Crystal structure of oxidized trimethylamine N -oxide reductase from Shewanella massilia at 2.5 å resolution 1 1Edited by R. Huber

104

The Escherichia coli NapA (periplasmic nitrate reductase) contains a [4Fe-4S] cluster and a Mo-bis-molybdopterin guanine dinucleotide cofactor. The NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). These proteins have been purified and studied by spectropotentiometry, and the structure of NapA has been determined. In contrast to the well characterized heterodimeric NapAB systems of ␣-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the ␥-proteobacterial E. coli NapA and NapB proteins purify independently and not as a tight heterodimeric complex. This relatively weak interaction is reflected in dissociation constants of 15 and 32 M determined for oxidized and reduced NapAB complexes, respectively. The surface electrostatic potential of E. coli NapA in the apparent NapB binding region is markedly less polar and anionic than that of the ␣-proteobacterial NapA, which may underlie the weaker binding of NapB. The molybdenum ion coordination sphere of E. coli NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo-O bond length is 2.6 Å , which is indicative of a water ligand. The potential range over which the Mo 6؉ state is reduced to the Mo 5؉ state in either NapA (between ؉100 and ؊100 mV) or the NapAB complex (؊150 to ؊350 mV) is much lower than that reported for R. sphaeroides NapA (midpoint potential Mo 6؉/5؉ > ؉350 mV), and the form of the Mo 5؉ EPR signal is quite distinct. In E. coli NapA or NapAB, the Mo 5؉ state could not be further reduced to Mo 4؉. We then propose a catalytic cycle for E. coli NapA in which nitrate binds to the Mo 5؉ ion and where a stable des-oxo Mo 6؉ species may participate.Bacterial nitrate reductases are molybdoenzymes that catalyze the two-electron reduction of nitrate to nitrite. They can be classified into three groups according to their localization and function, namely membrane-bound respiratory, periplasmic respiratory, or cytoplasmic assimilatory enzymes (1, 2). Bacterial respiratory membrane-bound nitrate reductases, such as Escherichia coli NarGHI, are generally integral membrane protein complexes that have an active site on the cytoplasmic face of the membrane and couple quinol oxidation by nitrate to the generation of a transmembrane proton electrochemical gradient (2). The catalytic subunit, NarG, contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) 3 cofactor and a [4Fe-4S] cluster (3, 4). Periplasmic nitrate reductases (Nap) are also linked to quinol oxidation in respiratory electron transport chains, but do not conserve the free energy of the QH 2 -nitrate couple. Nitrate reduction via Nap can be coupled to energy conservation if the primary quinone reductase, for example NADH dehydrogenase or formate dehydrogenase, generates a proton electrochemical gradient. Thus Nap systems have a range of physiological functions that include the disposal of reducing equivalents during aerobic growth on reduced carbon substrates and anaerob...

Section: Figure 3 Spectropotentiometric Properties Of the Momentioning

confidence: 99%

Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia coli

Jepson¹,

Mohan

Clarke³

et al. 2007

104

“…Considerable progress has already been made toward this objective. In addition to the Rhodobacter DMSO reductases (9,15,47,48), the crystal structures of Desulfovibrio gigas aldehyde oxidoreductase (14,49), E. coli formate dehydrogenase H (11), chicken liver sulfite oxidase (13), Shewanella massilia TMAO reductase (12), and the tungsten-containing Pyrococcus furiosus aldehyde oxidoreductase (50) have all been solved. However, of these, only R. sphaeroides DMSO reductase has been cloned, heterologously expressed in an active form, and purified.…”

Section: Figmentioning

confidence: 99%

“…An exciting recent development in the molybdoenzyme field is the elucidation of the x-ray crystallographic structures of a number of proteins (9,(11)(12)(13)(14)(15), including at least one from each of the three major families, setting the stage for detailed structure-function studies relating to substrate specificities, identification of active site residues, and delineation of internal electron transfer pathways in proteins containing multiple prosthetic groups. In particular, the effect of site-directed mutagenesis on the electronic and chemical properties of the molybdenum centers can be examined using kinetic techniques, analysis of substrate specificities, and a variety of spectroscopic techniques including EXAFS, RR, EPR, and x-ray crystallography.…”

mentioning

confidence: 99%

Re-design of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase

Hilton

Temple

Rajagopalan

1999

The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6؋ Histag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4 -3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me 2 SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4 -1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N 1 -oxide reductase activity increases by over 400%.

“…The other oxo group in this structure and the single oxo group in the R. sphaeroides Me 2 SO reductase crystal structure are hydrogen bonded to the OH of Tyr-114 (2, 4 -6). Although both of these residues are conserved in BSO reductase (Tyr-88 and Trp-90 (13)), only the tryptophan is conserved in all members of the Me 2 SO reductase family (10). This observation and the crystallographic evidence showing that it is the oxo group hydrogen bonded to the tryptophan that interacts with Me 2 S (6, 20) provide the rationale for preferring a hydrogen-bonding interaction with Trp-90 rather than Tyr-88.…”

mentioning

confidence: 94%

“…1a) (1)(2)(3). Although the recent proliferation of x-ray crystal structures for mononuclear molybdenum enzymes has revealed a common structure for the molybdopterin cofactor, they have also revealed considerable diversity in the molybdenum coordination environment (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). However, on the basis of the available crystallographic, spectroscopic, primary sequence, and cyanide inhibition data, these enzymes can be divided into three large families (Fig.…”

mentioning

confidence: 99%

Resonance Raman Characterization of Biotin Sulfoxide Reductase

Garton

Temple

Dhawan

et al. 2000

Resonance Raman spectroscopy has been used to define active site structures for oxidized Mo(VI) and reduced Mo(IV) forms of recombinant Rhodobacter sphaeroides biotin sulfoxide reductase expressed in Escherichia coli. On the basis of 18 With the exception of nitrogenase, molybdenum enzymes catalyze formal oxygen atom transfer between water and substrate and contain an active site in which the molybdenum is coordinated by the dithiolene side chain of one or two molybdopterins (Fig. 1a) (1-3). Although the recent proliferation of x-ray crystal structures for mononuclear molybdenum enzymes has revealed a common structure for the molybdopterin cofactor, they have also revealed considerable diversity in the molybdenum coordination environment (2-11). However, on the basis of the available crystallographic, spectroscopic, primary sequence, and cyanide inhibition data, these enzymes can be divided into three large families (Fig. 1b) (1). Hydroxylases, such as Desulfovibrio gigas aldehyde oxidoreductase, xanthine oxidase, and xanthine dehydrogenase, represent the xanthine oxidase family, characterized by a Mo(VI) active site with a single molybdopterin, as well as terminal oxo and sulfido groups. The oxotransferase class of molybdoenzymes catalyzes direct oxygen atom transfer between substrate and water and can be divided into two families. The sulfite oxidase family, as represented by sulfite oxidase and assimilatory nitrate reductase, contains Mo(VI) ligated by two oxo groups in addition to a single molybdopterin and a cysteine residue. The dimethyl sulfoxide (Me 2 SO) reductase family comprises mono-oxoMo(VI) sites ligated by two molybdopterins and a protein side chain ligand such as serine, cysteine, or selenocysteine. In addition to Me 2 SO reductase, this family also includes formate dehydrogenase, dissimilatory nitrate reductase, and biotin sulfoxide (BSO) 1 reductase. BSO reductase is found in bacterial systems, such as Rhodobacter sphaeroides and Escherichia coli, and catalyzes the reduction of d-biotin d-sulfoxide to d-biotin (Fig. 2). Although the precise role for BSO reductase in bacterial metabolism has yet to be defined, potential physiological functions include scavenging biotin sulfoxide from the environment, reducing bound intracellular biotin that has become oxidized in an aerobic environment, and protecting the cell from oxidative damage (12). Extensive characterization of this enzyme has been limited due to the low natural abundance of the protein and its constitutive expression (13). However, R. sphaeroides BSO reductase has recently been heterologously expressed in E. coli and characterized as containing the molybdopterin guanine dinucleotide form of the molybdopterin cofactor (14). Using either reduced methyl viologen or NADPH as electron donor, the enzyme was found to exhibit broad substrate specificity and was able to catalyze reduction of nicotinamide-N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide with varying efficiencies, in addition to the reduction of biotin ...