Dimethyl Sulfoxide Reductase Research Articles

Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues

Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

Oxygen Atom Transfer Catalysis with Homogenous and Polymer‐Supported N,N‐ and N,N,O‐Heteroscorpionate Dioxidomolybdenum(VI) Complexes

The N,N‐chelate ligand bis(3,5‐dimethyl‐4‐vinylpyrazol‐1‐yl)methane (bdmvpzm) and the N,N,O‐heteroscorpionate ligand bis(3,5‐dimethyl‐4‐vinylpyrazol‐1‐yl)acetic acid (Hbdmvpza), both with vinyl linkers suitable for copolymerisation, were synthesised under Vilsmeier–Haack conditions. The reactions of these ligands with [MoO2Cl2] yielded the corresponding molybdenum(VI) complexes [MoO2Cl2(bdmvpzm)] and [MoO2Cl(bdmvpza)], which were applied in oxygen atom transfer studies as mimics of dimethyl sulfoxide reductase (DMSO reductase). The catalytic properties of the homogeneous complexes and copolymers with embedded complexes were evaluated.

Identification of protein–protein interactions between the TatB and TatC subunits of the twin-arginine translocase system and respiratory enzyme specific chaperones

The Twin-arginine translocation (Tat) pathway serves for translocation of fully folded proteins across the cytoplasmic membrane in bacterial and chloroplast thylakoid membranes. The Escherichia coli Tat system consists of three core components: TatA, TatB, and TatC. The TatB and TatC subunits form the receptor complex for Tat dependent proteins. The TatB protein is composed of a single transmembrane helix and cytoplasmic domain. The structure of TatC revealed six transmembrane helices. Redox Enzyme Maturation Proteins (REMPs) are system specific chaperones, which play roles in the maturation of Tat dependent respiratory enzymes. Here we applied the in vivo bacterial two-hybrid technique to investigate interaction of REMPs with the TatBC proteins, finding that all but the formate dehydrogenase REMP dock to TatB or TatC. We focused on the NarJ subfamily, where DmsD – the REMP for dimethyl sulfoxide reductase in E. coli – was previously shown to interact with TatB and TatC. We found that these REMPs interact with TatC cytoplasmic loops 1, 2 and 4, with the exception of NarJ, that only interacts with 1 and 4. An in vitro isothermal titration calorimetry study was applied to confirm the evidence of interactions between TatC fragments and DmsD chaperone. Using a peptide overlapping array, it was shown that the different NarJ subfamily REMPs interact with different regions of the TatB cytoplasmic domains. The results demonstrate a role of REMP chaperones in targeting respiratory enzymes to the Tat system. The data suggests that the different REMPs may have different mechanisms for this task.

A Model for the Active-Site Formation Process in DMSO Reductase Family Molybdenum Enzymes Involving Oxido-Alcoholato and Oxido-Thiolato Molybdenum(VI) Core Structures.

New bis(ene-1,2-dithiolato)-oxido-alcoholato molybdenum(VI) and -oxido-thiolato molybdenum(VI) anionic complexes, denoted as [Mo(VI)O(ER)L2](-) (E = O, S; L = dimethoxycarboxylate-1,2-ethylenedithiolate), were obtained from the reaction of the corresponding dioxido-molybdenum(VI) precursor complex with either an alcohol or a thiol in the presence of an organic acid (e.g., 10-camphorsulfonic acid) at low temperature. The [Mo(VI)O(ER)L2](-) complexes were isolated and characterized, and the structure of [Mo(VI)O(OEt)L2](-) was determined by X-ray crystallography. The Mo(VI) center in [Mo(VI)O(OEt)L2](-) exhibits a distorted octahedral geometry with the two ene-1,2-dithiolate ligands being symmetry inequivalent. The computed structure of [Mo(VI)O(SR)L2](-) is essentially identical to that of [Mo(VI)O(OR)L2](-). The electronic structures of the resulting molybdenum(VI) complexes were evaluated using electronic absorption spectroscopy and bonding calculations. The nature of the distorted O(h) geometry in these [Mo(VI)O(EEt)L2](-) complexes results in a lowest unoccupied molecular orbital wave function that possesses strong π* interactions between the Mo(d(xy)) orbital and the cis S(p(z)) orbital localized on one sulfur donor from a single ene-1,2-dithiolate ligand. The presence of a covalent Mo-S(dithiolene) bonding interaction in these monooxido Mo(VI) compounds contributes to their low-energy ligand-to-metal charge transfer transitions. A second important d-p π bonding interaction derives from the ∼180° O(oxo)-Mo-E-C dihedral angle involving the alcoholate and thiolate donors, and this contributes to ancillary ligand contributions to the electronic structure of these species. The formation of [Mo(VI)O(OEt)L2](-) and [Mo(VI)O(SEt)L2](-) from the dioxidomolybdenum(VI) precursor may be regarded as a model for the active-site formation process that occurs in the dimethyl sulfoxide reductase family of pyranopterin molybdenum enzymes.

Dimethyl sulfoxide reduction by a hyperhermophilic archaeon Thermococcus onnurineus NA1 via a cysteine-cystine redox shuttle.

A variety of microbes grow by respiration with dimethyl sulfoxide (DMSO) as an electron acceptor, and several distinct DMSO respiratory systems, consisting of electron carriers and a terminal DMSO reductase, have been characterized. The heterotrophic growth of a hyperthermophilic archaeon Thermococcus onnurineus NA1 was enhanced by the addition of DMSO, but the archaeon was not capable of reducing DMSO to DMS directly using a DMSO reductase. Instead, the archaeon reduced DMSO via a cysteine-cystine redox shuttle through a mechanism whereby cystine is microbially reduced to cysteine, which is then reoxidized by DMSO reduction. A thioredoxin reductase-protein disulfide oxidoreductase redox couple was identified to have intracellular cystine-reducing activity, permitting recycle of cysteine. This study presents the first example of DMSO reduction via an electron shuttle. Several Thermococcales species also exhibited enhanced growth coupled with DMSO reduction, probably by disposing of excess reducing power rather than conserving energy.

Maturation of molybdoenzymes and its influence on the pathogenesis of non-typeable Haemophilus influenzae.

Mononuclear molybdenum enzymes of the dimethylsulfoxide (DMSO) reductase family occur exclusively in prokaryotes, and a loss of some these enzymes has been linked to a loss of bacterial virulence in several cases. The MobA protein catalyzes the final step in the synthesis of the molybdenum guanine dinucleotide (MGD) cofactor that is exclusive to enzymes of the DMSO reductase family. MobA has been proposed as a potential target for control of virulence since its inhibition would affect the activities of all molybdoenzymes dependent upon MGD. Here, we have studied the phenotype of a mobA mutant of the host-adapted human pathogen Haemophilus influenzae. H. influenzae causes and contributes to a variety of acute and chronic diseases of the respiratory tract, and several enzymes of the DMSO reductase family are conserved and highly expressed in this bacterium. The mobA mutation caused a significant decrease in the activities of all Mo-enzymes present, and also resulted in a small defect in anaerobic growth. However, we did not detect a defect in in vitro biofilm formation nor in invasion and adherence to human epithelial cells in tissue culture compared to the wild-type. In a murine in vivo model, the mobA mutant showed only a mild attenuation compared to the wild-type. In summary, our data show that MobA is essential for the activities of molybdenum enzymes, but does not appear to affect the fitness of H. influenzae. These results suggest that MobA is unlikely to be a useful target for antimicrobials, at least for the purpose of treating H. influenzae infections.

Transcriptional regulation of dimethyl sulfoxide respiration in a haloarchaeon, Haloferax volcanii.

The halophilic euryarchaeon Haloferax volcanii can grow anaerobically by DMSO respiration. DMSO reductase was induced by DMSO respiration not only under anaerobic growth conditions but also in denitrifying cells of H. volcanii. Deletion of the dmsR gene, encoding a putative regulator for the DMSO reductase, resulted in the loss of anaerobic growth by DMSO respiration. Reporter experiments revealed that only the anaerobic condition was essential for transcription of the dmsEABCD genes encoding DMSO reductase and that transcription was enhanced threefold by supplementation of DMSO. In the ∆dmsR mutant, transcription of the dmsEABCD genes induced by the anaerobic condition was not enhanced by DMSO, suggesting that DmsR is a DMSO-responsive regulator. Transcriptions of the dmsR and mgd genes for Mo-bisMGD biosynthesis were regulated in the same manner as the dmsEABCD genes. These results suggest that the genetic regulation of DMSO respiration in H. volcanii is controlled by at least two systems: one is the DMSO-responsive DmsR, and the other is an unknown anaerobic regulator.

Structural and mechanistic insights on nitrate reductases.

Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data.

Assessment of DFT methods for computing activation energies of Mo/W-mediated reactions.

Using high level ab initio coupled cluster calculations as reference, the performances of 15 commonly used density functionals (DFs) on activation energy calculations for typical Mo/W-mediated reactions have been systematically assessed for the first time in this work. The selected representative Mo/W-mediated reactions cover a wide range from enzymatic reactions to organometallic reactions, which include Mo-catalyzed aldehyde oxidation (aldehyde oxidoreductase), Mo-catalyzed dimethyl sulfoxide (DMSO) reduction (DMSO reductase), W-catalyzed acetylene hydration (acetylene hydratase), Mo/W-mediated olefin metathesis, Mo/W-mediated olefin epoxidation, W-mediated alkyne metathesis, and W-mediated C-H bond activation. Covering both Mo- and W-mediated reactions, four DFs of B2GP-PLYP, M06, B2-PLYP, and B3LYP are uniformly recommended with and without DFT empirical dispersion correction. Among these four DFs, B3LYP is notably improved in performance by DFT empirical dispersion correction. In addition to the absolute value of calculation error, if the trend of DFT results is also a consideration, B2GP-PLYP, B2-PLYP, and M06 keep better performance than other functionals tested and constitute our final recommendation of DFs for both Mo- and W-mediated reactions.

Influence of GTP on system specific chaperone – Twin arginine signal peptide interaction

Many bacterial respiratory redox enzymes depend on the twin-arginine translocase (Tat) system for translocation and membrane insertion. Tat substrates contain an N-terminal twin-arginine (SRRxFLK) motif serving as the targeting signal towards the translocon. Many Tat substrates have a system specific chaperone – redox enzyme maturation protein (REMP) – for final folding and assembly prior to Tat binding. The REMP DmsD strongly interacts with the twin-arginine motif of the DmsA signal sequence of dimethyl sulfoxide (DMSO) reductase. In this study, we have utilized the in vitro protein–protein interaction technique of an affinity pull down assay, as well as protein thermal stability measurement via differential scanning fluorimetry (DSF) to investigate the interaction of guanosine nucleotides (GNPs) with DmsD. Here we have shown highly cooperative binding of DmsD with GTP. A dissociative ligand-binding style isotherm was generated upon GTP titration into the DmsD:DmsAL interaction, yielding sigmoidal release of DmsD with a Hill coefficient of 2.09 and a dissociation constant of 0.99 mM. DSF further illustrated the change in thermal stability upon DmsD interaction with DmsAL and GTP. These results imply the possibility of DmsD detection and binding of GTP during the DMSO protein maturation mechanism, from ribosomal translation to membrane targeting and final assembly. Conceivably, GTP is shown to act as a molecular regulator in the biochemical pathway.

Periplasmic Export of Bile Salt Hydrolase in Escherichia coli by the Twin-Arginine Signal Peptides

Bile salt hydrolase (BSH, EC 3.5.1.24) is considered as an ideal way with lower cost and less side effects to release the risk of coronary heart disease caused by hypercholesterolemia. As bile salt hydrolase from Lactobacillus plantarum BBE7 could not be efficiently exported by PelB signal peptide of the general secretory (Sec) pathway, three twin-arginine signal peptides from twin-arginine translocation (Tat) pathway were synthesized, fused with bsh gene, inserted into expression vectors pET-20b(+) and pET-22b(+), and transformed into four different Escherichia coli hosts, respectively. Among the 24 recombinant bacteria obtained, E. coli BL21 (DE3) pLysS (pET-20b(+)-dmsA-bsh) showed the highest BSH activity in periplasmic fraction, which was further increased to 1.21 ± 0.03 U/mL by orthogonal experimental design. And, signal peptide dimethyl sulfoxide reductase subunit DmsA (DMSA) had the best activity of exported BSH. More importantly, the presence of BSH in the periplasm had proven to be caused by the export rather than cell leakage. For the first time, we report the periplasmic expression of BSH by signal peptides from the Tat pathway. This will lay a solid foundation for the purification and biochemical characterization of BSH from the supernatant, and strategies adopted here could be used for the periplasmic expression of other proteins in E. coli.

NarJ subfamily system specific chaperone diversity and evolution is directed by respiratory enzyme associations.

BackgroundRedox enzyme maturation proteins (REMPs) describe a diverse family of prokaryotic chaperones involved in the biogenesis of anaerobic complex iron sulfur molybdoenzyme (CISM) respiratory systems. Many REMP family studies have focused on NarJ subfamily members from Escherichia coli: NarJ, NarW, DmsD, TorD and YcdY. The aim of this bioinformatics study was to expand upon the evolution, distribution and genetic association of these 5 REMP members within 130 genome sequenced taxonomically diverse species representing 324 Prokaryotic sequences. NarJ subfamily member diversity was examined at the phylum-species level and at the amino acid/nucleotide level to determine how close their genetic associations were between their respective CISM systems within phyla.ResultsThis study revealed that NarJ members possessed unique motifs that distinguished Gram-negative from Gram-positive/Archaeal species and identified a strict genetic association with its nitrate reductase complex (narGHI) operon compared to all other members. NarW appears to be found specifically in Gammaproteobacteria. DmsD also showed close associations with the dimethylsulfoxide reductase (dmsABC) operon compared to TorD. Phylogenetic analysis revealed that YcdY has recently evolved from DmsD and that YcdY has likely diverged into 2 subfamilies linked to Zn- dependent alkaline phosphatase (ycdX) operons and a newly identified operon containing part of Zn-metallopeptidase FtsH complex component (hflC) and NADH-quinone dehydrogenase (mdaB). TorD demonstrated the greatest diversity in operon association. TorD was identifed within operons from either trimethylamine-N-oxide reductase (torAC) or formate dehydrogenase (fdhGHI), where each type of TorD had a unique motif. Additionally a subgroup of dmsD and torD members were also linked to operons with biotin sulfoxide (bisC) and polysulfide reductase (nrfD) indicating a potential role in the maturation of diverse CISM.ConclusionExamination of diverse prokaryotic NarJ subfamily members demonstrates that the evolution and genetic association of each member is uniquely biased by its CISM operon association.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0412-3) contains supplementary material, which is available to authorized users.

A Computational Comparison of Oxygen Atom Transfer Catalyzed by Dimethyl Sulf­oxide Reductase with Mo and W

AbstractA thorough computational study has been performed to investigate oxygen atom transfer (OAT) reactions catalyzed by dimethyl sulfoxide reductase (DMSOR) with a catalytic molybdenum or tungsten ion. Thirteen different density functional theory (DFT) methods have been employed to obtain structural parameters along the reaction pathway, and single‐point energies were computed with local correlation coupled‐cluster methods [LCCSD(T0)]. For both Mo and W, most DFT methods indicate that the enzyme follows a two‐step mechanism with a stable intermediate in which a DMSO molecule coordinates to the metal ion in the +IV oxidation state, and this is also confirmed by the LCCSD(T0) energies. The W‐substituted models have a 26–30 kJ/mol lower activation barrier for the OAT reaction, and the reaction is 63–70 kJ/mol more exothermic than that with Mo. Different DFT methods give widely different activation and reaction energies, which roughly depend on the amount of exact exchange in the method; these differences are also reflected in the structures, especially for the rate‐limiting transition state. Consequently, there is quite a large variation in energies and various energy corrections (thermal, solvation, dispersion, and relativistic; up to 39 kJ/mol) depending on which DFT method is used to obtain the geometries. Therefore, a mechanism predicted by a single method should be viewed with caution.

Unusual pairing between assistants: Interaction of the twin-arginine system-specific chaperone DmsD with the chaperonin GroEL

DmsD is a system-specific chaperone that mediates the biogenesis and maturation of DMSO reductase in Escherichia coli. It is required for DmsAB holoenzyme formation and its targeting to the cytoplasmic membrane for translocation by the twin-arginine translocase. Previous studies suggested that DmsD also interacts with general molecular chaperones to assist in folding of the reductase subunits. Here, the interaction between DmsD and GroEL was further characterized to understand the role of GroEL in DMSO reductase maturation. The inherently weak interaction between the two was strengthened in vivo under growth conditions that induce DMSO reductase expression, and the DmsD–GroEL complex showed negligible change in hydrodynamic diameter by dynamic light scattering when cross-linked. Mapping the cross-linked sites on DmsD shows that the GroEL binding site is in close proximity to the previously characterized DmsA leader binding site. These findings support a role of GroEL in DMSO reductase maturation that likely involves its chaperonin function for assisting in folding of the DmsA preprotein.

Electrochemically mediated enantioselective reduction of chiral sulfoxides.

The respiratory DMSO reductase from Rhodobacter capsulatus catalyzes the reduction of dimethyl sulfoxide to dimethyl sulfide. Herein, we have utilized this Mo enzyme as an enantioselective catalyst to generate optically pure sulfoxides (methyl p-tolyl sulfoxide, methyl phenyl sulfoxide and phenyl vinyl sulfoxide) from racemic starting materials. A hexaaminecobalt coordination compound in its divalent oxidation state was employed as the mediator of electron transfer between the working electrode and DMSO reductase to continually reactivate the enzyme after turnover. In all cases, chiral HPLC analysis of the reaction mixture revealed that the S-sulfoxide was reduced more rapidly leading to enrichment or isolation of the R isomer.

Bis-Molybdopterin guanine dinucleotide is required for persistence of Mycobacterium tuberculosis in guinea pigs.

Mycobacterium tuberculosis is able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo to bis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of the mobA homolog in M. tuberculosis was confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in a mobA deletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or more bis-MGD-dependent enzymes in the persistence of M. tuberculosis in guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen.

Local Hybrid QM/QM Calculations of Reaction Pathways in Metallobiosites.

The accurate calculation of activation barriers is fundamental for the modeling of reaction pathways. However, the computational cost of describing electronic correlation at metal sites can be a deterrent. A possible solution to this problem is the use of hybrid QM/QM approaches, which combine different levels of theory in a single calculation. In this way, the metal and its direct vicinity can be treated, for example, with a coupled cluster method, with the remaining system at the MP2 level. We present calculations on two selected molybdenum complexes modeling the active sites of sulfite oxidase and dimethyl sulfoxide reductase. Use is made of the LMOMO scheme, a hybrid QM/QM model that enables the partition of the system directly at the orbital level, thus avoiding the use of model systems. The results show that even when the high-level calculations are restricted to the orbitals directly located at the metal atom, most of the electronic correlation effects are captured.

Mysteries of metals in metalloenzymes.

Natural metalloenzymes are often the most proficient catalysts in terms of their activity, selectivity, and ability to operate at mild conditions. However, metalloenzymes are occasionally surprising in their selection of catalytic metals, and in their responses to metal substitution. Indeed, from the isolated standpoint of producing the best catalyst, a chemist designing from first-principles would likely choose a different metal. For example, some enzymes employ a redox active metal where a simple Lewis acid is needed. Such are several hydrolases. In other cases, substitution of a non-native metal leads to radical improvements in reactivity. For example, histone deacetylase 8 naturally operates with Zn(2+) in the active site but becomes much more active with Fe(2+). For β-lactamases, the replacement of the native Zn(2+) with Ni(2+) was suggested to lead to higher activity as predicted computationally. There are also intriguing cases, such as Fe(2+)- and Mn(2+)-dependent ribonucleotide reductases and W(4+)- and Mo(4+)-dependent DMSO reductases, where organisms manage to circumvent the scarcity of one metal (e.g., Fe(2+)) by creating protein structures that utilize another metal (e.g., Mn(2+)) for the catalysis of the same reaction. Naturally, even though both metal forms are active, one of the metals is preferred in every-day life, and the other metal variant remains dormant until an emergency strikes in the cell. These examples lead to certain questions. When are catalytic metals selected purely for electronic or structural reasons, implying that enzymatic catalysis is optimized to its maximum? When are metal selections a manifestation of competing evolutionary pressures, where choices are dictated not just by catalytic efficiency but also by other factors in the cell? In other words, how can enzymes be improved as catalysts merely through the use of common biological building blocks available to cells? Addressing these questions is highly relevant to the enzyme design community, where the goal is to prepare maximally efficient quasi-natural enzymes for the catalysis of reactions that interest humankind. Due to competing evolutionary pressures, many natural enzymes may not have evolved to be ideal catalysts and can be improved for the isolated purpose of catalysis in vitro when the competing factors are removed. The goal of this Account is not to cover all the possible stories but rather to highlight how variable enzymatic catalysis can be. We want to bring up possible factors affecting the evolution of enzyme structure, and the large- and intermediate-scale structural and electronic effects that metals can induce in the protein, and most importantly, the opportunities for optimization of these enzymes for catalysis in vitro.

Characterization of a P1-like bacteriophage carrying an SHV-2 extended-spectrum β-lactamase from an Escherichia coli strain.

P1 bacteriophages lysogenize bacteria as independent plasmid-like elements. We describe here a P1-like bacteriophage, RCS47, carrying a blaSHV-2 gene, isolated from a clinical strain of Escherichia coli from phylogroup B1, and we report the prevalence of P1-like prophages in natural E. coli isolates. We found that 70% of the sequence of RCS47, a 115-kb circular molecule, was common to the reference P1 bacteriophage under GenBank accession no. AF234172.1, with the shared sequences being 99% identical. RCS47 had acquired two main foreign DNA fragments: a 9,636-bp fragment mobilized by two IS26 elements containing a blaSHV-2 gene, and an 8,544-bp fragment mobilized by two IS5 elements containing an operon encoding a dimethyl sulfoxide reductase. The reference P1 prophage plasmid replication gene belonged to the IncY incompatibility group, whereas that of RCS47 was from an unknown group. The lytic capacity of RCS47 and blaSHV-2 gene transduction, through the lysogenization of RCS47 in the recipient E. coli strains, were not demonstrated. The prevalence of P1-like prophages in various animal and human E. coli strain collections, as determined by the PCR detection of repL, the lytic replication gene, was 12.6%. No differences in the prevalences of these prophages were found between extended-spectrum β-lactamase (ESBL)-producing and non-ESBL-producing strains (P = 0.69), but this prevalence was lower in phylogroup B2 than in the other phylogroups (P = 0.008), suggesting epistatic interactions between P1 family phages and the genetic background of E. coli strains. P1-like phages are part of the mobile elements that carry antibiotic resistance. The high prevalence of P1-like prophages suggests their role may be underestimated.

Sulfur K-edge X-ray absorption spectroscopy and density functional theory calculations on monooxo Mo(IV) and bisoxo Mo(VI) bis-dithiolenes: insights into the mechanism of oxo transfer in sulfite oxidase and its relation to the mechanism of DMSO reductase.

Sulfur K-edge X-ray absorption spectroscopy (XAS) and density functional theory (DFT) calculations have been used to determine the electronic structures of two complexes [MoIVO(bdt)2]2– and [MoVIO2(bdt)2]2– (bdt = benzene-1,2-dithiolate(2−)) that relate to the reduced and oxidized forms of sulfite oxidase (SO). These are compared with those of previously studied dimethyl sulfoxide reductase (DMSOr) models. DFT calculations supported by the data are extended to evaluate the reaction coordinate for oxo transfer to a phosphite ester substrate. Three possible transition states are found with the one at lowest energy, stabilized by a P–S interaction, in good agreement with experimental kinetics data. Comparison of both oxo transfer reactions shows that in DMSOr, where the oxo is transferred from the substrate to the metal ion, the oxo transfer induces electron transfer, while in SO, where the oxo transfer is from the metal site to the substrate, the electron transfer initiates oxo transfer. This difference in reactivity is related to the difference in frontier molecular orbitals (FMO) of the metal–oxo and substrate–oxo bonds. Finally, these experimentally related calculations are extended to oxo transfer by sulfite oxidase. The presence of only one dithiolene at the enzyme active site selectively activates the equatorial oxo for transfer, and allows facile structural reorganization during turnover.