Engineering Pseudomonas aeruginosa arylsulfatase for hydrolysis of α-configured steroid sulfates.

Steroid sulfate esters are important metabolites for anti-doping efforts in sports, pathology and research. Analysis of these metabolites is facilitated by hydrolysis using either acid or enzymatic catalysis. Although enzymatic hydrolysis is preferred for operating at neutral pH, no known enzyme is capable of hydrolyzing all steroid sulfate metabolites. Pseudomonas aeruginosa arylsulfatase (PaS) is ideal for the hydrolysis of ß-configured steroid sulfates but like other known class I sulfatases it is inefficient at hydrolyzing α-configured steroid sulfates. We have used directed evolution with liquid chromatography mass spectrometry screening to find variants capable of hydrolyzing a α-configured steroid sulfate: etiocholanolone sulfate (ECS). After targeting two regions of PaS, four residues were identified and optimized to yield a final variant with a total of seven mutations (DRN-PaS) capable of hydrolyzing ECS ~80 times faster than the best PaS variant previously available. This DRN-PaS also shows improved activity for other α-configured steroid sulfates. Simultaneous mutagenesis was essential to obtain DRN-PaS due to complementarity between targeted residues.

Evolving a lipase for hydrolysis of natural triglycerides along with enhanced tolerance towards a protease and surfactants.

In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS). In addition, all the variants had very low levels of activity with triglycerides with long aliphatic chains and with naturally occurring oils, like olive oil. Directed evolution was used to select variants with enhanced properties. In the first 10 rounds of evolution, the primary screen was selected for variants capable of hydrolyzing olive oil whereas the secondary screen was selected for enhanced tolerance towards a protease and SDS. In the final six rounds of evolution, the primary and secondary screens identified variants that retained activity after treatment with SDS. Sixteen cycles of evolution gave variants with greatly enhanced lipolytic activity on substrates that had both long (C16 and C18) as well as short (C3 and C8) chains. We found variants that were stable for more than 3 hours in protease concentrations that rapidly degrade the wild-type enzyme. Enhanced tolerance towards SDS was found in variants that could break down naturally occurring lipid and resist protease attack. The amino acid changes that gave enhanced properties were concentrated in the cap domain responsible for substrate binding.

Replacing PAPS: In vitro phase II sulfation of steroids with the liver S9 fraction employing ATP and sodium sulfate.

In vitro technologies provide the capacity to study drug metabolism where in vivo studies are precluded due to ethical or financial constraints. The metabolites generated by in vitro studies can assist anti-doping laboratories to develop protocols for the detection of novel substances that would otherwise evade routine screening efforts. In addition, professional bodies such as the Association of Official Racing Chemists (AORC) currently permit the use of in-vitro-derived reference materials for confirmation purposes providing additional impetus for the development of cost effective in vitro metabolism platforms. In this work, alternative conditions for in vitro phase II sulfation using human, equine or canine liver S9 fraction were developed, with adenosine triphosphate (ATP) and sodium sulfate in place of the expensive and unstable co-factor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), and employed for the generation of six representative steroidal sulfates. Using these conditions, the equine in vitro phase II metabolism of the synthetic or so-called designer steroid furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17ß-ol) was investigated, with ATP and Na2 SO4 providing comparable metabolism to reactions using PAPS. The major in vitro metabolites of furazadrol matched those observed in a previously reported equine in vivo study. Finally, the equine in vitro phase II metabolism of the synthetic steroid superdrol (methasterone, 17ß-hydroxy-2α,17α-dimethyl-5α-androstan-3-one) was performed as a prediction of the in vivo metabolic profile.

Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione, and dihydrotestosterone misuse by means of carbon isotope ratio mass spectrometry.

In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted. Copyright © 2017 John Wiley & Sons, Ltd.

Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates.

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3ß,17ß-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation.

In vitro directed evolution of enzymes expressed by E. coli in microtiter plates.

A method is described for using 96-well plates to prepare libraries of Escherichia coli cultures for screening a library of gene variants. This approach bypasses colony-picking to allow standard molecular biology laboratories to carry out directed evolution efficiently with a 96-well plate-reader and multichannel pipettes. Initial screens are applied to cultures that are rapidly prepared by diluting transformed cells so that an average of four cells starts each culture. Subsequent screens are used to isolate individual enzyme-expressing clones that exhibit activity higher than the parental clone. The outlined method also includes guidelines for preparing a library of gene variants and for optimizing a screening method.

Pinpointing P450s associated with pyrethroid metabolism in the dengue vector, Aedes aegypti: developing new tools to combat insecticide resistance.

BACKGROUND: Pyrethroids are increasingly used to block the transmission of diseases spread by Aedes aegypti such as dengue and yellow fever. However, insecticide resistance poses a serious threat, thus there is an urgent need to identify the genes and proteins associated with pyrethroid resistance in order to produce effective counter measures. In Ae. aegypti, overexpression of P450s such as the CYP9J32 gene have been linked with pyrethroid resistance. Our aim was to confirm the role of CYP9J32 and other P450s in insecticide metabolism in order to identify potential diagnostic resistance markers. METHODOLOGY/PRINCIPAL FINDINGS: We have expressed CYP9J32 in Escherichia coli and show that the enzyme can metabolize the pyrethroids permethrin and deltamethrin. In addition, three other Ae. aegypti P450s (CYP9J24, CYP9J26, CYP9J28) were found capable of pyrethroid metabolism, albeit with lower activity. Both Ae. aegypti and Anopheles gambiae P450s (CYP's 6M2, 6Z2, 6P3) were screened against fluorogenic and luminescent substrates to identify potential diagnostic probes for P450 activity. Luciferin-PPXE was preferentially metabolised by the three major pyrethroid metabolisers (CYP9J32, CYP6M2 and CYP6P3), identifying a potential diagnostic substrate for these P450s. CONCLUSIONS/SIGNIFICANCE: P450s have been identified with the potential to confer pyrethroid resistance in Ae.aegypti. It is recommended that over expression of these enzymes should be monitored as indicators of resistance where pyrethroids are used.

Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana.

In the last decade there have been marked reductions in malaria incidence in sub-Saharan Africa. Sustaining these reductions will rely upon insecticides to control the mosquito malaria vectors. We report that in the primary African malaria vector, Anopheles gambiae sensu stricto, a single enzyme, CYP6M2, confers resistance to two classes of insecticide. This is unique evidence in a disease vector of cross-resistance associated with a single metabolic gene that simultaneously reduces the efficacy of two of the four classes of insecticide routinely used for malaria control. The gene-expression profile of a highly DDT-resistant population of A. gambiae s.s. from Ghana was characterized using a unique whole-genome microarray. A number of genes were significantly overexpressed compared with two susceptible West African colonies, including genes from metabolic families previously linked to insecticide resistance. One of the most significantly overexpressed probe groups (false-discovery rate-adjusted P < 0.0001) belonged to the cytochrome P450 gene CYP6M2. This gene is associated with pyrethroid resistance in wild A. gambiae s.s. populations) and can metabolize both type I and type II pyrethroids in recombinant protein assays. Using in vitro assays we show that recombinant CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in the presence of solubilizing factor sodium cholate.

Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme.

Each of the twelve enzymes for glycolytic fermentation, eleven from Escherichia coli and one from Saccharomyces cerevisiae, have been over-expressed in E. coli and purified with His-tags. Simple assays have been developed for each enzyme and they have been assembled for fermentation of glucose to ethanol. Phosphorus-31 NMR revealed that this in vitro reaction accumulates fructose 1,6-bisphosphate while recycling the cofactors NAD(+) and ATP. This reaction represents a defined ATP-regeneration system that can be tailored to suit in vitro biochemical reactions such as cell-free protein synthesis. The enzyme from S. cerevisiae, pyruvate decarboxylase 1 (Pdc1; EC 4.1.1.1), was identified as one of the major 'flux controlling' enzymes for the reaction and was replaced with an evolved version of Pdc1 that has over 20-fold greater activity under glycolysis reaction conditions. This substitution was only beneficial when the ratio of glycolytic enzymes was adjusted to suit greater Pdc1 activity.

Cytochrome P450 6M2 from the malaria vector Anopheles gambiae metabolizes pyrethroids: Sequential metabolism of deltamethrin revealed.

Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is a major threat to malaria control programmes. Cytochome P450-mediated detoxification is an important resistance mechanism. CYP6M2 is over-expressed in wild populations of permethrin resistant A. gambiae but its role in detoxification is not clear. CYP6M2 was expressed in Escherichia coli and a structural model was produced to examine its role in pyrethroid metabolism. Both permethrin and deltamethrin were metabolized. Rates were enhanced by A. gambiae cytochrome b(5) with kinetic parameters of K(M)=11±1µM and k(cat)=6.1±0.4 per min for permethrin (1:1 cis-trans) and K(M)=2.0±0.3µM and k(cat)=1.2±0.1 per min for deltamethrin. Mass spectrometry and NMR analysis identified 4'-hydroxy deltamethrin and hydroxymethyl deltamethrin as major and minor deltamethrin metabolites respectively. Secondary breakdown products included cyano(3-hydroxyphenyl)methyl deltamethrate and deltamethric acid. CYP6M2 was most highly transcribed in the midgut and Malpighian tubules of adult A. gambiae, consistent with a role in detoxification. Our data indicates that CYP6M2 plays an important role in metabolic resistance to pyrethroids and thus an important target for the design of new tools to combat malaria.

Structural model and functional characterization of the Bemisia tabaci CYP6CM1vQ, a cytochrome P450 associated with high levels of imidacloprid resistance.

The neonicotinoid imidacloprid is one of the most important insecticides worldwide. It is used extensively against the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), an insect pest of eminent importance globally, which was also the first pest to develop high levels of resistance against imidacloprid and other neonicotinoids in the field. Recent reports indicated that in both the B and Q biotypes of B. tabaci, the resistant phenotype is associated with over-expression of the cytochrome P450 gene CYP6CM1. In this study, molecular docking and dynamic simulations were used to analyze interactions of imidacloprid with the biotype Q variant of the CYP6CM1 enzyme (CYP6CM1vQ). The binding mode with the lowest energy in the enzyme active site, the key amino acids involved (i.e. Phe-130 and Phe-226), and the putative hydroxylation site (lowest distance to carbon 5 of the imidazolidine ring system of imidacloprid) were predicted. Heterologous expression of the CYP6CM1vQ confirmed the accuracy of our predictions and demonstrated that the enzyme catalyses the hydroxylation of imidacloprid to its less toxic 5-hydroxy form (K(cat) = 3.2 pmol/min/pmol P450, K(m) = 36 microM). The data identify CYP6CM1vQ as a principle target for inhibitor design, aimed at inactivating insecticide-metabolizing P450s in natural insect pest populations.

Field-caught permethrin-resistant Anopheles gambiae overexpress CYP6P3, a P450 that metabolises pyrethroids.

Insects exposed to pesticides undergo strong natural selection and have developed various adaptive mechanisms to survive. Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is receiving increasing attention because it threatens the sustainability of malaria vector control programs in sub-Saharan Africa. An understanding of the molecular mechanisms conferring pyrethroid resistance gives insight into the processes of evolution of adaptive traits and facilitates the development of simple monitoring tools and novel strategies to restore the efficacy of insecticides. For this purpose, it is essential to understand which mechanisms are important in wild mosquitoes. Here, our aim was to identify enzymes that may be important in metabolic resistance to pyrethroids by measuring gene expression for over 250 genes potentially involved in metabolic resistance in phenotyped individuals from a highly resistant, wild A. gambiae population from Ghana. A cytochrome P450, CYP6P3, was significantly overexpressed in the survivors, and we show that the translated enzyme metabolises both alpha-cyano and non-alpha-cyano pyrethroids. This is the first study to demonstrate the capacity of a P450 identified in wild A. gambiae to metabolise insecticides. The findings add to the understanding of the genetic basis of insecticide resistance in wild mosquito populations.

Directed evolution of yeast pyruvate decarboxylase 1 for attenuated regulation and increased stability.

Five generations of directed evolution resulted in yeast pyruvate decarboxylase 1 (Pdc1) variants with improved activity for 1 mM pyruvate at pH 7.5 in the presence of phosphate. The best variant, named 5LS30, contained the following mutations: A143T, T156A, Q367H, N396I, and K478R. In comparison with native Pdc1, 5LS30 had the substrate concentration required for half-saturation reduced by almost 3-fold at pH 7.5 and the phosphate inhibition reduced by 4-fold at pH 6.0. The apparent cooperativity for pyruvate displayed by 5LS30 was also reduced since it appeared to be activated by pyruvate more easily than the native enzyme. The temperature at which half of the Pdc1 activity was irreversibly lost in 5 min increased from 52.6 degrees C, seen with the native form, to 61.8 degrees C for 5LS30. Curiously, the optimal temperature for Pdc1 activity was found to be dependent upon pyruvate concentration. In 1 mM pyruvate, native Pdc1 performed optimally at 30 degrees C and 5LS30 at 40 degrees C, whereas in 25 mM pyruvate native activity peaked at 45 degrees C and 5LS30 at 55 degrees C. Two screening processes were developed for directed evolution of Pdc1 expressed in Escherichia coli: colony screening and culture screening. The latter proved to be an ideal method for isolating PCR-generated variants of the pdc1 gene with the desired phenotype. In this process, cultures were diluted and partitioned within 96-well plates such that each culture aliquot contained an average of two unique genotypes. This allowed rapid preparation of libraries for analysis of activity in crude lysates and can be applied to other directed evolution projects.

The Aedes aegypti glutathione transferase family.

In this report, we describe the glutathione transferase (GST) gene family in the dengue vector Aedes aegypti and suggest a novel role for a new class of mosquito GSTs. Twenty-six GST genes are present in Ae. aegypti, two of which are alternatively spliced to give a total of 29 transcripts for cytosolic GSTs. The six classes identified in other insect species are all represented and, as in Anopheles gambiae, the majority of the mosquito GSTs belong to the insect-specific Delta and Epsilon classes with eight members each. Sixteen secure 1:1 orthologs were identified between GSTs in Ae. aegypti and An. gambiae, but only four of these have recognisable orthologs in Drosophila melanogaster. Three mosquito-specific GSTs were identified which did not belong to any previously recognised GST classes. One of these, GSTx2, has been previously implicated in conferring 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) resistance in Ae. aegypti from South America. However, we found no evidence for increased levels of this GST protein in DDT/pyrethroid-resistant populations from Thailand. Furthermore, we show that the recombinant GSTX2-2 protein is unable to metabolise DDT. Interestingly, GSTX2-2 showed an affinity for hematin, and this, together with the restricted distribution of this class to haematophagous insects, may indicate a role for these enzymes in protecting mosquitoes against heme toxicity during blood feeding.