Arsinothricin, an arsenic-containing non-proteinogenic amino acid analog of glutamate, is a broad-spectrum antibiotic.

The emergence and spread of antimicrobial resistance highlights the urgent need for new antibiotics. Organoarsenicals have been used as antimicrobials since Paul Ehrlich's salvarsan. Recently a soil bacterium was shown to produce the organoarsenical arsinothricin. We demonstrate that arsinothricin, a non-proteinogenic analog of glutamate that inhibits glutamine synthetase, is an effective broad-spectrum antibiotic against both Gram-positive and Gram-negative bacteria, suggesting that bacteria have evolved the ability to utilize the pervasive environmental toxic metalloid arsenic to produce a potent antimicrobial. With every new antibiotic, resistance inevitably arises. The arsN1 gene, widely distributed in bacterial arsenic resistance (ars) operons, selectively confers resistance to arsinothricin by acetylation of the α-amino group. Crystal structures of ArsN1 N-acetyltransferase, with or without arsinothricin, shed light on the mechanism of its substrate selectivity. These findings have the potential for development of a new class of organoarsenical antimicrobials and ArsN1 inhibitors.

An integrated portable system for single chip simultaneous measurement of multiple disease associated metabolites.

Metabolites, the small molecules that underpin life, can act as indicators of the physiological state of the body when their abundance varies, offering routes to diagnosis of many diseases. The ability to assay for multiple metabolites simultaneously will underpin a new generation of precision diagnostic tools. Here, we report the development of a handheld device based on complementary metal oxide semiconductor (CMOS) technology with multiple isolated micro-well reaction zones and integrated optical sensing allowing simultaneous enzyme-based assays of multiple metabolites (choline, xanthine, sarcosine and cholesterol) associated with multiple diseases. These metabolites were measured in clinically relevant concentration range with minimum concentrations measured: 25 µM for choline, 100 µM for xanthine, 1.25 µM for sarcosine and 50 µM for cholesterol. Linking the device to an Android-based user interface allows for quantification of metabolites in serum and urine within 2 min of applying samples to the device. The quantitative performance of the device was validated by comparison to accredited tests for cholesterol and glucose.

Microfluidic Devices for Drug Assays.

In this review, we give an overview of the current state of microfluidic-based high-throughput drug assays. In this highly interdisciplinary research field, various approaches have been applied to high-throughput drug screening, including microtiter plate, droplets microfluidics as well as continuous flow, diffusion and concentration gradients-based microfluidic drug assays. Therefore, we reviewed over 100 recent publications in the field and sorted them according to their microfluidic approach. As a result, we are showcasing, comparing and discussing broadly applied approaches as well as singular promising ones that might contribute to shaping the future of this field.

A disulfide-bond cascade mechanism for arsenic(III) S-adenosylmethionine methyltransferase.

Methylation of the toxic metalloid arsenic is widespread in nature. Members of every kingdom have arsenic(III) S-adenosylmethionine (SAM) methyltransferase enzymes, which are termed ArsM in microbes and AS3MT in animals, including humans. Trivalent arsenic(III) is methylated up to three times to form methylarsenite [MAs(III)], dimethylarsenite [DMAs(III)] and the volatile trimethylarsine [TMAs(III)]. In microbes, arsenic methylation is a detoxification process. In humans, MAs(III) and DMAs(III) are more toxic and carcinogenic than either inorganic arsenate or arsenite. Here, new crystal structures are reported of ArsM from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508 (CmArsM) with the bound aromatic arsenicals phenylarsenite [PhAs(III)] at 1.80 Šresolution and reduced roxarsone [Rox(III)] at 2.25 Šresolution. These organoarsenicals are bound to two of four conserved cysteine residues: Cys174 and Cys224. The electron density extends the structure to include a newly identified conserved cysteine residue, Cys44, which is disulfide-bonded to the fourth conserved cysteine residue, Cys72. A second disulfide bond between Cys72 and Cys174 had been observed previously in a structure with bound SAM. The loop containing Cys44 and Cys72 shifts by nearly 6.5 Šin the arsenic(III)-bound structures compared with the SAM-bound structure, which suggests that this movement leads to formation of the Cys72-Cys174 disulfide bond. A model is proposed for the catalytic mechanism of arsenic(III) SAM methyltransferases in which a disulfide-bond cascade maintains the products in the trivalent state.

Pathway of human AS3MT arsenic methylation.

A synthetic gene encoding human As(III) S-adenosylmethionine (SAM) methyltransferase (hAS3MT) was expressed, and the purified enzyme was characterized. The synthetic enzyme is considerably more active than a cDNA-expressed enzyme using endogenous reductants thioredoxin (Trx), thioredoxin reductase (TR), NADPH, and reduced glutathione (GSH). Each of the seven cysteines (the four conserved residues, Cys32, Cys61, Cys156, and Cys206, and nonconserved, Cys72, Cys85, and Cys250) was individually changed to serine. The nonconserved cysteine derivates were still active. None of the individual C32S, C61S, C156S, and C206S derivates were able to methylate As(III). However, the C32S and C61S enzymes retained the ability to methylate MAs(III). These observations suggest that Cys156 and Cys206 play a different role in catalysis than that of Cys32 and Cys61. A homology model built on the structure of a thermophilic orthologue indicates that Cys156 and Cys206 form the As(III) binding site, whereas Cys32 and Cys61 form a disulfide bond. Two observations shed light on the pathway of methylation. First, binding assays using the fluorescence of a single-tryptophan derivative indicate that As(GS)3 binds to the enzyme much faster than inorganic As(III). Second, the major product of the first round of methylation is MAs(III), not MAs(V), and remains enzyme-bound until it is methylated a second time. We propose a new pathway for hAS3MT catalysis that reconciles the hypothesis of Challenger ((1947) Sci. Prog., 35, 396-416) with the pathway proposed by Hayakawa et al. ((2005) Arch. Toxicol., 79, 183-191). The products are the more toxic and more carcinogenic trivalent methylarsenicals, but arsenic undergoes oxidation and reduction as enzyme-bound intermediates.

Purification and properties of Amycolatopsis mediterranei DSM 43304 lipase and its potential in flavour ester synthesis.

An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in a 398-fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33 kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of K(m)(app) and V(max)(app) for p-nitrophenyl palmitate (p-NPP) at the optimal temperature (60°C) and pH (8.0) were 0.099±0.010 mM and 2.53±0.06 mmol/min mg, respectively. The purified AML displayed significant activity towards a range of short and long chain triglyceride substrates and p-nitrophenyl esters. Hydrolysis of glycerol ester bonds occurred non-specifically. The purified AML displayed significant stability in the presence of organic solvents (40%, v/v) and catalyzed the synthesis of the flavour ester isoamyl acetate in free and immobilized states.

Influence of cultivation conditions on the production of a thermostable extracellular lipase from Amycolatopsis mediterranei DSM 43304.

Among several lipase-producing actinomycete strains screened, Amycolatopsis mediterranei DSM 43304 was found to produce a thermostable, extracellular lipase. Culture conditions and nutrient source modification studies involving carbon sources, nitrogen sources, incubation temperature and medium pH were carried out. Lipase activity of 1.37 +/- 0.103 IU/ml of culture medium was obtained in 96 h at 28 degrees C and pH 7.5 using linseed oil and fructose as carbon sources and a combination of phytone peptone and yeast extract (5:1) as nitrogen sources. Under optimal culture conditions, the lipase activity was enhanced 12-fold with a twofold increase in lipase specific activity. The lipase showed maximum activity at 60 degrees C and pH 8.0. The enzyme was stable between pH 5.0 and 9.0 and temperatures up to 60 degrees C. Lipase activity was significantly enhanced by Fe(3+) and strongly inhibited by Hg(2+). Li(+), Mg(2+) and PMSF significantly reduced lipase activity, whereas other metal ions and effectors had no significant effect at 0.01 M concentration. A. mediterranei DSM 43304 lipase exhibited remarkable stability in the presence of a wide range of organic solvents at 25% (v/v) concentration for 24 h. These features render this novel lipase attractive for potential biotechnological applications in organic synthesis reactions.