Omega oxygenases: nonheme-iron enzymes and P450 cytochromes

Enzymes that effect with ease one of the most difficult chemical reactions, hydroxylation of an unfunctionalized alkyl group, are of particular interest because highly reactive intermediates must be produced. A typical example, the hydroxylation of fatty acids in the omega position, is now known to occur widely in nature. The catalysts, which can be called "omega-oxygenases," also insert molecular oxygen into a variety of other substrates at positions removed from activating functional groups, as in steroids, eicosanoids, and numerous drugs and other xenobiotics. Progress in the characterization of bacterial nonheme-iron enzymes, and plant, bacterial, and mammalian P450 cytochromes that catalyze fatty acid omega-oxidation, and evidence for multiple functional oxidants are summarized.     

Evidence for two enzymatic pathways for omega-oxidation of docosanoic acid in rat liver microsomes

We studied the omega-oxidation of docosanoic acid (C22:0) in rat liver microsomes. C22:0 and 22-hydroxy-docosanoic acid (omega-hydroxy-C22:0) were used as substrates, and the reaction products were analyzed by electrospray ionization mass spectrometry. In the presence of NADPH, omega-oxidation of C22:0 produced not only the hydroxylated product, omega-hydroxy-C22:0, but also the dicarboxylic acid of C22:0, docosanedioic acid (C22:0-DCA). When rat liver microsomes were incubated with omega-hydroxy-C22:0 in the presence of either NAD+ or NADPH, C22:0-DCA was formed readily. Formation of C22:0-DCA from either C22:0 or omega-hydroxy-C22:0 with NADPH as cofactor was inhibited strongly by miconazole and disulfiram, whereas no inhibition was found with NAD+ as cofactor. Furthermore, omega-oxidation of C22:0 was reduced significantly when molecular oxygen was depleted. The high sensitivity toward the more specific cytochrome P450 inhibitors ketoconazole and 17-octadecynoic acid suggests that hydroxylation of C22:0 and omega-hydroxy-C22:0 may be catalyzed by one or more cytochrome P450 hydroxylases belonging to the CYP4A and/or CYP4F subfamily. This study demonstrates that C22:0 is a substrate for the omega-oxidation system in rat liver microsomes and that the product of the first hydroxylation step, omega-hydroxy-C22:0, may undergo further oxidation via two distinct pathways driven by NAD+ or NADPH.     

An enzymatic method for the production of 6-oxohexanoic acid from 6-aminohexanoic acid by an enzyme oxidizing ω-amino compounds from Phialemonium sp. AIU 274

An enzymatic method for 6-oxohexanoic acid production was developed using 6-aminohexanoic acid and an ω-amino group-oxidizing enzyme (ω-AOX) from Phialemonium sp. AIU 274. 6-Oxohexanoic acid was produced from 6-aminohexanoic acid with 100% yield by incubation with 0.3 U of the ω-AOX and 20 U of catalase at 30 °C for 30 h in 0.1 M potassium phosphate buffer (pH 7.0).     

Mutagenicity of BCNU and related chloroethylnitrosoureas in Drosophila.

BCNU and 10 related chloroethylnitrosoureas were tested for their ability to induce sex-linked recessive lethals in Drosophila spermatozoa. All chloroethylnitrosoureas tested were potent mutagens. Among the substances with one chloroethylnitrosourea group, chlorozotocin, BCNU and methanesulfonyloxyethyl chloroethylnitrosourea exhibited the strongest mutagenic effects. Two hydroxyalkyl chloroethylnitrosoureas behaved as potent mutagens too, although the mutation frequencies obtained were one order of magnitude lower relative to the other substances. Among the compounds with two chloroethylnitrosourea groups, bisCNU-ethane and bisCNU-diphenylmethane were most active. When the interconnecting polymethylene chain was elongated from 2 methylene groups (bisCNU-ethane) to 6 methylene groups (bisCNU-hexane), the mutagenic activity decreased by a factor of 2. The mutagenic activity of polymethylene bischloroethylnitrosoureas with connecting chains of intermediate length was not different from bisCNU-hexane. Differences in mutagenic activity were supposed to reflect different concentrations reaching the target cells, possibly in part as a result of differences in transportability of the substances.     

Antibacterial properties of bile acid oxazoline compounds

Chenooxazoline³ (50–100 μM) inhibited (>50%) both 7α and 7β-dehydroxylase activities in whole cells and cell extracts of $\text{Eubacterium}$ sp. V.P.I. 12708. Chenooxazoline (>50 μM) and methylchenooxazoline (>25 μM) but not lithooxazoline (≤100 μM) inhibited growing cultures of $\text{Eubacterium}$ sp. V.P.I. 12708. Chenooxazoline (100 μM) also inhibited the growth of certain members of the genera $\text{Eubacterium}$, $\text{Clostridium}$, $\text{Bacteroides}$ and $\text{Staphylococcus}$ but not $\text{Pseudomonas}$, $\text{Escherichia}$, $\text{Salmonella}$ or the eucaryotic microorganism, $\text{Saccharomyces cerevisiae}$ (_< 400 μM).     

Identity of Phosphodiesterase and Phosphomonoesterase Activities with Nuclease P1 (a Nuclease from Penicillium citrinum)

Enzymatic properties of a purified Penicillium nuclease (designated as nuclease P₁) were investigated. The enzyme activities for RNA, heat-denatured DNA, native DNA, 3′-AMP and 2′-AMP showed a great degree of similarity with respect to the following properties: a) Range of stable pH (5~8), b) temperature optima (at around 70°C), c) thermostability (about 50% inactivation at 67°C, pH 6.0 for 15 min, d) effect of metal ions and SH inhibitors, e) requirement of Zn²⁺, f) protection from the heat-inactivation by albumin and Zn²⁺, g) inactivation on standing in the cold and reactivation on heating, h) sensitivity to protease, and i) competitive relationship between substrates in the enzyme reaction. Moreover, the ratio of enzyme activities in several mutants of Penicillium citrinum was constant. From these results, together with constant ratio of the specific activities throughout purification, it is concluded that a single enzyme might be responsible for both phosphodiesterase and phosphomonoesterase functions.     

Synthesis of (R)- or (S)-valinol using ω-transaminases in aqueous and organic media

Valinol is part of numerous pharmaceuticals and has various other important applications. Optically pure valinol (ee >99%) was prepared employing different ω-transaminases from the corresponding prochiral hydroxy ketone. By the choice of the enzyme the (R)- as well as the (S)-enantiomer were accessible. Reductive amination was performed in organic solvent (MTBE) using 2-propyl amine as amine donor whereas alanine was applied in or in aqueous medium. Transformations in phosphate buffer were successfully performed even at 200 mM substrate concentration (20.4 g/L) leading to 99% (R) and 94% (S) conversion with perfect optical purity (>99% ee).     

Reduction of Nomega-hydroxy-L-arginine to L-arginine by pig liver microsomes, mitochondria, and human liver microsomes

Nomega-Hydroxy-L-arginine, the intermediate in nitric oxide formation from L-arginine catalyzed by NO synthase, can be released into the extracellular space. It has been suggested that it can circulate and exert paracrine effects. Since it cannot only be used as substrate by NO synthases, but can also be oxidized by cytochrome P450 and other hemoproteins in a superoxide-dependent manner, it has been proposed that it can serve as NO donor. In the present study, the in vitro reduction of Nomega-hydroxy-L-arginine was examined. Pig and human liver microsomes as well as pig liver mitochondria were capable of reducing Nomega-hydroxy-L-arginine to L-arginine in an oxygen-insensitive enzymatic reaction. These results demonstrate that this metabolic pathway has to be considered when suggesting Nomega-hydroxy-L-arginine as NO-precursor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme, the benzamidoxime reductase, was unable to replace microsomes to produce L-arginine from Nomega-hydroxy-L-arginine.     

Glycerol-derived ester oligomers from cork suberin

The cork suberin polyester was partially depolymerized by a methanolysis reaction catalyzed by calcium hydroxide. The methanolisate was analysed by ESI-MS/MS in the form of [M+Li](+) adduct-ions. This reaction solubilized a mixture of monomers and oligomers, including a set of glycerol-derived dimeric and trimeric esters. Four types of glycerol esters were identified: monoacylglycerols of alpha,omega-diacids, of omega-hydroxyacids and of monoacids; diglycerol diesters of alpha,omega-diacids; diacylglycerols of alpha,omega-diacids; monoacylglycerols of linear dimeric esters of alpha,omega-diacids and omega-hydroxyacids. The alpha,omega-diacids and omega-hydroxyacids found as monomer residues in the glycerol esters are the main ones found as cork suberin monomers. It is concluded that suberin is a glycerol-derived lipid of polymeric dimensions. Due to the protective and insulating role that it plays in plants, suberin should be considered together with the other known glycerolipids that build up biological membranes.