Biological degradation of Polyethylene terephthalate (PET) plastic and assimilation of the corresponding monomers ethylene glycol and terephthalate (TPA) into central metabolism offers an attractive route for bio-based molecular recycling and bioremediation applications. A key step is the cellular uptake of the non-permeable TPA into bacterial cells which has been shown to be dependent upon the presence of the key tphC gene. However, little is known from a biochemical and structural perspective about the encoded solute binding protein, TphC. Here, we report the biochemical and structural characterisation of TphC in both open and TPA-bound closed conformations. This analysis demonstrates the narrow ligand specificity of TphC towards aromatic para-substituted dicarboxylates, such as TPA and closely related analogues. Further phylogenetic and genomic context analysis of the tph genes reveals homologous operons as a genetic resource for future biotechnological and metabolic engineering efforts towards circular plastic bio-economy solutions.

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.

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.

The emergence and spread of bacterial 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 (AST), a mimetic of the herbicidal antibiotic phosphinothricin (PT). We demonstrate that AST is an effective broad-spectrum antibiotic, showing that bacteria have acquired the ability to utilize environmental arsenic to produce a potent antimicrobial. With every new antibiotic, resistance inevitably arises. The arsN gene, widely distributed in ars operons, confers PT resistance. The functional linkage of arsN to arsenic detoxification was unclear. Here we show that arsN selectively confers resistance to AST. Crystal structures of the ArsN N-acetyltransferase shed light on the mechanism of selectivity. These results may lead to development of a new class of antimicrobials and ArsN inhibitors.

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.

Amine transaminases offer an environmentally sustainable synthesis route for the production of pure chiral amines. However, their catalytic efficiency toward bulky ketone substrates is greatly limited by steric hindrance and therefore presents a great challenge for industrial synthetic applications. We hereby report an example of rational transaminase enzyme design to help alleviate these challenges. Starting from the Vibrio fluvialis amine transaminase that has no detectable catalytic activity toward the bulky aromatic ketone 2-acetylbiphenyl, we employed a rational design strategy combining in silico and in vitro studies to engineer the transaminase enzyme with a minimal number of mutations, achieving an high catalytic activity and high enantioselectivity. We found that, by introducing two mutations W57G/R415A, detectable enzyme activity was achieved. The rationally designed variant, W57F/R88H/V153S/K163F/I259M/R415A/V422A, showed an improvement in reaction rate by more than 1716-fold toward the bulky k...

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 Å resolution and reduced roxarsone [Rox(III)] at 2.25 Å resolution. 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 Å in 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.

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.

ADVERTISEMENT RETURN TO ISSUEPREVLetters to the Edito...Letters to the EditorNEXTResponse to the Letter to the Editor Regarding Our Article (Dheeman et al., 2014)Dharmendra S. Dheeman, Charles Packianathana, Jitesh K. Pillai, and Barry P. Rosen*View Author Information College of Medicine, Florida International University, Miami, Florida 33199, United States*Phone: 305-348-0657; E-mail: [email protected]Cite this: Chem. Res. Toxicol. 2014, 27, 12, 2012Publication Date (Web):November 10, 2014Publication History Published online10 November 2014Published inissue 15 December 2014https://pubs.acs.org/doi/10.1021/tx500439ehttps://doi.org/10.1021/tx500439eletterACS PublicationsCopyright © 2014 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views397Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (110 KB) Get e-AlertscloseSUBJECTS:Anatomy,Metabolism,Oxidation reactions,Oxidation state,Peptides and proteins Get e-Alerts

A newly isolated fungal strain, Penicillium sp. DS-39 (DSM 23773), was found to produce an inducible extracellular lipase when grown in a medium containing 1.0% (v/v) olive oil. Maximum lipase activity was obtained after 120h of incubation at 28°C. The lipase was purified 129-fold with a final specific activity of 308.73IU/mg. The molecular weight of the homogeneous lipase was 43kDa as determined by SDS-PAGE. It was optimally active at pH 5.5 and 45°C. The lipase was most active on triolein and exhibited a broad substrate range with a preference for triacylglycerols containing long chain unsaturated fatty acids. It showed no regio-specificity for the ester bond in triolein. It was activated by Ca2+ and Mn2+, while significant inhibition was observed with Hg2+ and Zn2+. The lipase showed significant stability and activation in the presence of organic solvents with log P≥2.0. These features render Penicillium sp. DS-39 lipase (PEL) a potential biocatalyst for applications such as biodiesel production, enzymatic restructuring, by interesterification of different oils and fats, and biodegradation of oil spills in the environment.

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.

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.