CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85°C. UVvisible and resonance Raman show the enzyme is in the low spin state and only modestly shifts to the high spin state at higher temperatures. Styrene only causes a small spin state shift, but T 1 NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H 2 O 2 -dependent epoxidation of styrene, cis-␤-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80°C for 90 min. Site-specific mutagenesis shows that Thr-213 is catalytically important and Thr-214 helps to control the iron spin state. Topological analysis by reaction with aryldiazenes shows that Thr-213 lies above pyrrole rings A and B and is close to the iron atom, whereas Thr-214 is some distance away. CYP119 is very slowly reduced by putidaredoxin and putidaredoxin reductase, but these proteins support catalytic turnover of the Thr-214 mutants. Protein melting curves indicate that the thermal stability of CYP119 does not depend on the iron spin state or the active site architecture defined by the threonine residues. Independence of thermal stability from active site structural factors should facilitate the engineering of novel thermostable catalysts.

Marine red algae (Rhodophyta) are a rich source of bioactive halogenated natural products, including cyclic terpenes. The biogenesis of certain cyclic halogenated marine natural products is thought to involve marine haloperoxidase enzymes. Evidence is presented that vanadium bromoperoxidase (V-BrPO) isolated and cloned from marine red algae that produce halogenated compounds (e.g., Plocamium cartilagineum, Laurencia pacifica, Corallina officinalis) can catalyze the bromination and cyclization of terpenes and terpene analogues. The V-BrPO-catalyzed reaction with the monoterpene nerol in the presence of bromide ion and hydrogen peroxide produces a monobromo eight-membered cyclic ether similar to laurencin, a brominated C15 acetogenin, from Laurencia glandulifera, along with noncyclic bromohydrin, epoxide, and dibromoproducts; however, reaction of aqueous bromine with nerol produced only noncyclic bromohydrin, epoxide, and dibromoproducts. The V-BrPO-catalyzed reaction with geraniol in the presence of bromide ion and hydrogen peroxide produces two singly brominated six-membered cyclic products, analogous to the ring structures of alpha and beta snyderols, brominated sesquiterpenes from Laurencia, spp., along with noncyclic bromohydrin, epoxide, and dibromoproducts; again, reaction of geraniol with aqueous bromine produces only noncyclic bromohydrin, epoxide, and dibromoproducts. Thus, V-BrPO can direct the electrophilic bromination and cyclization of terpenes.

Globin-centered radicals at tyrosine and tryptophan residues and a peroxyl radical at an unknown location have been reported previously as products of the reaction of metmyoglobin with hydrogen peroxide. The peroxyl radical is shown here to be localized on tryptophan through the use of recombinant sperm whale myoglobin labeled with 13C at the indole ring C-3. Peroxyl radical formation was not prevented by site-directed mutations that replaced all three tyrosines, the distal histidine, or tryptophan 7 with non-oxidizable residues. In contrast, mutation of tryptophan 14 prevents peroxyl radical formation, implicating tryptophan 14 as the specific site of the peroxidation.

Vanadium haloperoxidase enzymes catalyze the oxidation of halide ions by hydrogen peroxide, producing an oxidized intermediate, which can halogenate an organic substrate or react with a second equivalent of hydrogen peroxide to produce dioxygen. Haloperoxidases are thought to be involved in the biogenesis of halogenated natural products isolated from marine organisms, including indoles and terpenes, of which many are selectively oxidized or halogenated. Little has been shown concerning the ability of the marine haloperoxidases to catalyze regioselective reactions. Here we report the regiospecific bromoperoxidative oxidation of 1,3-di-tert-butylindole by V-BrPO from the marine algae Ascophyllum nodosum and Corallina officinalis. Both enzymes catalyze the regiospecific oxidation of 1,3-di-tert-butylindole in a reaction requiring both H(2)O(2) and Br(-) as substrates, but which produce the unbrominated 1,3-di-tert-butyl-2-indolinone product exclusively, in near quantitative yield (i.e. one H(2)O(2) consumed per product). By contrast, reactions with the controlled addition of aqueous bromine solution (HOBr = Br(2) = Br(3)(-)) produce three monobromo and one dibromo-2-indolinone products, all of which differ from the V-BrPO-catalyzed product. Further, reactivities of 1,3-di-tert-butyl-2-indolinone with both aqueous bromine and V-BrPO differ significantly and shed light onto the possible nature of the oxidizing intermediate. This is the first example of a regiospecific bromination by a vanadium haloperoxidase and further extends their usefulness as catalysts.

Cytochrome P450 eryF (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450 eryF by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala 245 by a threonine enables the oxidation of alternative substrates using either H 2 O 2 or O 2 /spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11␣-, 12-, and 16␣-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450 eryF -androstenedione complex. P450 eryF is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450 eryF mutants are also attractive templates for the engineering of novel P450 activities. P450 eryF1 (CYP107A1) catalyzes the stereospecific 6(S)-hydroxylation of deoxyerythronolide B (6-DEB) in the biosynthesis of erythromycin by Saccharopolyspora erythraea ( Fig. 1) (1-3). The genetic manipulation of macrocyclic antibiotic biosynthetic pathways, including that of erythromycin, is currently under investigation as a route for the production of novel antibiotics (4). Hydroxylations catalyzed by P450 enzymes play key roles in these biosynthetic pathways, and modification of the substrate and regiospecificity of appropriate P450 enzymes is therefore of considerable interest. In an early example, targeted disruption of the gene encoding P450 eryF in S. erythraea yielded a strain that was unable to hydroxylate 6-DEB and which therefore produced 6-deoxyerythromycin (3).P450 eryF , a soluble 45-kDa protein, has been crystallized, and its structure has been determined in complexes with both the natural substrate 6-DEB and alternative ligands (5, 6). Two endogenous proteins able to provide electrons for turnover of P450 eryF have been cloned and expressed (7,8), although spinach ferredoxin and FNR function as acceptable surrogate electron donors (9). The structure of P450 eryF reveals two particularly interesting features of the enzyme. One is that the active site is much larger than the active sites of the other structurally defined bacterial P450 enzymes, as expected from the size of its macrocyclic substrate. The second is that the highly conserved threonine, Thr 252 in P450 cam (CYP101), is replaced in P450 eryF by Ala 245 (5, 6). The conserved threonine is thought to be required for dioxygen bond cleavage in the activation of molecular oxy...