygen ligand is coordinated to the Mo ion (oxo, hydroxo or aqua) [4,6,9,10,12,13]. Since al- Key words: Dimethylsulfoxide reductase; Molybdenum most all Moco-containing enzymes catalyze an oxygen atom enzyme; Site-directed mutagenesis; Electrochemistry; transfer it seems likely that di¡erences in the Mo active sites Rhodobacter capsulatus represent the tuning of their thermodynamic and structural properties to allow distinct substrates to be used. A further level of diversity in the DMSO reductase family 1. Introduction can be seen in the interaction between the Mo active site and nearby amino acid residues which are not directly coordinated The dimethylsulfoxide (DMSO) reductase family of molyb- to the Mo center. In the crystal structure of DMSO reductase denum enzymes is one of four families of enzyme that contain it has been observed that a tryptophan (residue 116) hydro- a form of pterin molybdenum cofactor (Moco) [1]. In all gen-bonds to an oxo group coordinated to the Mo ion [5,6,8]. enzymes of this superfamily a molybdenum atom coordinated This tryptophan residue is conserved in TMAO reductase, by one or two ene-dithiolate ligands is provided by an organic which is almost structurally identical to DMSO reductase component known as molybdopterin (MPT). A distinctive [12]. In DMSO reductase another amino acid side chain pro- feature of the DMSO reductase family of MPT-containing vided by Y114 is also located close to the Mo active site [8]. enzymes is that the Mo is bound by four thiolate ligands Although recent studies have indicated that this residue does provided by molybdopterin guanine dinucleotide (MGD) moi- not H-bond to the Mo-oxo group [8] two recent studies have eties [1,2]. Enzymes of the DMSO reductase family are thus shown that Y114 plays a role in de¢ning the substrate spec- far limited to prokaryotes where they play an important role i¢city and kinetic parameters of DMSO reductase [14,15].In in anaerobic respiration and lithotrophy [3]. the present paper we report the characterization of a The X-ray crystal structure of several members of the W116CF mutant DMSO reductase from Rhodobacter capsu- DMSO reductase family has been determined. These include latus. This enabled us to investigate the e¡ect of the removal of the hydrogen bonding interaction between the NO1 hydro- gen of W116 and the Mo-oxo group. *Corresponding author. Fax: (61)-7-33654620. E-mail address: [email protected] (A.G. McEwan). 2. Materials and methods Abbreviations: MPT, molybdopterin; DMSO, dimethylsulfoxide; MGD, molybdopterin guanine dinucleotide; NAP, periplasmic nitrate 2.1. Mutagenesis and expression of DMSO reductase reductase; FDH, formate dehydrogenase; ASO, arsenite oxidase; The mutagenesis of the dorA gene encoding DMSO reductase (ac- TMAO, trimethylamine-N-oxide; MV, methyl viologen; DMS, di- cession number U49506) in Rb. capsulatus was carried out essentially methylsul¢de; DCPIP, dichlorophenol indophenol; DDAB, didode- as described in [15]. Brie£y, complementary primers were designed cyldimethylammonium bromide; CSIRO, Commonwealth Scienti¢c against the dorA nucleotide sequence containing the appropriate and Industrial Research Organization; ICP-MS, inductively coupled changes to introduce the W116F mutation (numbering follows [5], plasma mass spectrometry accession no. 1DMS). The primers used were W116Ffwd, 5P-GGC- 0014-5793 / 04 / $30.00 ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/S0014-5793(04)00301-1 FEBS 28256 29-3-04 198 J.P. Ridge et al./FEBS Letters 563 (2004) 197^202 TCCTATGGCTTCAAAAGCCCCGGG-3P, and W116Frev, 5P-CCC- 3. Results GGGGCTTTTGAAGCCATAGGAGCC-3P. These primers were then used to mutate the construct, pUCJRDor [15], using the Quikchange XL site-directed mutagenesis kit (Stratagene) following the manufac- 3.1. UV/visible spectroscopy turer’s protocol. The entire fragment of the dor operon contained in Fig. 1 shows a comparison of the UV/visible absorption pUCJRDor was then sequenced to ensure that no non-speci¢c muta- spectra of the resting MoVI forms of native and W116F tions had occurred. This fragment was then cloned into pJP5603 to DMSO reductase between 300 nm and 800 nm. The most form the construct pJP5603W116F. This construct was then conju- gated into the Rb. capsulatus strain 37b4vdorA as described previously striking feature of the spectrum of the W116F enzyme was [15]. The expression of mutant DMSO reductase was con¢rmed by the absence of the long-wavelength transition which in the Western blotting. The genomic DNA of clones expressing DMSO native enzyme is centered at 720 nm. The two transitions reductase was screened as described previously to con¢rm the pres- present in the spectrum of the native enzyme at 480 and 550 ence of the mutation [15]. One clone was then selected for further nm were still present in the spectrum of W116F DMSO re- study and named 37b4W116F. ductase (Fig. 1) but their intensity was increased. The elec- 2.2. Puri¢cation of W116F DMSO reductase tronic transition centered at 380 nm was more de¢ned in the Strain 37b4W116F was grown under phototrophic condition in spectrum of the W116F DMSO reductase compared to the RCV medium supplemented with the appropriate antibiotics until native enzyme due in part to the deepening of the trough the culture reached late log/early stationary phase. DMSO reductase production was then induced with the addition of 15 mM DMSO and centered at 350 nm in the mutant enzyme. Addition of growth for a further 12 h. DMSO reductase was then puri¢ed from DMS to native DMSO reductase causes the formation of a 37b4W116F as described previously [15]. Purity of the sample was pink species with a characteristic spectrum [7]. This has been con¢rmed by sodium dodecyl sulfate^polyacrylamide gel electropho- shown to be a MoIV complex with DMSO bound [19]. Bray resis analysis. and co-workers have also shown that addition of DMSO to 2.3. UV/visible spectroscopy and steady-state kinetic analysis the resting native enzyme also causes a change in the spectrum All UV/visible spectroscopy was carried out using a Hitachi U-3000 [20]. However, the addition of DMSO or DMS at concentra- spectrophotometer. Steady-state kinetics of dithionite-reduced meth- tions of up to 28 mM and 25 mM respectively caused no ylviologen (MV):DMSO oxidoreductase activity and PES-dependent dimethylsul¢de (DMS):dichlorophenol indophenol (DCPIP) oxidore- appreciable changes in the spectrum of the W116F form of ductase activity were measured as described previously [16,17]. Deter- DMSO reductase. mination of kcat and Km values were carried out in triplicate. Values of kcat are expressed as moles of DCPIP or reduced MV consumed per 3.2. Mo content of W116F DMSO reductase 31 mole of enzyme per second giving units of s . Observation of the loss of the long-wavelength transition in 2.4. Electrochemistry and electrode preparation the optical spectrum for the W116F DMSO reductase imme- Electrochemical measurements were performed with a BAS100B/W diately led to concern over the integrity of the active site and workstation employing a conventional three electrode system compris- whether the Mo atom had been lost. This seemed unlikely ing an edge plane pyrolytic graphite working, Pt wire counter and Ag/ because of the presence of absorption bands in the visible AgCl reference electrodes. Experiments were performed at 25‡C with- region which almost certainly arise from ligand-to-metal in a Belle Technology glovebox under an atmosphere of N2 (O2 con- centration less than 2 ppm).
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