Search

MycG is a P450 monooxygenase that catalyzes the sequential hydroxylation and epoxidation of mycinamicin IV (M-IV), the last two steps in the biosynthesis of mycinamicin II, a macrolide antibiotic isolated from Micromonospora griseorubida. The crystal structure of MycG with M-IV bound was previously determined but showed the bound substrate in an orientation that did not rationalize the observed regiochemistry of M-IV hydroxylation. Nuclear magnetic resonance paramagnetic relaxation enhancements provided evidence of an orientation of M-IV in the MycG active site more compatible with the observed chemistry, but substrate-induced changes in the enzyme structure were not characterized. We now describe the use of amide 1H-15N residual dipolar couplings as experimental restraints in solvated "soft annealing" molecular dynamics simulations to generate solution structural ensembles of M-IV-bound MycG. Chemical shift perturbations, hydrogen-deuterium exchange, and 15N relaxation behavior provide insight into the dynamic and electronic perturbations in the MycG structure in response to M-IV binding. The solution and crystallographic structures are compared, and the possibility that the crystallographic orientation of bound M-IV represents an inhibitory mode is discussed.

The human steroidogenic cytochrome P450 CYP17A1 catalyzes two types of reactions in the biosynthetic pathway leading from pregnenolone to testosterone and several other steroid hormones. The first is the hydroxylation of pregnenolone or progesterone to the corresponding 17α-hydroxy steroid, followed by a lyase reaction that converts these 17α-hydroxy intermediates to the androgens dehydroepiandrosterone and androstenedione, respectively. cytochrome b5 (cytb5) is known to act as both an effector and electron donor for the lyase oxidations, markedly stimulating the rate of the lyase reaction in its presence relative to the rate in its absence. Extensive sequential backbone 1H,15N and 13C nuclear magnetic resonance assignments have now been made for oxidized CYP17A1 bound to the prostate cancer drug and inhibitor abiraterone. This is the first eukaryotic P450 for which such assignments are now available. These assignments allow more complete interpretation of the structural perturbations observed upon cytb5 addition. Possible mechanism(s) for the effector activity of cytb5 are discussed in light of this new information. [ABSTRACT FROM AUTHOR]

Research has been conducted on the acireductone dioxygenases, enzymes involved in cell cycle regulation. The structure of the catalytic nickel center in the nickel-acireductone dioxygenases and the enzyme-substrate complex has been investigated via the use of the X-ray absorption spectroscopy and the details are discussed.

Research has been conducted on the vertebrate-type ferredoxins, small acidic proteins acting as electron shuttles between NAD(P)H-dependent reductases and cytochromes. The comparison of the functional C-terminal domains of these ferredoxins has been carried out via the use of the nuclear magnetic resonance spectroscopy and the results are reported.

The two dioxygenases of Klebsiella pneumoniae though bind different metal ions exhibit similar polypeptide sequence and defined mechanisms. The binding of a dianion dethio analogue of the aci-reductone intermediate forms a four- and five- membered cyclic peroxide intermediates. Data indicate that cyclopropyl substrate analogue inhibits both dioxygenases.

Results reveal that the metal cluster binding loop of the ferredoxin putidaredoxin primarily determines redox-dependent conformational selection in Pseudomonas putida. Nuclear magnetic resonance spectroscopic data on conformational exchange property of Putidaredoxin indicate that reduced protein form exhibits fewer conformational substates than the oxidized protein form.

The study presents nuclear magnetic resonance methods used to characterize solution structure of ion pairs that are formed by quarternary ammonium ions derived from quinine alkaloids. Results include investigation of the asymmetric induction origin in reductions catalyzed by the cations.

The motions of the tetrabutylammonium (TBA) cation and ion aggregates were characterized in solutions of TBA tetrahydridoborate and TBA chloride in nonpolar solvents. Findings suggest that aggregate size is limited to fewer than five ion pairs even in the fairly concentrated solutions of ion pairs used in the experiment. Although self-diffusion is a translational rather than a rotational phenomenon, the size of the aggregate formed appears to determine both rotational and translational motion. This suggests that the aggregates are roughly spherical.

Homonuclear NMR methods develop a model for the solution structure of oxidized putidaredoxin (Pdx). Ferredoxin transferring electron to monooxygenases Pdx has an identical folding topology to other ferredoxins but a more compact structure in the C-terminal region. Site-directed mutagenesis and model compounds explain the metallic group environment of ferredoxins.

Two dimensional H NMR spectral analysis suggests a model for the solution structure of oxidized and reduced forms of putidaredoxin (Pdx) proteins, based on H resonances. Free electron spin density increases on the metal cluster. Pdx encounters a broadening of H resonances by the metal cluster, indicating the position of the metal cluster in the protein. A slow electron self-exchange is noted in the chemical shift time scale of half-reduced Pdx.