Elucidation of the ipso-Substitution Mechanism for Side-Chain Cleavage of α-Quaternary 4-Nonylphenols and 4-t-Butoxyphenol in Sphingobium xenophagum Bayram

Recently we showed that degradation of several nonylphenol isomers with α-quaternary carbon atoms is initiated by ipso-hydroxylation in Sphingobium xenophagum Bayram (F. L. P. Gabriel, A. Heidlberger, D. Rentsch, W. Giger, K. Guenther, and H.-P. E. Kohler, J. Biol. Chem. 280:15526-15533, 2005). Here, we demonstrate with ¹⁸O-labeling experiments that the ipso-hydroxy group was derived from molecular oxygen and that, in the major pathway for cleavage of the alkyl moiety, the resulting nonanol metabolite contained an oxygen atom originating from water and not from the ipso-hydroxy group, as was previously assumed. Our results clearly show that the alkyl cation derived from the α-quaternary nonylphenol 4-(1-ethyl-1,4-dimethyl-pentyl)-phenol through ipso-hydroxylation and subsequent dissociation of the 4-alkyl-4-hydroxy-cyclohexadienone intermediate preferentially combines with a molecule of water to yield the corresponding alcohol and hydroquinone. However, the metabolism of certain α,α-dimethyl-substituted nonylphenols appears to also involve a reaction of the cation with the ipso-hydroxy group to form the corresponding 4-alkoxyphenols. Growth, oxygen uptake, and ¹⁸O-labeling experiments clearly indicate that strain Bayram metabolized 4-t-butoxyphenol by ipso-hydroxylation to a hemiketal followed by spontaneous dissociation to the corresponding alcohol and p-quinone. Hydroquinone effected high oxygen uptake in assays with induced resting cells as well as in assays with cell extracts. This further corroborates the role of hydroquinone as the ring cleavage intermediate during degradation of 4-nonylphenols and 4-alkoxyphenols.     

ipso-substitution: a general biochemical and biodegradation mechanism to cleave alpha-quaternary alkylphenols and bisphenol A

Sphingobium xenophagum Bayram is capable of metabolizing 4-alkoxyphenols and endocrine disruptive alpha-quaternary 4-nonylphenols by an ipso-substitution mechanism that involves ring hydroxylation at the site of the substituent. Here, we show that Bayram's ipso-hydroxylating activity was able to transform also bisphenol A (= dimethyl-4,4'-methylenediphenol; BPA) and 4-isopropylphenol. We identified six metabolites when resting cells of strain Bayram were incubated with BPA. They were unambiguously characterized by HPLC-UV, HPLC/MS, and HPLC/MS/MS as hydroquinone, 2-(4-hydroxyphenyl)isopropanol, 4-isopropenylphenol, 4-isopropylphenol, 4-hydroxy-4-isopropenylcyclohexa-2,5-dien-1-one, and 4-hydroxy-4-isopropylcyclohexa-2,5-dien-1-one. In experiments with 4-isopropylphenol as a substrate, 4-hydroxy-4-isopropylcyclohexa-2,5-dien-1-one, one of the metabolites from BPA, accumulated to a high degree. We could rationalize the formation of all metabolites by invoking ipso-hydroxylation and ipso-substitution mechanisms. On closer view, also classical bacterial metabolism of BPA can be well rationalized by an ipso-substitution mechanism, albeit with ipso-attack of an internal alkyl radical instead of an activated oxygen species. This highlights the important role of ipso-substitution as a versatile degradative principle utilized by diverse organisms to degrade alpha-quaternary 4-nonylphenols, 4-alkoxyphenols, and BPA.     

Elucidation of the mechanism producing menaquinone-4 in osteoblastic cells

Vitamin K is an essential nutrient and a cofactor for the carboxylation of specific glutamyl residues of proteins to γ-glutamyl residues, which activates osteocalcin related to bone formation. Among vitamin K homologues, menaquinone-4 (MK-4) is the most active biologically, up-regulating the gene expression of bone markers, and thus has been clinically used in the treatment of osteoporosis in Japan. Recently, we confirmed that MK-4 was converted from dietary phylloquinone (PK), and then accumulated in various tissues at high concentrations. This system should play an important role in biological functions including bone formation, however, the pathway by which MK-4 is converted remains unclear. In this study, we studied the mechanism of MK-4’s conversion with chemical techniques using deuterated analogues.     

Formation and functioning of fused cholesterol side-chain cleavage enzymes

We studied the properties of various fused combinations of the components of the mitochondrial cholesterol side-chain cleavage system including cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). When recombinant DNAs encoding these constructs were expressed in Escherichia coli, both cholesterol side-chain cleavage activity and sensitivity to intracellular proteolysis of the three-component fusions depended on the species of origin and the arrangement of the constituents. To understand the assembly of the catalytic domains in the fused molecules, we analyzed the catalytic properties of three two-component fusions: P450scc-Adx, Adx-P450scc, and AdR-Adx. We examined the ability of each fusion to carry out the side-chain cleavage reaction in the presence of the corresponding missing component of the whole system and examined the dependence of this reaction on the presence of exogenously added individual components of the double fusions. This analysis indicated that the active centers in the double fusions are either unable to interact or are misfolded; in some cases they were inaccessible to exogenous partners. Our data suggest that when fusion proteins containing P450scc, Adx, and AdR undergo protein folding, the corresponding catalytic domains are not formed independently of each other. Thus, the correct folding and catalytic activity of each domain is determined interactively and not independently.     

Bacterial metabolism of 4-chloro-2-methylphenoxyacetate. Formation of glyoxylate by side-chain cleavage

Crude extracts of Pseudomonas sp. grown on 4-chloro-2-methylphenoxyacetate as sole source of carbon were shown to oxidize 4-chloro-2-methylphenoxyacetate to 5-chloro-o-cresol and glyoxylate. A labelled 2,4-dinitrophenylhydrazone was isolated from an incubation mixture in which 4-chloro-2-methylphenoxy[carboxy-¹⁴C]acetate was used. The hydrazone was shown to behave identically on thin-layer chromatograms with the authentic 2,4-dinitrophenylhydrazone of glyoxylate. Radioactivity assay showed that 82% of the side chain of 4-chloro-2-methylphenoxyacetate was recovered as glyoxylate.     

Cholesterol side-chain cleavage activity in the outer and inner zones of the adrenal cortex

Cholesterol side-chain cleavage activity in mitochondria isolated from the outer and inner zones of the guinea pig adrenal cortex was evaluated in order to clarify the role of the zona reticularis in steroidogenesis. It was found that side-chain cleavage activity was three times higher in the outer zone. In addition, ether stress increased side-chain cleavage activity in the outer zone but not the inner zone. The concentration of total and free cholesterol was also found to be higher in the outer zone. However, when exogenous cholesterol was added to mitochondria, there was no enhancement in side-chain cleavage activity in either zone.     

Specificity of the cholesterol side-chain cleavage enzyme system of adrenal cortex

Incubation of lanosta-8, 24-dien-3β-o1-1,2- ³H and lanost-8-en-3β-o1-1, 2-³H with an adrenocortical bovine mitochondrial acetone-dried preparation did not yield any significant ( < 0.01%) 3β-hydroxy-4, 4, 14-trimethyl-5α-pregn-8-en-20-one. Under the same conditions cholesterol-1,2-³H yielded 8.3% pregnenolone. Incubation of (20S?) — 17α, 20-dihydroxycholesterol-7-³ H yielded 0.6 to 1.6% (20SS?, 22R?) — 17α, 20, 22-trihydroxycholesterol, 1.0 to 3.2% of 17α-hydroxypregnenolone, but no significant ( < 0.02%) (20S, 22S)-17α, 20, 22-trihydroxycholesterol. In another experiment incubation of cholesterol-1, 2-³H yielded 5% pregnenolone, 0.5% 17α-hydroxypregnenolone, 0.2% (20R?,22R?)-20, 22-dihydroxy-cholesterol, but no significant ( < 0.01%) 17α-hydroxy-cholesterol, (20S?) -17α, 20-dihydroxycholesterol or (20S?, 22R?)-17α, 20, 22-trihydroxycholesterol.     

Trifluoperazine inhibits ovarian mitochondrial side-chain cleavage enzyme activity in the absence of calcium

In a previous report we described the inhibitory effect of trifluoperazine (TFP) on steroidogenesis in avian granulosa cells. To clarify the possible site of action of TFP we measured the cholesterol side-chain cleavage enzyme (CSCC) activity in a mitochondrial preparation of granulosa cells isolated from mature and developing ovarian follicles. Using a calcium free medium, TFP inhibited CSCC in a dose related manner with an IC50 of 50 microM. Kinetic parameters (apparent Km and Vmax) obtained in the presence of TFP are indicative of uncompetitive inhibition of CSCC. Moreover, enzyme activity increased during follicular maturation while the efficacy of TFP was similar in both young and mature follicles. Because the inhibitory effects of TFP were manifest in medium from which calcium was omitted, it is suggested that the drug acts independently of the calcium-calmodulin system to suppress CSCC activity.     

Clomiphene and tamoxifen inhibit the cholesterol side-chain cleavage enzyme activity in hen granulosa cells

A radiochemical assay was utilized to study the inhibitory effects of clomiphene and tamoxifen on the cholesterol side-chain cleavage enzyme activity in a mitochondrial preparation of granulosa cells isolated from mature ovarian follicles of laying hens. At saturating substrate concentrations, both clomiphene and tamoxifen were able to suppress enzyme activity in a dose-related manner (IC50 1.8 X 10(-5) M). Double reciprocal plots of kinetic data show that the inhibition is mixed, exhibiting competitive kinetics at low concentrations, whereas at high concentrations, the inhibition is of a non-competitive nature. The competitive inhibition constants as determined from Dixon plots are 2 X 10(-5) M for clomiphene and 2.3 X 10(-5) M for tamoxifen. It is concluded that, in granulosa cells, clomiphene and tamoxifen directly inhibit the mitochondrial cholesterol side-chain cleavage activity. This inhibition may represent an important aspect of the mode of action of clomiphene and tamoxifen.     

Differences between the regulation of cholesterol side-chain cleavage in Leydig cells from mice and rats

A Leydig cell culture system has been used to study the in vitro modulation by luteinizing hormone (LH) of steroidogenesis in Leydig cells isolated from mice and immature rats. Mouse Leydig cells precultured for 24 h in the presence of increasing concentrations of LH (1 ng-1 microgram/ml) showed a dose-dependent decrease of the maximal LH-stimulated testosterone production. After pretreatment with 1 microgram LH/ml, maximal LH-stimulated testosterone production. After production in the presence of excess 20 alpha-hydroxycholesterol (a cholesterol side-chain cleavage substrate) were reduced to approx. 50% of control values. The possible site of action of LH is probably prior to pregnenolone, because testosterone production in the presence of excess pregnenolone was not affected by the LH pretreatment. Immature rat Leydig cells showed no decrease of maximal steroid production after 24 h culture in the presence of 1 microgram LH/ml. These results indicate that the regulation of the cholesterol side-chain cleavage activity during long-term LH action is different in mouse and rat Leydig cells. The properties of the cholesterol side-chain cleavage enzyme in mouse and rat Leydig cells were further investigated with different hydroxylated cholesterol derivatives as substrates. Steroid production by mouse Leydig cells in the presence of (22R)-22 hydroxycholesterol was similar as in the presence of LH. In contrast, steroidogenesis in rat Leydig cells in the presence of (22R)-22 hydroxycholesterol was at least 10-fold higher than in the presence of LH. It is concluded that the cholesterol side-chain cleaving enzyme in the mouse Leydig cell operates at its maximal capacity during short-term LH stimulation and can be inhibited after long-term LH action, whereas in the rat Leydig cell only a fraction of the potential activity is used during short-term LH stimulation, which is not affected during long-term LH action.     

Stimulation by endozepine of the side-chain cleavage of cholesterol in a reconstituted enzyme system

Addition of endozepine in nanomolar concentrations to a system for side-chain cleavage reconstituted from highly purified P-450scc and electron carriers (adrenodoxin reductase and adrenodoxin) stimulates the conversion of cholesterol to pregnenolone (side-chain cleavage). This response is concentration and time-dependent and specific to the extent that a second steroidogenic P-450 located in the inner mitochondrial membrane (ie 11 beta-hydroxylase) was not stimulated by endozepine. Homogeneous endozepine prepared from bovine brain, the corresponding genetically engineered peptide and des(glu-ilu)-endozepine isolated from bovine adrenal cortex are all approximately equipotent in this system. Moreover, endozepine accelerates the rate of reduction of P-450scc by NADPH and the electron carriers. The results suggest that endozepine acts directly on P-450 and hence the rate of side-chain cleavage.     

Elucidation of the antimicrobial mechanism of mutacin 1140

Mutacin 1140 and nisin A are peptide antibiotics that belong to the lantibiotic family. N-Terminal rings A and B of nisin A and mutacin 1140 (lipid II-binding domain) share many structural and sequence similarities. Nisin A binds lipid II and thus disrupts cell wall synthesis and also forms transmembrane pores. Very little is known about mutacin 1140 in this regard. We performed fluorescence-based studies using a bacteria-mimetic membrane system. The results indicated that lipid II monomers are arranged differently in the mutacin 1140 complex than in the nisin A complex. These differences in complex formation may be attributed to the fact that nisin A uses lipid II to form a distinct pore complex, while mutacin 1140 does not form pores in this membrane system. Further experiments demonstrated that the mutacin 1140-lipid II and nisin A-lipid II complexes are very stable and capable of withstanding competition from each other. Transmembrane electrical potential experiments using a Streptococcus rattus strain, which is sensitive to mutacin 1140, demonstrated that mutacin 1140 does not form pores in this strain even at a concentration 8 times higher than the minimum inhibitory concentration (MIC). Circular complexes of mutacin 1140 and nisin A were observed by electron microscopy, providing direct evidence for a lateral assembly mechanism for these antibiotics. Mutacin 1140 did exhibit a membrane disruptive function in another commonly used artificial bacterial membrane system, and its disruptive activity was enhanced by increasing amounts of anionic phospholipids.