Homogeneous assays based on deoxyribozyme catalysis.

We report herein the first homogeneous assays based on the ribonuclease activity of a deoxyribozyme. The previously reported deoxyribozyme was covalently modified with biotin and used to assay biotin-binding interactions through changes in fluorescence upon substrate turnover. Deoxyribozymes with fluorescence-based reporting have the potential to serve as general analytical tools.

Application of artificial enzymes to the problem of cocaine.

Cocaine mediates its reinforcing and toxic actions through a "loss of function" effect at multiple receptors. The difficulties inherent in blocking a pleiotropic blocker pose a great obstacle for the classical receptor-antagonist approach and have contributed to the failure-to-date to devise specific treatments for cocaine overdose and addiction. As an alternative, we have embarked on an investigation of catalytic antibodies, a programmable class of artificial enzyme, as "peripheral blockers"--agents designed to bind and degrade cocaine in the circulation before it partitions into the central nervous system to exert reinforcing or toxic effects. We synthesized transition-state analogs of cocaine's hydrolysis at its benzoyl ester, immunized mice, prepared hybridomas, and developed the first anti-cocaine catalytic antibodies with the capacity to degrade cocaine to non-reinforcing, non-toxic products. We subsequently identified several families of anti-cocaine catalytic antibodies and found that out most potent antibody, Mab15A10, possessed sufficient activity to block cocaine-induced reinforcement and sudden death in rodent models of addiction and overdose, respectively. With the potential to promote cessation of use, prolong abstinence, and provide a treatment for acute overdose, the artificial enzyme approach comprehensively responds to the problem of cocaine.

Purification and characterization of two extracellular alkaline phosphatases from a psychrophilic arthrobacter isolate.

Two extracellular, heat-labile alkaline phosphatases were purified from a psychrophilic Arthrobacter isolate, D10. The enzymes were active over different pH ranges, used distinct substrates, and had different kinetic properties. Each enzyme reacted specifically to its own antibody during immunoblot analysis. One had both monophosphatase and diesterase activities.

Purification and characterization of a novel 17 alpha-hydroxysteroid dehydrogenase from an intestinal Eubacterium sp. VPI 12708.

A novel steroid-inducible 17 alpha-hydroxysteroid dehydrogenase (17 alpha-HSDH) has been purified over 850-fold from an intestinal Eubacterium sp. VPI 12708. The purified protein has a subunit molecular mass of 42,000 daltons and a native molecular weight (M(r)) of 160,000 as estimated by gel filtration chromatography. Enzyme activity was induced by growth in the presence of androstenedione or cholic acid, but not deoxycholic acid. Enzymatic activity required anaerobic conditions and was highly specific for NADP+ and the 17 alpha-hydroxy group of C-19 steroids. Estimated Km values were 31 microM and 70 microM for androstenedione and epitestosterone, respectively. Vmax values were estimated to be 3250 nmol/min per mg protein and 1800 nmol/min per mg protein for the reductive and oxidative reactions, respectively. The pH optima for both the oxidative and reductive reactions ranged between 5.5 and 7.0. Treatment with EDTA completely inactivated 17 alpha-HSDH activity but activity was partially restored by the addition of either Mg2+ (1 mM) or Zn2+ (10 mM). The N-terminal amino acid sequence analysis of purified enzyme suggests that 17 alpha-HSDH may belong to a disulfide reductase gene family.

Production of two extracellular alkaline phosphatases by a psychrophilic arthrobacter strain.

We surveyed our collection of psychrophilic bacteria to determine the types of phosphatases they produce and whether any had heat-labile activities with potential applications. Assays at different temperatures showed that the activity from one isolate was optimal at 45(deg)C and decreased dramatically above 55(deg)C. This isolate, D10, had the rod-coccus morphological cycle and cell wall amino acids associated with members of the Arthrobacter genus. Interestingly, we found that this strain made two extracellular phosphatases that could be separated by ammonium sulfate fractionation and migration during polyacrylamide gel electrophoresis. One enzyme, designated D10A, hydrolyzed both X-phos (5-bromo-4-chloro-3-indolyl phosphate) and para-nitrophenyl phosphate as substrates and had activity over a broad pH range of 7 to 11. The second enzyme, D10B, lacked activity against X-phos and had a narrow pH range of about 8 to 9. In addition, the D10B enzyme required calcium for activity. The levels of activity of both enzymes decreased for cells grown in media containing more than 100 (mu)M P(infi). These results not only demonstrate the existence of different enzymes from one Arthrobacter strain but also suggest ways in which other studies may have missed phosphatases with unknown requirements.

Purification and characterization of a microbial, NADP-dependent bile acid 7 alpha-hydroxysteroid dehydrogenase.

A constitutively expressed 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) has been purified over 1200-fold, to apparent homogeneity, from an intestinal anaerobic bacterium. The purified protein had a subunit molecular mass of 32 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sepharose CL-6B gel filtration gave a native molecular mass estimate of 124 kDa, suggesting that this enzyme existed as a tetramer of identical subunits. Sulfhydryl reactive compounds were potent inhibitors of 7 alpha-HSDH activity, however, metal ion chelators had no effect upon catalytic activity. The purified enzyme was highly NADP-dependent. Bile acid substrate utilization studies revealed that the enzyme was specific for the oxidation of an unhindered 7 alpha-hydroxyl group. A wide variety of bile acids and analogs were used as substrates including glycine and taurine conjugates, and methyl esters, amines, and bile alcohols. The purified 7 alpha-HSDH obeyed Michaelis-Menten kinetics. Hanes plots of substrate saturation kinetics revealed that most bile acid substrates had Km values ranging from 4 to 20 microM, while Vmax was 601 and 674 mumol/min/mg in the direction of bile acid oxidation and reduction, respectively. Primary kinetic plots and product inhibition patterns were consistent with an ordered sequential mechanism, with NADP(H) binding first. The N-terminal amino acid sequence analysis of the purified enzyme revealed a striking homology to several short, non-zinc alcohol/polyol dehydrogenases and a putative, cholate-inducible, hydroxysteroid dehydrogenase from the same organism. The high specific activity together with the stability, substrate range, and ease of purification, make this enzyme an excellent candidate for use in quantitating primary bile acids both in laboratory and clinical samples. Spectrofluorometry allowed for the quantitation of as little as 10 nM of both free and conjugated primary bile acids.

Catalytic molecular beacons.

We have constructed catalytic molecular beacons from a hammerhead-type deoxyribozyme by a modular design. The deoxyribozyme was engineered to contain a molecular beacon stem-loop module that, when closed, inhibits the deoxyribozyme module and is complementary to a target oligonucleotide. Binding of target oligonucleotides opens the beacon stem-loop and allosterically activates the deoxyribozyme module, which amplifies the recognition event through cleavage of a doubly labeled fluorescent substrate. The customized modular design of catalytic molecular beacons allows for any two single-stranded oligonucleotide sequences to be distinguished in homogenous solution in a single step. Our constructs demonstrate that antisense conformational triggers based on molecular beacons can be used to initiate catalytic events. The selectivity of the system is sufficient for analytical applications and has potential for the construction of deoxyribozyme-based drug delivery tools specifically activated in cells containing somatic mutations.

Aptamer-based folding fluorescent sensor for cocaine.

We adapted in two steps a deoxyribonucleotide-based aptamer to signal the recognition of cocaine: an instability was engineered in one stem of a three-way junction that forms the cocaine-binding pocket and the resulting short stem was end labeled with a fluorophore and a quencher. In the absence of cocaine, two stems are open, but in its presence they close and the three-way junction forms. This major structural change brings fluorophore and quencher together thereby signaling the presence and concentration of ligand. The sensor is selective for cocaine over its metabolites, can operate in serum, and is useful for the screening of cocaine hydrolases.

Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10.

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.