ABSTRACT
Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and longchain fatty acids at the omega-1, omega-2, and omega-3 positions. A continuous spectrophotometric assay for P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycarboxylic acids and the chromophore p-nitrophenolate was reported recently. However, this pNCA assay procedure contained steps that limited its application in high throughput screening, including expression of P450 BM-3 variant F87A in 4-ml cultures, centrifugation, resuspension of the cell pellet, and cell lysis. We have shown that permeabilization of the outer membrane of Escherichia coli DH5alpha with polymyxin B sulfate, EDTA, polyethylenimine, or sodium hexametaphosphate results in rapid conversion of 12-pNCA. A NADPH-generating system consisting of NADP(+), D/L-isocitric acid, and the D/L-isocitrate dehydrogenase of E. coli DH5alpha reduced the cofactor expense more than 10-fold. By avoiding cell lysis, resuspension, and centrifugation, the high throughput protocol allows screening of thousands of samples per day.
Subject(s)
Bacillus megaterium/enzymology , Drug Evaluation, Preclinical/methods , Escherichia coli/enzymology , Microbial Sensitivity Tests/methods , Mixed Function Oxygenases/analysis , Mixed Function Oxygenases/biosynthesis , Automation , Catalysis , Dose-Response Relationship, Drug , Hydroxylation , Isocitrate Dehydrogenase/metabolism , Light , NADP/metabolism , Spectrophotometry , Time Factors , Ultraviolet RaysABSTRACT
Laboratory evolutionists continue to generate better enzymes for industrial and research applications. Exciting developments include new biocatalysts for enantioselective carbon-carbon bond formation and fatty acid production in plants. Creative contributions to the repertoire of evolutionary methods will ensure further growth in applications and expand the scope and complexity of biological design problems that can be addressed. Researchers are also starting to elucidate mechanisms of enzyme adaptation and natural evolution by testing evolutionary scenarios in the laboratory.
Subject(s)
Enzymes/chemistry , Enzymes/genetics , Mutagenesis, Site-Directed , Oxygen/chemistry , Bacterial Proteins/chemistry , Carbon/chemistry , DNA-Directed DNA Polymerase/genetics , Peptide Library , Software , TemperatureABSTRACT
A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [Fe(S-Cys)4](-1),(-2). The design criteria for the target site included taking backbone movements into account, tetrahedral metal-binding, and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(II) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal-free and Cd(II)-bound forms of the variant are folded correctly and are stable. The Fe(III) complex of the metal-binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(III) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.
Subject(s)
Bacterial Proteins/chemistry , Iron/chemistry , Protein Engineering , Rubredoxins/chemistry , Amino Acid Sequence , Binding Sites , Cadmium/chemistry , Circular Dichroism , Cobalt/chemistry , Cysteine/genetics , Electron Spin Resonance Spectroscopy , Magnetic Resonance Spectroscopy , Metalloproteins/chemistry , Models, Molecular , Molecular Sequence Data , Mutagenesis/genetics , Recombinant Proteins/chemistry , SpectrophotometrySubject(s)
Gastrointestinal Diseases/chemically induced , Histamine H1 Antagonists/adverse effects , Intestinal Obstruction/chemically induced , Loratadine/adverse effects , Administration, Oral , Adult , Aged , Delayed-Action Preparations , Esophageal Stenosis/complications , Female , Gastrointestinal Diseases/diagnosis , Histamine H1 Antagonists/administration & dosage , Humans , Intestinal Obstruction/diagnosis , Loratadine/administration & dosageABSTRACT
UNLABELLED: We have studied the incidence of thromboembolic complications in 46 patients. Twenty-one patients had a Medtronic-Hall prosthesis implanted in the mitral position (group A) and 25 patients had the same prosthesis implanted in the aortic position (group B). Several factors were defined initially as "thromboembolic risk factors" and we have tried to establish a correlation between those factors and the actual thromboembolic complications. Group A: 21 patients presented 5 thromboembolic accidents (23.8%). Preeminent factors were: preoperative thromboembolic phenomena, previous heart operations, atrial fibrillation and the presence of congestive heart failure and/or low outpout syndrome in the postoperative period. Group B: 25 patients presented 6 thromboembolic accidents (24%). If we exclude the presence of low outpout syndrome in the postoperative period, there was no correlation between other risk factors and the incidence of thromboembolic complications. We must mention that the incidence of thromboembolic complications in this group diminished significantly (26% to 16%) when oral anticoagulation was continued with aspirin. COMMENTARY: these figures of thromboembolic complications appear too high at first sight if we consider only their absolute value, but are justified when one bears in mind the clinical circumstances of the patients who suffered from thromboembolic complications.
