Purification and characterization of dimethylsulfide monooxygenase from Hyphomicrobium sulfonivorans

Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase from Hyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. It consists of two subunits: DmoA, a 53-kDa FMNH₂-dependent monooxygenase, and DmoB, a 19-kDa NAD(P)H-dependent flavin oxidoreductase. Enzyme kinetics were investigated with a range of substrates and inhibitors. The enzyme had a K_m of 17.2 (± 0.48) μM for DMS (k_cat = 5.45 s⁻¹) and a V_max of 1.25 (± 0.01) μmol NADH oxidized min⁻¹ (mg protein⁻¹). It was inhibited by umbelliferone, 8-anilinonaphthalenesulfonate, a range of metal-chelating agents, and Hg²⁺, Cd²⁺, and Pb²⁺ ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH₂-dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.Dimethylsulfide (DMS) is a volatile organosulfur compound, important in the biogeochemical cycling of sulfur and global climate regulation (4, 9). Bacterial metabolism of DMS is an important sink of the compound in nature and is thought to account for degradation of over 80% of the DMS produced in the marine environment. Although bacterial pathways of DMS degradation have been studied previously in Hyphomicrobium spp. and in Thiobacillus spp. (12, 36), they remain poorly characterized, and few enzymes of DMS metabolism have been purified (see reference 32). DMS monooxygenase was first reported from an assay of NADH-dependent oxygen uptake in the presence of DMS by cell extracts of Hyphomicrobium S (12), an activity also demonstrated in cell extracts of other Hyphomicrobium, Thiobacillus, and Arthrobacter isolates (6, 7, 34), with specific activities around 30 nmol NADH oxidized min⁻¹ mg protein⁻¹. The enzyme has not previously been purified or characterized.The aims of this study were to purify and characterize the DMS monooxygenase enzyme from a member of the genus Hyphomicrobium. Since Hyphomicrobium S is no longer available, studies were undertaken using the type strain of H. sulfonivorans. The strain was originally isolated from garden soil and grows on DMS, as well as the related compounds dimethyl sulfoxide (DMSO) and dimethylsulfone (DMSO₂). During growth on DMSO₂, H. sulfonivorans first reduces DMSO₂ to DMSO by a dimethylsulfone reductase, and subsequently a DMSO reductase converts DMSO to DMS, which is further oxidized to methanethiol and formaldehyde by a DMS monooxygenase. Oxidation of methanethiol to formaldehyde by methanethiol oxidase yields another mole of formaldehyde, which is either assimilated into biomass or oxidized to carbon dioxide to provide reducing equivalents (Fig. ​(Fig.1).1). DMS monooxygenase activity is present in the soluble protein fraction during growth on these compounds (6, 7). A 53-kDa polypeptide was previously observed in organisms grown on DMS, DMSO, and DMSO₂ (6, 7), but its significance in the metabolism of these compounds was unknown.Open in a separate windowFIG. 1.Pathway and enzymes of dimethylsulfone degradation in Hyphomicrobium sulfonivorans S1. Reduction of dimethylsulfone [DMSO₂; (CH₃)₂SO₂] to dimethyl sulfoxide [DMSO; (CH₃)₂SO] and further reduction of DMSO to dimethylsulfide provides the substrate for DMS monooxygenase. Formaldehyde is either assimilated (via the serine cycle) or oxidized to CO₂ providing reducing equivalents. Sulfide is oxidized to sulfate; see reference 7 for further details.     

Re-orienting in space: do animals use global or local geometry strategies?

Here we compare whether birds encode surface geometry using principal axes, medial axes or local geometry. Birds were trained to locate hidden food in two geometrically identical corners of a rectangular arena and subsequently tested in an L-shaped arena. The chicks showed a primary local geometry strategy, and a secondary medial axes strategy, whereas the pigeons showed a medial axes strategy. Neither species showed behaviour supportive of the use of principal axes. This is, to our knowledge, the first study to directly examine these three current theories of geometric encoding.     

Evaluation of attractant flavours for use in oral vaccine baits for badgers (<Emphasis Type="Italic">Meles meles</Emphasis>)

European badgers (Meles meles) are a wildlife reservoir for Mycobacterium bovis infection (tuberculosis) in Ireland and the UK and are implicated in the transmission of infection to livestock. Vaccination of badgers with the human BCG vaccine (Bacille Calmette Guerin) is considered as an important strategy to reduce the burden of disease in this species, and a pragmatic approach is likely to involve oral vaccination. In this study, we evaluated nine different flavours for use as attractants in a prototype oral vaccine bait for European badgers (M. meles): aniseed, apple, cocoa powder, carob powder, curry, fish, garlic, peanut and strawberry. The bait matrix was composed of a natural lipid formulation, developed as a vehicle for oral vaccination against tuberculosis in wildlife. A ‘food for work’ paradigm was employed during the trials to ensure the animals were actively seeking the baits. The trials showed carob and cocoa powders were equally attractive and more attractive than any of the other candidates. Carob and cocoa show potential as bait attractants for badgers and might form part of a novel vaccine delivery system.     

Production of steroids and release of prostaglandins by spherical pig blastocysts in vitro

Levels of pregnenolone and progesterone in spherical pig blastocysts (near 4 and 15 microM respectively) exceeded respective levels in histotroph by about 400-fold. When blastocysts were cultured for 5 days in a synthetic medium containing pregnenolone sulfate (1 microM), daily rates of release of pregnenolone, progesterone, androstenedione, testosterone, oestrone and oestradiol were determined to be near 320, 45, 26, 27, 0.8 and 9.2 fmol per blastocyst respectively. Daily outputs of progesterone and testosterone (fmol per blastocyst) diminished (P less than 0.05) to 1.3 and undetectable levels (less than 2) respectively in the presence of Trilostane (94 microM). Increasing the content of pregnenolone sulfate in the culture medium (to 4.5 microM) resulted in higher daily rates of release of pregnenolone and progesterone (to near 1740 and 380 fmol per blastocyst respectively), verifying activity of 3 beta-hydroxy-delta 5-steroid dehydrogenase, and of arylsulfatase, in tissues of intact spherical pig blastocysts. Prostaglandin E2 was the predominant prostaglandin (PG) released by cultured blastocysts (about 1 fmol per blastocyst per hour), hourly rates of release of PGH2 (derived) and PGF2 alpha being near 0.1 and less than 0.06 fmol per blastocyst respectively. The data establish a capacity for spherical pig blastocysts to release a range of steroids and PGs of possible significance to embryonic growth and development in vivo.     

A twin study of auditory processing indicates that dichotic listening ability is a strongly heritable trait

We administered tests commonly used in the diagnosis of auditory processing disorders (APDs) to twins recruited from the general population. We observed significant correlations in test scores between co-twins. Our analyses of test score correlations among 106 MZ and 33 DZ twin pairs indicate that dichotic listening ability is a highly heritable trait. Dichotic listening is the ability to identify and distinguish different stimuli presented simultaneously to each ear. Deficits in dichotic listening skills indicate a lesion or defect in interhemispheric information processing. Such defects or lesions can be prominent in elderly listeners, language-impaired children, stroke victims, and individuals with PAX6 mutations. Our data indicates that other auditory processing abilities are influenced by shared environment. These findings should help illuminate the etiology of APDs, and help to clarify the relationships between auditory processing abilities and learning/language disorders associated with APDs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA^Gln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA^Glu and Glu-tRNA^Gln. The Glu-tRNA^Gln is then converted to Gln-tRNA^Gln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA^Glu and tRNA^Gln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA^Glu/tRNA^Gln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA^Gln complex reveals the structural determinants responsible for specific tRNA^Gln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.     

Heparin binds 8 kDa gelsolin cross-β-sheet oligomers and accelerates amyloidogenesis by hastening fibril extension

Glycosaminoglycans (GAGs) are highly sulfated linear polysaccharides prevalent in the extracellular matrix, and they associate with virtually all amyloid deposits in vivo. GAGs accelerate the aggregation of many amyloidogenic peptides in vitro, but little mechanistic evidence is available to explain why. Herein, spectroscopic methods demonstrate that GAGs do not affect the secondary structure of the monomeric 8 kDa amyloidogenic fragment of human plasma gelsolin. Moreover, monomerized 8 kDa gelsolin does not bind to heparin under physiological conditions. In contrast, 8 kDa gelsolin cross-β-sheet oligomers and amyloid fibrils bind strongly to heparin, apparently because of electrostatic interactions between the negatively charged polysaccharide and a positively charged region of the 8 kDa gelsolin assemblies. Our observations are consistent with a scaffolding mechanism whereby cross-β-sheet oligomers, upon formation, bind to GAGs, accelerating the fibril extension phase of amyloidogenesis, possibly by concentrating and orienting the oligomers to more efficiently form amyloid fibrils. Notably, heparin decreases the 8 kDa gelsolin concentration necessary for amyloid fibril formation, likely a consequence of fibril stabilization through heparin binding. Because GAG overexpression, which is common in amyloidosis, may represent a strategy for minimizing cross-β-sheet oligomer toxicity by transforming them into amyloid fibrils, the mechanism described herein for GAG-mediated acceleration of 8 kDa gelsolin amyloidogenesis provides a starting point for therapeutic strategy development. The addition of GAG mimetics, small molecule sulfonates shown to reduce the amyloid load in animal models of amyloidosis, to a heparin-accelerated 8 kDa gelsolin aggregation reaction mixture neither significantly alters the rate of amyloidogenesis nor prevents oligomers from binding to GAGs, calling into question their commonly accepted mechanism.     

Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.     

Long continuous actin bundles in Drosophila bristles are constructed by overlapping short filaments

The actin bundles essential for Drosophila bristle elongation are hundreds of microns long and composed of cross-linked unipolar filaments. These long bundles are built from much shorter modules that graft together. Using both confocal and electron microscopy, we demonstrate that newly synthesized modules are short (1-2 microm in length); modules elongate to approximately 3 microm by growing over the surface of longitudinally adjacent modules to form a graft; the grafted regions are initially secured by the forked protein cross-bridge and later by the fascin cross-bridge; actin bundles are smoothed by filament addition and appear continuous and without swellings; and in the absence of grafting, dramatic alterations in cell shape occur that substitutes cell width expansion for elongation. Thus, bundle morphogenesis has several components: module formation, elongation, grafting, and bundle smoothing. These actin bundles are much like a rope or cable, made by overlapping elements that run a small fraction of the overall length, and stiffened by cross-linking.