Paul R. Ortiz de Montellano

AMP-activated protein kinase phosphorylation of endothelial NO synthase

The AMP‐activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl‐coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co‐immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser‐1177 in the presence of Ca2+‐calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+‐calmodulin, AMPK also phosphorylates eNOS at Thr‐495 in the CaM‐binding sequence, resulting in inhibition of eNOS activity but Thr‐495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.

Cytochrome P-450

ing species placed symmetrically between the endo and exo hydrogens could result in a G value of I, with subsequent asymmetric placement of the oxygen atom selectively at the exo face. The specificity of oxygen atom transfer from the exo face of the camphor skeleton is further demonstrated by the epoxidation of 5 ,6-dehydrocamphor.211 High-resolution NMR and 180rlabeling studies indicate that the product is completely the exo isomer and one atom from molecular oxygen is inserted to form the epoxide. This further emphasizes the apparent geometry of the activated oxygen species as being more closely situated at the exo face of camphor. As for hydroxylation of the natural substrate, the stereochemistry of the epoxidation reaction is the same for the NADH-, iodosobenzene-, and HzOz-driven turnovers. Epoxidations in model systems have been well documented. 234 As with the hydroxylation reactions, differences in the stereochemistry of epoxidation by Mn(III)TPP versus Fe(IIl)TPP may reflect differences in the lifetime of the radical species generated. The absolute stereoselectivity of oxygen insertion at the exo face of camphor is very reminiscent of the rigorous stereochemical integrity of product formation observed with the P-450scc enzyme. Here, cholesterol is hydroxylated at the 22and 20-position to afford (20R, 22R)-dihydroxyBACfERIAL P-450 ENZYMES 477 cholesterol as an intermediate in the production of pregnenolone.235-237 No other stereoisomeric products are obtained with those steroid-metabolizing activities. Nevertheless, the stereochemical parallel to P-450cam cannot be extended to the hydrogen abstraction step since evidence for such a chemical step is tenuous at best in these systems. When 22R-[223H]cholesterol was incubated with a side-chain cleavage preparation, the 22R-[22-3H]-22-hydroxycholesterol intermediate had negligible loss of radioactivity. This suggests selectivity in the hydrogen abstraction if it occurs. On the other hand, the loss of even a small amount of tritium may in fact suggest possible abstraction of either enantiotopic hydrogen in the natural cholesterol, especially when one consdiers the large isotopic selectivity expected for hydrogen rather than tritium. So, whereas, the insertion of the oxygens into the cholesterol side chain demonstrates strict stereochemical selectivity, a systematic investigation of the stereochemistry of the putative hydrogen abstraction steps has not been performed. As mentioned previously, when norbornane is hydroxylated by P-450LM20 both the 2-exoand 2-endo-alcohols are produced. 193 Also, the four exo-hydrogens were replaced by deuterium, and the deuterium-containing product alcohols were examined. It was found that the exo-2norborneol contained four deuteriums, whereas the endo-2-norborneol contained three deuteriums. This demonstrates an interesting contrast to the P-450cam stereochemistry, assuming both P-450LM2 and P-450cam proceed via a similar hydrogen-abstracted substrate species. These norborneol products demonstrate endo abstraction followed by exo rebound, and exo abstraction followed by endo rebound. Thus, both P-450LM2 and P-450cam are capable of endo or exo hydrogen abstraction, although P450cam displays absolute stereochemical control in the oxygen transfer step. This lack of complete stereochemical integrity of oxygen insertion is perhaps explained by a great degree of substrate motion at the active site of P-450LM2 as compared to P-450cam. A comparison of substrate regioselection has been made directly between these two isozymes using camphor, adamantanone, and adamantane as substrates. 154 As expected, P-450LM2 gave both 5-exoand 5-endo-hydroxycamphor, a result analogous to what was observed with norbornane. Also evident were 3-exoand 3-endo-hydroxycamphor. The greater stereoselectivity of oxygen insertion by P-450cam was observed with the other substrates as well (Table V). With P-450cam as the catalyst, 5-hydroxyadamantanone and 1-adamantanol were produced from the nonhydroxylated substrates. As the 5and 1-carbons of these substrates are tertiary, only one isomeric alcohol is possible for these products. With P-450LM2 , adamantanone was processed to 4-antiand 5-hydroxyadamantanone, while adamantane was hydroxylated at both the 1and 2-positions. The authors predict which

Hydrocarbon Hydroxylation by Cytochrome P450 Enzymes

In chemical terms, the regioand stereoselective hydroxylation of hydrocarbon C-H bonds is a very difficult transformation. Nevertheless, these reactions are deftly catalyzed by a variety of metalloenzymes, among which the most diverse are the many members of the cytochrome P450 family. Cytochrome P450 enzymes are found in most classes of organisms, including bacteria, fungi, plants, insects, and mammals. Thousands of such proteins are now known (http://drnelson.utmem.edu/cytochromeP450.html), including 57 in the human genome (1), 20 in Mycobacterium tuberculosis (2), 272 in Arabidopsis (3), and the amazing number of 457 in rice (4). The nomenclature for these enzymes is based on their sequence similarity when appropriately aligned, a somewhat arbitrary similarity cutoff (approximately >40% identity) being used to define members of a family and a higher cutoff (approximately >55% identity) members of a subfamily (5). Thus CYP3A4 corresponds to the fourth enzyme in family 3, subfamily A. This nomenclature allows the naming of enzymes without regard to their origin or specific properties.

Crystal structure of human heme oxygenase-1

Heme oxygenase catalyzes the first step in the oxidative degradation of heme. The crystal structure of heme oxygenase-1 (HO-1) reported here reveals a novel helical fold with the heme sandwiched between two helices. The proximal helix provides a heme iron ligand, His 25. Conserved glycines in the distal helix near the oxygen binding site allow close contact between the helix backbone and heme in addition to providing flexibility for substrate binding and product release. Regioselective oxygenation of the α-meso heme carbon is due primarily to steric influence of the distal helix.

Oxidizing species in the mechanism of cytochrome P450

This review discusses the mechanisms of oxygen activation by cytochrome P450 enzymes, the possible catalytic roles of the various iron—oxygen species formed in the catalytic cycle, and progress in understanding the mechanisms of hydrocarbon hydroxylation, heteroatom oxidation, and olefin epoxidation. The focus of the review is on recent results, but earlier work is discussed as appropriate. The literature through to February 2002 is surveyed, and 175 referenced are cited. Covering: up to February 2002.

Crystal structure of a thermophilic cytochrome P450 from the archaeon Sulfolobus solfataricus.

The structure of the first P450 identified in Archaea, CYP119 from Sulfolobus solfataricus, has been solved in two different crystal forms that differ by the ligand (imidazole or 4-phenylimidazole) coordinated to the heme iron. A comparison of the two structures reveals an unprecedented rearrangement of the active site to adapt to the different size and shape of ligands bound to the heme iron. These changes involve unraveling of the F helix C-terminal segment to extend a loop structure connecting the F and G helices, allowing the longer loop to dip down into the active site and interact with the smaller imidazole ligand. A comparison of CYP119 with P450cam and P450eryF indicates an extensive clustering of aromatic residues may provide the structural basis for the enhanced thermal stability of CYP119. An additional feature of the 4-phenylimidazole-bound structure is a zinc ion tetrahedrally bound by symmetry-related His and Glu residues.

Inhibition of the HIV-1 and HIV-2 proteases by curcumin and curcumin boron complexes

Curcumin, a relatively non-toxic natural product isolated from Curcuma longa, is a modest inhibitor of the HIV-1 (IC50 = 100 microM) and HIV-2 (IC50 = 250 microM) proteases. Simple modifications of the curcumin structure raise the IC50 value but complexes of the central dihydroxy groups of curcumin with boron lower the IC50 to a value as low as 6 microM. The boron complexes are also time-dependent inactivators of the HIV proteases. The increased affinity of the boron complexes may reflect binding of the orthogonal domains of the inhibitor in interesecting sites within the substrate-binding cavity of the enzyme, while activation of the alpha, beta-unsaturated carbonyl group of curcumin by chelation to boron probably accounts for time-dependent inhibition of the enzyme.

The mechanism of heme oxygenase.

Major advances have been made in determining the structure of heme oxygenase and the relationship between its structure and catalytic activity. The nature of the first step in the reaction sequence, heme alpha-meso-hydroxylation, is now clear, although the mechanisms that control the alpha-regiospecificity remain elusive. Hypothetical mechanisms can be written for the steps that convert alpha-meso-hydroxyheme to biliverdin, but these mechanisms must be validated before this complex reaction sequence can be fully understood. The salient conclusion appears to be that the heme-oxygenase reaction reflects the absence of interactions that channel the reaction towards a ferryl species, rather than the presence of interactions that specifically promote heme oxidation.

THE ANTITUBERCULOSIS DRUG ETHIONAMIDE IS ACTIVATED BY A FLAVOPROTEIN MONOOXYGENASE

Ethionamide (ETA), a prodrug that must undergo metabolic activation to exert its cytotoxic effects, is a second line drug against tuberculosis, a disease that infects more than a third of the worlds population. It has been proposed, on the basis of genetic experiments, that ETA is activated in Mycobacterium tuberculosis by the protein encoded by the gene Rv3854c (DeBarber, A. E., Mdluli, K., Bosman, M., Bekker, L.-G., and Barry, C. E., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9677–9682; Baulard, A. R., Betts, J. C., Engohang-Ndong, J., Quan, S., McAdam, R. A., Brennan, P. J., Locht, C., and Besra, G. S. (2000) J. Biol. Chem.275, 28326–28331). We report here the expression, purification, and characterization of the protein encoded by this gene. Our results establish that the enzyme (EtaA) is an FAD-containing enzyme that oxidizes ETA to the corresponding S-oxide. TheS-oxide, which has a similar biological activity as ETA, is further oxidized by EtaA to 2-ethyl-4-amidopyridine, presumably via the unstable doubly oxidized sulfinic acid intermediate. This flavoenzyme also oxidizes thiacetazone, thiobenzamide, and isothionicotinamide and thus is probably responsible, as suggested by the observation of crossover resistance, for the oxidative activation of other thioamide antitubercular drugs.

Substrate Oxidation by Cytochrome P450 Enzymes

Cytochrome P450 mechanisms continue to surprise and delight, although the field is growing to maturity and the completely unexpected is less frequently encountered. Experimentally, the past few years have seen major progress in characterizing the intermediates that are formed as molecular oxygen is activated to the final oxidizing species. All the intermediates, with the exception of the critical ferryl species, have now been directly observed by various spectroscopic and crystallographic methods. The ferric peroxo anion has been found to act as the oxidizing agent with a growing range of highly electrophilic substrates. In contrast, the proposed role for the ferric hydroperoxo complex as an electrophilic oxidizing agent remains a matter of debate, as the evidence advanced in support of the proposal is circumstantial and contradictory. Although the ferryl species remains elusive, it is increasingly clear that it plays the predominant role as the oxidizing agent in the P450 catalytic cycle. A second area that has recently received considerable attention is the mechanism of hydrocarbon hydroxylation, the key question being whether the radical rebound mechanism that has held sway for three decades is in fact valid. The contradictory results obtained with radical and cation probes, which have provided most of the new evidence, must be resolved by further experimentation in order for this quest ionto be settled. The development of a two-state model for the catalytic action of P450 enzymes may be one of the most important recent advances in the field, as it provides a ready explanation for a variety of otherwise contradictory data, some of which argues for concerted and some for nonconcerted oxidation mechanisms. No doubt, the next few years will uncover novel aspects of P450 function and will lead to deeper and more sophisticated understanding of the catalytic mechanisms of the amazing family of P450 enzymes.