Directed evolution and characterization of atrazine chlorohydrolase variants with enhanced activity

Atrazine chlorohydrolase (AtzA, EC 3.8.1.8) has attracted widespread interests as it catalyzes conversion of toxic atrazine to nontoxic hydroxyatrazine and can be used in the biodegradation of atrazine. To facilitate this application, a Haematococcus pluvialis-based method was applied to screen AtzA variants from a random mutagenesis library. Eight variants with enhanced enzyme activity were obtained. They showed 2.7- to 5.0-fold increase in specific activity compared with the wild type. Sequencing revealed that the two most active variants contained single substitution at Val12 and Leu395, respectively, while several improved variants contained substitutions at the four sites of Met315, His399, Asn429, and Val466 simultaneously, indicating that these residues contribute to the enzyme activity of AtzA. Kinetic analysis showed that five variants decreased the K _m value 0.6- to 0.9-fold, whereas all the variants increased the catalytic efficiency (k _cat/K _m value) 2.5- to 4.1-fold compared to the wild type. The modeled three-dimensional structure showed that AtzA is comprised of a typical (β/α)₈ domain of the amidohydrolase superfamily and a dual β-sheet domain. An iron ion and five ligand-binding residues are located in the β-barrel core of the (β/α)₈ domain. Some substituted residues are involved in hydrogen bond formation in the (β/α)₈-neighboring β-sheet.     

cAMP binds with high affinity and specificity to the androgen receptor from murine skeletal muscle

cAMP binding of the androgen receptor (AR) from murine skeletal muscle was studied. Testosterone affinity chromatography yielded androgen receptor with about 4000-fold purification. Determination of the cAMP binding in the affinity eluate, by adsorption of protein-cAMP complexes to cellulose ester filters or removal of unbound cAMP by dextran-coated charcoal, was not possible, as the observed binding was not stable during the assays. Displacement studies suggest that this is due to a very fast dissociation kinetics of the binding. The problem could be solved by assaying the components of affinity eluate immobilized to a testosterone affinity resin that stabilizes the cAMP-protein complexes. The cAMP binding found in the affinity eluate shows an upward concave Scatchard plot and is compatible with a model containing two independent binding sites with dissociation constants of 7 and 58 nM. However, a larger number of binding sites or negative cooperativity cannot be excluded. Sixteen cAMP binding sites were observed per testosterone binding site. The binding affinity of cAMP exceeds that of cGMP 200-fold, that of cCMP 2000-fold, and that of AMP and 2',3'-cAMP more than 10,000-fold. Results indicate that cAMP is bound by the AR, although it only represents about 1% of the total protein in the affinity eluate: (i) Specific testosterone and cAMP binding of affinity eluate was copurified by affinity chromatography, density gradient centrifugation, and gel filtration. The ratio of cAMP to testosterone binding in each peak was about 16:1, identical with that found in the total affinity eluate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Directed evolution of the lambda receptor of Escherichia coli through affinity chromatographic selection

Two low-resolution three-dimensional maps of the structure of crystalline ribosomes from the oocytes of the lizard, Lacerta sicula, have been obtained by electron microscopy and image processing. One map, derived from sheets contrasted with gold-thioglucose, shows the whole ribosome in outline. The other map, based on sheets embedded in glucose, shows predominantly the RNA in the ribosome.The distribution of RNA-rich and protein-rich regions within the ribosome was assessed by comparing both maps. The RNA forms a dense central core, while the ribosomal protein is located mainly at the periphery and constitutes most of the ribosome surface. The RNA appears to be accessible at several sites on the surface. The two subunits of the ribosome are not resolved, indicating that they are in close contact with one another. The subunit interface cuts through a region of the ribosome that is particularly rich in RNA.     

Directed evolution of lectins with sugar-binding specificity for 6-sulfo-galactose

6-sulfo-galactose (6S-Gal) is a prevalent motif observed in highly sulfated keratan sulfate, which is closely associated with the glioblastoma malignancy while acting as a critical determinant for endogenous lectins. However, facile detection of this unique glycoepitope is greatly hampered because of a lack of appropriate probes. We have previously reported tailoring an α2-6-linked sialic acid-binding lectin from a ricin-B chain-like galactose-binding protein, EW29Ch, by a reinforced ribosome display system following an error-prone PCR. In this study, we challenged the creation of novel lectins to recognize 6S-Gal-terminated glycans by incorporating a high-throughput screening system with a glycoconjugate microarray. After two rounds of selection procedures, 20 mutants were obtained and 12 were then successfully expressed in Escherichia coli, 8 of which showed a significant affinity for 6'-Sulfo-LN (6-O-sulfo-Galβ1-4GlcNAc), which the parental EW29Ch lacked. Analysis of two representative mutants by frontal affinity chromatography revealed a substantial affinity (K(d) ～3 μm) for a 6S-Gal-terminated glycan. On the basis of the observation that all eight mutants have a common mutation at Glu-20 to Lys, site-directed mutagenesis experiments were performed focusing on this aspect. The results clearly indicated that the E20K mutation is necessary and sufficient to acquire the specificity for 6S-Gal. We also confirmed a difference in binding between E20K and EW29Ch to CHO cells, in which enzymes to catalyze the synthesis of 6S-Gal were overexpressed. The results clearly demonstrate that these mutants have potential to distinguish between cells containing different amounts of 6S-Gal-terminated glycans. This new technology will be used to provide novel tools essential for sulfoglycomics.     

Concentration and cellular distribution of androgen receptor in human prostatic neoplasia: can estrogen treatment increase androgen receptor content?

The concentration of androgen receptor in cytosol (free and total sites) and nuclear fractions from benign (28 specimens) and malignant prostatic tissue from treated (16 specimens) and untreated patients (10 specimens) were assayed using [3H]methyltrienolone (3H R-1881) as ligand under conditions which stabilize AR and prevent binding of 3H R-1881 to progesterone receptor. It was found that optimum results were obtained when sodium molybdate (10 mM) was added after separation of the nuclear pellet rather than during tissue homogenization; when cytosol and nuclear exchange assays were carried out at 15 degrees C rather than at 0 degrees C; and when hydroxylapatite was used to separate free and bound steroid in the nuclear assay. Although AR values were variable in both BPH and carcinoma tissue, certain patterns of concentration, occupancy, and cellular distribution were observed in different patient groups. In BPH and untreated carcinoma tissue, the mean occupancy of cytosol AR by endogenous androgens was high, but the mean nuclear AR concentration was higher in BPH than in carcinoma tissue. Androgen receptor concentrations in tissue from orchiectomized patients were consistent with the effects of androgen deprivation: total cell AR was depleted, and a higher proportion was present as free cytosol AR. However, in tissue from most patients who had been treated with diethylstilbestrol (DES) on a long-term basis, total cell AR values were high. Although most of the AR was present as free cytosol AR, in three of four patients who had been treated with both orchiectomy and DES, the concentrations of bound cytosol AR and nuclear AR were similar to those in untreated patients.     

The pathophysiological role of estrogen receptor variants in human breast cancer

The accumulated evidence supports the expression of estrogen receptor variants at both the mRNA and protein levels. The relative level of expression of some estrogen receptor variant mRNAs and possibly progesterone receptor variant mRNAs is altered during breast tumorigenesis and breast cancer progression. The altered expression of estrogen receptor variants may effect estrogen signal transduction as well as the interpretation of assays where the estimation of estrogen receptor levels is used as a guide to treatment strategies and prognosis.     

Directed evolution of LuxI for enhanced OHHL production

Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.     

Directed evolution of recombinase specificity by split gene reassembly

The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding β-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications.     

Characterization of steroid binding specificity of the androgen receptor in human foreskin fibroblasts

Our objective was to evaluate a convenient in vitro model for measuring steroid affinities to the human androgen receptor. The ability of unlabeled analogues of dihydrotestosterone (DHT) to compete with [3H]DHT for binding to the receptor in human fibroblasts was measured and expressed relative to DHT. The C-3 ketone group and the planar configuration of the A and B rings were critical for binding. Absence of the 10 beta-methyl group increased affinity of the androstane compounds for the receptor. The 17 beta-hydroxyl group was also essential for high affinity binding and addition of a 17 alpha-methyl group enhanced binding. Binding of steroids with a delta 4 double bond was consistently less than that of the 5 alpha-reduced steroids. This was true of both the androstene and estrene series. We conclude that human foreskin fibroblasts offer a useful model for in vitro studies characterizing the effects of steroid structural modifications on binding to the human androgen receptor.     

Target specificity of selective estrogen receptor modulators within human endometrial cancer cells

Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands that function as antagonists in some tissues, but have either partial or full agonist activity in others. SERMs often display variable partial agonist activity in uterine tissues and this activity can be displayed in uterine cell lines such as the human Ishikawa endometrial adenocarcinoma cell line. In this study, we compared the effects of several ER ligands including some SERMs on alkaline phosphatase (AP) activity and the expression of an ER target gene, the progesterone receptor (PR), in Ishikawa cells. As expected, estradiol (E2) was a potent and efficacious activator of both AP activity and PR mRNA expression. 4-Hydroxytamoxifen (4OHT) stimulated AP activity to a level 47% of that of E2 (100nM), while CP 336156 (lasofoxifene) increased AP activity 18%. A benzothiophene, such as LY 117018, a raloxifene analog, stimulated AP even less with values approximately 11% of E2-stimulated levels. A pure antiestrogen, ICI 182,780 did not stimulate AP activity. Interestingly, when we examined the ability of these compounds to increase the expression of the ER target gene, PR, a different rank order of efficacy was detected. After E2, CP 336156 was the most efficacious in increasing PR mRNA with a maximal stimulation of 20% of E2 levels, while 4OHT stimulated only 17%. LY 117018 increased PR mRNA expression 8% while ICI 182,780 did not increase PR mRNA expression at all. These data illustrate the target specificity that a SERM is able to display within a single cell type independent of "tissue specificity" and differential levels of expression of various cofactors. While 4OHT is 160% more active than CP 336156 in terms of inducing AP activity in the Ishikawa cells, CP 336156 has equivalent activity as 4OHT when one examines the ability of these SERMs to induce PR mRNA expression. Since the stimulation of Ishikawa cells by ER ligands is often used to assess the potential in vivo uterotrophic activity, these data indicate that examination of several endpoints in these cells may be necessary in order to fully characterize the activity of SERMs.     

Affinity engineering of maltoporin: variants with enhanced affinity for particular ligands

Affinity-chromatographic selection on immobilized starch was used to selectively enhance the affinity of the maltodextrin-specific pore protein ( maltoporin , LamB protein, or lambda receptor protein) in the outer membrane of E. coli. Selection strategies were established for rare bacteria in large populations producing maltoporin variants with enhanced affinities for both starch and maltose, for starch but not maltose and for maltose but not starch. Three classes of lamB mutants with up to eight-fold increase in affinity for particular ligands were isolated. These mutants provide a unique range of modifications in the specificity of a transport protein.     

Social dominance regulates androgen and estrogen receptor gene expression

In Astatotilapia burtoni, dominant males have higher levels of sex steroid hormones than subordinate males. Because of the complex regulatory interactions between steroid hormones and receptors, we asked whether dominance is also associated with variation in sex steroid receptor gene expression. Using quantitative PCR, we compared the expression of specific subtypes of androgen (AR) and estrogen (ER) receptor genes between dominant and subordinated males in 3 divisions of the brain, the pituitary, and the testes. We measured mRNA levels of AR-alpha, AR-beta, ER-alpha, ER-betaa, and ER-betab, gonadotropin-releasing hormone 1 (GnRH1), and GnRH receptor 1 (GnRH-R1) relative to 18S rRNA. In the anterior part of the brain, we found that dominant males had higher mRNA expression of AR-alpha, AR-beta, ER-betaa, and ER-betab, but not ER-alpha, compared to subordinate males. This effect of dominance was reflected in a positive correlation between testes size and AR-alpha, AR-beta, ER-betaa, and ER-betab in the anterior brain. In addition, mRNA levels of all ARs and ERs in the anterior brain were positively correlated with mRNA level of GnRH1. In the middle and posterior portions of the brain, as well as the testes, steroid receptor mRNA levels were similar among dominants and subordinates. In the pituitary, ER-alpha mRNA level was positively correlated with testes size and AR-alpha mRNA was positively correlated with GnRH-R1 mRNA level. These data suggest that dominant male brains could be more sensitive to sex steroids, which may contribute to the increased complexity of the behavioral repertoires of dominant males.     

Imaging analysis of subcellular correlation of androgen receptor and estrogen receptor alpha in single living cells using green fluorescent protein color variants

Androgen and estrogen act not only in a sex-specific manner but also interactively and synergistically. In the present study, to examine the possible interaction between androgen receptor (AR) and estrogen receptor-alpha (ERalpha), we investigated the subcellular dynamics of AR and ERalpha fused with green fluorescent protein color variants in single living cells using time-lapse microscopy and the technique of fluorescence recovery after photobleaching. AR and ERalpha showed punctate colocalization in the nucleus with estrogen, but not androgen. N-terminal AR deletion mutant did not form a nuclear punctate pattern with either androgen or estrogen. In the presence of AR, but not ERalpha, N-terminal AR deletion mutant formed a punctate nuclear pattern with androgen. AR had different mobility depending on the ligand and the presence of ERalpha. On the other hand, AR had little effect on the stability of ERalpha. ERalpha mutant that does not bind coactivators did not alter the mobility of AR. Taken together, using an imaging technique, we clarified that possible homo/hetero dimerization between AR and ERalpha could be attributed to androgen-estrogen interaction in living cells.     

Structural plasticity and the evolution of antibody affinity and specificity

The germline precursor to the ferrochelatase antibody 7G12 was found to bind the polyether jeffamine in addition to its cognate hapten N-methylmesoporphyrin. A comparison of the X-ray crystal structures of the ligand-free germline Fab and its complex with either hapten or jeffamine reveals that the germline antibody undergoes significant conformational changes upon the binding of these two structurally distinct ligands, which lead to increased antibody-ligand complementarity. The five somatic mutations introduced during affinity maturation lead to enhanced binding affinity for hapten and a loss in affinity for jeffamine. Moreover, a comparison of the crystal structures of the germline and affinity-matured antibodies reveals that somatic mutations not only fix the optimal binding site conformation for the hapten, but also introduce interactions that interfere with the binding of non-hapten molecules. The structural plasticity of this germline antibody and the structural effects of the somatic mutations that result in enhanced affinity and specificity for hapten likely represent general mechanisms used by the immune response, and perhaps primitive proteins, to evolve high affinity, selective receptors for so many distinct chemical structures.     

Estrogen and androgen receptor binding affinity of 10 beta-chloro-estrenen derivatives

10 beta-Chloroestradien-3-one and its derivatives with chlorine substitution in ring A have been prepared. Efficient synthetic methods for 2-chloro- and 4-chloroestradiol are described. The binding affinity of these chlorinated estrogens to the uterine estrogen receptor was measured by a competitive binding assay using [3H]estradiol as ligand. 4-Chloroestradiol showed high binding affinity for the receptor (110% of that of estradiol). 2-Chloroestradiol, 10 beta-chloroestradien-3-one and 4,10 beta-dichloroestradien-3-one had moderate binding affinity. The structures of 10 beta-chloroestradien-3-one and androst-1,4-dien-3-one are very similar and can almost be superimposed. However, their binding affinities to the estrogen and androgen receptor were different. Androst-1,4-dien-3-one displayed no measurable affinity for the estrogen receptor and measurable affinity for the androgen receptor whereas 10 beta-chloroestradien-3-one had very low affinity for the androgen receptor.     

Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde

Mutants of transketolase (TK) with improved substrate specificity towards the non-natural aliphatic aldehyde substrate propionaldehyde have been obtained by directed evolution. We used the same active-site targeted saturation mutagenesis libraries from which we previously identified mutants with improved activity towards glycolaldehyde, which is C2-hydroxylated like all natural TK substrates. Comparison of the new mutants to those obtained previously reveals distinctly different subsets of enzyme active-site mutations with either improved overall enzyme activity, or improved specificity towards either the C2-hydroxylated or non-natural aliphatic aldehyde substrate. While mutation of phylogenetically variant residues was found previously to yield improved enzyme activity on glycolaldehyde, we show here that these mutants in fact gave improved activity on both substrate types. In comparison, the new mutants were obtained at conserved residues which interact with the C2-hydroxyl group of natural substrates, and gave up to 5-fold improvement in specific activity and 64-fold improvement in specificity towards propionaldehyde relative to glycolaldehyde. This suggests that saturation mutagenesis can be more selectively guided for evolution towards either natural or non-natural substrates, using both structural and sequence information.