ABSTRACT
BACKGROUND: Structural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11. METHODS: A series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast. RESULTS: Most of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F'/G' cassette role and substrate access channels. CONCLUSIONS: Some sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity. GENERAL SIGNIFICANCE: The result provides a new basis for determining the structural elements that control functions in complex enzymes.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP2B6/metabolism , Steroid Hydroxylases/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/chemistry , Aryl Hydrocarbon Hydroxylases/genetics , Coumarins/metabolism , Cyclophosphamide/metabolism , Cytochrome P-450 CYP2B6/chemistry , Cytochrome P-450 CYP2B6/genetics , Cytochrome P450 Family 2 , Dogs , Humans , Kinetics , Molecular Docking Simulation , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Steroid Hydroxylases/chemistry , Steroid Hydroxylases/genetics , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Extensive knowledge of the protein components of the senile plaques, one of the hallmark lesions of Alzheimer's disease, has been acquired over the years, but their lipid composition remains poorly known. Evidence suggests that cholesterol contributes to the pathogenesis of Alzheimer's disease. However, its presence within senile plaques has never been ascertained with analytic methods. Senile plaques were microdissected from sections of the isocortex in three Braak VI Alzheimer's disease cases and compared with a similar number of samples from the adjoining neuropil, free of amyloid-beta peptide (A beta) deposit. Two cases were apo epsilon 4/apo epsilon 3, and one case was apo epsilon 3/apoepsilon3. A known quantity of (13)C-labeled cholesterol was added to the samples as a standard. After hexane extraction, cholesterol content was analyzed by liquid chromatography coupled with electrospray ionization mass spectrometry. The mean concentration of free cholesterol was 4.25 +/- 0.1 attomoles/microm(3) in the senile plaques and 2.2 +/- 0.49 attomoles/microm(3) in the neuropil (t = 4.41, P < 0.0009). The quantity of free cholesterol per senile plaque (67 +/- 16 femtomol) is similar to the published quantity of A beta peptide. The highly significant increase in the cholesterol concentration, associated with the increased risk of Alzheimer's disease linked to the apo epsilon 4 allele, suggests new pathogenetic mechanisms.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Cholesterol/metabolism , Microdissection , Plaque, Amyloid/metabolism , Amyloid beta-Peptides/metabolism , Apolipoproteins E/metabolism , Calibration , Cholesterol/analysis , Cholesterol/chemistry , Humans , Mass Spectrometry , Neuropil/metabolismABSTRACT
Combinatorial libraries coding for mosaic enzymes with predefined crossover points constitute useful tools to address and model structure-function relationships and for functional optimization of enzymes based on multivariate statistics. The presented method, called sequence-independent generation of a chimera-ordered library (SIGNAL), allows easy shuffling of any predefined amino acid segment between two or more proteins. This method is particularly well adapted to the exchange of protein structural modules. The procedure could also be well suited to generate ordered combinatorial libraries independent of sequence similarities in a robotized manner. Sequence segments to be recombined are first extracted by PCR from a single-stranded template coding for an enzyme of interest using a biotin-avidin-based method. This technique allows the reduction of parental template contamination in the final library. Specific PCR primers allow amplification of two complementary mosaic DNA fragments, overlapping in the region to be exchanged. Fragments are finally reassembled using a fusion PCR. The process is illustrated via the construction of a set of mosaic CYP2B enzymes using this highly modular approach.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Combinatorial Chemistry Techniques/methods , Gene Library , Oxidoreductases, N-Demethylating/genetics , Steroid Hydroxylases/genetics , Algorithms , Amino Acid Sequence , Animals , Aryl Hydrocarbon Hydroxylases/metabolism , Base Sequence , Biotinylation , Cloning, Molecular , Cytochrome P-450 CYP2B6 , Cytochrome P450 Family 2 , DNA/chemistry , DNA Primers/genetics , DNA Shuffling , Dogs , Humans , Molecular Sequence Data , Nucleic Acid Amplification Techniques , Open Reading Frames , Oxidoreductases, N-Demethylating/metabolism , Plasmids , Polymerase Chain Reaction , Sequence Homology, Amino Acid , Steroid Hydroxylases/metabolism , Templates, GeneticABSTRACT
The beta(3) adrenergic receptor (beta(3)AR) is the predominant beta subtype in human brown adipocytes and is essential for regulating thermogenic lipolysis. To establish a novel experimental system for the biochemical analysis of this protein, we engineered several yeast strains. We show that the sterol background of the host strain greatly modulates the beta(3)AR expression but not in the same way as it modulates the beta(2) adrenergic receptor (beta(2)AR), the other main studied adipocyte subtype. The human beta(3)AR expressed in yeast is N-glycosylated but not phosphorylated. This latter characteristic distinguishes it from the beta(2)AR. We showed that both beta(2)AR and beta(3)AR follow the secretory pathway to the yeast plasma membrane (PM) and are degraded in the vacuole. In the yeast strains used in this work, the two receptors also share a common mechanism of direct signal transduction through the yeast G(alpha) protein, Gpa1p. These strains thus appear to be useful for biochemical and structural studies of the human beta(3)AR in an in vivo reconstitution system.
