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A J Fulco

Publications and source records attributed to A J Fulco.

At least 37 records · Page 2Linked to original sources
PubMed1987

Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium.

In a previous publication (Narhi, L. O. and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a soluble 119,000-dalton P-450 cytochrome (P-450BM-3) that was induced by barbiturates in Bacillus megaterium. This single polypeptide contained 1 mol each of FAD and FMN/mol of heme and, in the presence of NADPH and O2, catalyzed the oxygenation of long-chain fatty acids without the aid of any other protein. We have now utilized limited trypsin proteolysis in the presence of substrate to cleave P-450BM-3 into two polypeptides (domains) of about 66,000 and 55,000 daltons. The 66-kDa domain contains both FAD and FMN but no heme, reduces cytochrome c in the presence of NADPH, and is derived from the C-terminal portion of P-450BM-3. The 55-kDa domain is actually a mixture of three discrete peptides (T-I, T-II, and T-III) separable by high performance liquid chromatography. All three contain heme and show a P-450 absorption peak in the presence of CO and dithionite. The major component, T-I (Mr = 55 kDa), binds fatty acid substrate and has an N-terminal amino acid sequence identical to that of intact P-450BM-3, an indication that this domain constitutes the N-terminal portion of the 119-kDa protein. T-II (54 kDa) is the same as T-I except that it is missing the first nine N-terminal amino acids and does not bind substrate. T-III (Mr = 53.5 kDa) has lost the first 15 N-terminal residues and does not bind substrate. Since trypsin digestion of P-450BM-3 carried out in the absence of substrate yields T-II and T-III but no T-I, it appears that 1 or more residues of the first nine N-terminal amino acids of this protein are intimately involved in substrate binding. Although both the heme- and flavin-containing tryptic peptides retain their original half-reactions, fatty acid monooxygenase activity cannot be reconstituted after proteolysis, and the two domains, once separated, show no affinity for each other. In most respects, the reductase domain of P-450BM-3 more closely resembles the mammalian microsomal P-450 reductases than it does any known bacterial protein.

Amino Acid Sequence↗
PubMed1987

Occurrence of a barbiturate-inducible catalytically self-sufficient 119,000 dalton cytochrome P-450 monooxygenase in bacilli.

In a recent publication (Narhi, L.O. and Fulco, A.J.[1986] J. Biol. Chem. 261, 7160-7169) we described the characterization of a catalytically self-sufficient 119,000 Dalton cytochrome P-450 fatty acid monooxygenase (P-450BM-3) induced by barbiturates in Bacillus megaterium ATCC 14581. We have now examined cell-free preparations from 12 distinct strains of B. megaterium and from one or two strains each of B. alvei, B. brevis, B. cereus, B. licheniformis, B. macerans, B. pumilis and B. subtilis for the presence of this inducible enzyme. Using Western blot analyses in combination with assays for fatty acid hydroxylase activity and cytochrome P-450, we were able to show that 11 of the 12 B. megaterium strains contained not only a strongly pentobarbital-inducible fatty acid monooxygenase identical to or polymorphic with P-450BM-3 but also significant levels of two smaller P-450 cytochromes that were the same as or similar to cytochromes P-450BM-1 and P-450BM-2 originally found in ATCC 14581. Unlike the 119,000 Dalton P-450, however, the two smaller P-450s were generally easily detectable in cultures grown to stationary phase in the absence of barbiturates and, with some exceptions, were not strongly induced by pentobarbital. None of the non-megaterium species of Bacillus tested exhibited significant levels of either fatty acid monooxygenase activity or cytochrome P-450. The one strain of B. megaterium that lacked inducible P-450BM-3 was also negative for BM-1 and BM-2. However, this strain (ATCC 13368) did contain a small but significant level of another P-450 cytochrome that others have identified as the oxygenase component of a steroid 15-beta-hydroxylase system. Our evidence suggests that the BM series of P-450 cytochromes is encoded by chromosomal (rather than by plasmid) DNA.

Bacillus↗
PubMed1986

Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium.

A unique cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 is strongly induced by phenobarbital (Narhi, L. O., and Fulco, A. J. (1982) J. Biol. Chem. 257, 2147-2150) and many other barbiturates (Kim, B.-H., and Fulco, A. J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). This monooxygenase has now been purified to homogeneity from pentobarbital-induced bacteria as a single polypeptide with a molecular weight of 119,000 +/- 5,000 daltons. In the presence of NADPH and O2, it can catalyze the oxygenation of long chain fatty acids without the aid of any other protein. The enzyme has a catalytic center activity of 4,600 nmol of fatty acid oxygenated per nmol of P-450 (the highest activity yet reported for a P-450-dependent monooxygenase) and also functions as a highly active cytochrome c reductase in the presence of NADPH. The purified holoenzyme is a soluble protein containing 40 mol % hydrophobic amino acid residues and 1 mol each of FAD and FMN/mol of heme. It is isolated and purified in the low spin form but is converted to the high spin form in the presence of long chain fatty acids. The enzyme, which catalyzes the omega-2 hydroxylation of saturated fatty acids and the hydroxylation and epoxidation of unsaturated fatty acids has its highest affinity (Km = 2 +/- 1 microM) for the C15 and C16 chain lengths.

Amino Acid Sequence↗
PubMed1985

Purification and characterization of pentobarbital-induced cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581.

When Bacillus megaterium ATCC 14581 is grown in the presence of barbiturates, a cytochrome P-450-dependent fatty acid monooxygenase (Mr 120000) is induced (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). Gel filtration chromatography of a crude monooxygenase preparation from pentobarbital-induced B. megaterium indicated that not all of the induced cytochrome P-450 present in the extract was accounted for by this high-molecular-weight component. Further purification revealed the presence of two additional but smaller cytochrome P-450 species. The minor component, designated cytochrome P-450BM-2, had a molecular mass of about 46 kDa, but has not yet been completely purified or further characterized. The major component, designated cytochrome P-450BM-1, was obtained in pure form, exhibited fatty acid monooxygenase activity in the presence of iodosylbenzenediacetate, and has been extensively characterized. Its Mr of 38000 makes it the smallest cytochrome P-450 yet purified to homogeneity. Although it is a soluble protein, a complete amino acid analysis indicated that it contains 42% hydrophobic residues. By the dansyl chloride procedure the NH2-terminal amino acid is proline; the penultimate NH2-terminal residue is alanine. The absolute absorption spectra of cytochrome P-450BM-1 show maxima in the same general regions as do P-450 cytochromes from mammalian or other bacterial sources, but they differ in detail. The oxidized form of P-450BM-1 has absorption maxima at 414, 533 and 567 nm, while the reduced form has peaks at 410 and 540 nm. The absorption maxima for the CO-reduced form of P-450BM-1 are found at 415, 448 and 550 nm. Antisera from rabbits immunized with pure P-450BM-1 strongly reacted with and precipitated this P-450, but showed no detectable affinity for either the 46 kDa P-450 or the 120 kDa fatty acid monooxygenase.

Amino Acids↗
PubMed1985

Induction of a cytochrome P-450-dependent fatty acid monooxygenase in Bacillus megaterium by a barbiturate analog, 1-[2-phenylbutyryl]-3-methylurea.

In previous publications from our laboratory, we reported that a soluble, cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 can be induced by phenobarbital and a variety of other barbiturates. The tested barbiturates showed an excellent correlation between increasing lipophilicity and increasing inducer potency (Kim BH, Fulco AJ; Biochem Biophys Res Commun 116: 843-850, 1983). The only exception proved to be mephobarbital (N-methylphenobarbital) which, although more lipophilic than phenobarbital, is not an inducer of fatty acid monooxygenase activity. We have now found that 1-[2-phenylbutyryl]-3-methylurea (PBMU), an acylurea that can be derived from mephobarbital by hydrolytic cleavage of the barbiturate ring, is an excellent inducer of this activity. Paradoxically, the addition of mephobarbital to the bacterial growth medium containing PBMU significantly enhances the apparent potency of the acylurea to induce fatty acid monooxygenase activity as measured in cell-free extracts. When cell-free extracts of cells grown separately in PBMU or mephobarbital are mixed no enhancement of activity is seen. This finding suggests that the effect of mephobarbital is to somehow increase the efficiency of PBMU as an inducer of the P-450-dependent fatty acid monooxygenase rather than to induce an activator of this enzyme or a rate-limiting component of the monooxygenase system. Finally, both mephobarbital and PBMU induce the synthesis of total cytochrome P-450 in B. megaterium although PBMU is a much more potent P-450 inducer. For cytochrome P-450 induction, however, there is no synergistic or even additive effect when mephobarbital and PBMU are used together in the bacterial growth medium.

Bacillus megaterium↗
PubMed1984

Acylureas: a new class of barbiturate-like bacterial cytochrome P-450 inducers.

The soluble, cytochrome P-450-dependent fatty acid monooxygenase of Bacillus megaterium ATCC 14581 is induced by phenobarbital and at least twelve other barbiturates (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). We have since found that the inducer potency of phenobarbital and of six other of these barbiturates was enhanced by adding them to the growth medium prior to sterilization by autoclaving. A similar 'activation' was effected simply by autoclaving these barbiturates in distilled water at pH 8.0. When the hydrolytic products resulting from such treatment of phenobarbital were identified and screened for inducer activity, the major product, 2-phenylbutyrylurea, was found to be 3-5-times more potent than phenobarbital itself. The racemic mixture, (+/-)-2-phenylbutyrylurea was somewhat more active as an inducer than was either of the enantiomers [+/-) or (-] tested singly. Of the other hydrolytic products of phenobarbital, only 2-phenylbutyramide had significant inducer activity (about the same as phenobarbital). Among other ureides tested, two monosubstituted acetylureas (phenylacetylurea and dodecanoylurea) were inactive as inducers, but six of seven disubstituted acetylureas were better inducers than 2-phenylbutyrylurea.

Anthraquinones↗
PubMed1983

Induction by barbiturates of a cytochrome P-450-dependent fatty acid monooxygenase in Bacillus megaterium: relationship between barbiturate structure and inducer activity.

In a recent communication (Narhi, L. and Fulco, A.J. [1982] J. Biol. Chem. 257, 2147-2150) we found that a soluble cytochrome P-450-dependent fatty acid monooxygenase isolated from Bacillus megaterium ATCC 14581 could be induced about 28-fold by phenobarbital. We have now examined 19 barbiturates and found that 13 significantly induce the specific monooxygenase activity. Of these, 11 are more active than phenobarbital and three (secobarbital, thiamylal and methohexital) are more than 30 times as active on a molar basis. The dialkyl barbiturates without exception show an excellent correlation between increasing lipophilicity and increasing potency as inducers as do most of the barbiturates containing an aromatic substituent. Nevertheless, it is apparent that certain structural features involving factors other than lipophilicity are also necessary for induction. Our finding that barbiturates can cause the non-substrate induction of a cytochrome P-450-dependent monooxygenase in a prokaryote represents a unique discovery that may provide a relatively simple model for apparently similar induction systems in higher animals.

Bacillus megaterium↗
PubMed1983

Partial characterization of a barbiturate-induced cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium.

A soluble cytochrome P-450-dependent fatty acid monooxygenase activity obtained from Bacillus megaterium ATCC 14581 can be induced by at least 13 different barbiturates. In general, the potency of these compounds as inducers increases with their increasing lipophilicity. We have now shown that at least 4 of these barbiturates (phenobarbital, secobarbital, pentobarbital and methohexital) seem to induce the same active cytochrome P-450-containing enzyme by a non-substrate type mechanism. The partially purified enzymes obtained from cultures induced with each of the 4 barbiturates tested were all of similar molecular size (Mr = 130,000 +/- 10,000) and had similar turnover numbers (1400-1800 +/- 300) with either palmitoleate or myristate as substrates. None of the tested barbiturates served as substrates, activators or inhibitors of any of the monooxygenase preparations, nor did they appear to interact in any way with the monooxygenase enzyme or the P-450 component.

Bacillus megaterium↗
PubMed1983

Cytochrome P-450 revealed: the effect of the respiratory cytochromes on the spectrum of bacterial cytochrome P-450.

Soluble extracts of Bacillus megaterium ATCC 14581 prepared by centrifuging a sonicated cell suspension at 40,000 xg for 30 min apparently contained no cytochrome P-450 unless the culture had been grown in the presence of an inducer: a reduced+CO minus reduced spectrum was used to measure cytochrome P-450 concentration. When the 40,000 xg supernatants from the uninduced cultures were recentrifuged at 105,000 xg the respiratory cytochromes, including one like cytochrome a1, were sedimented, and cytochrome P-450 was observed to be 100 nM or 30 +/- 9 p mol cytochrome P-450/mg protein (n=9). Measurements of cytochrome P-450 in cultures induced with phenobarbital were always higher after ultracentrifugation. There was soluble cytochrome o in all extracts. When cytochrome a1 was present a deep trough at 441 nm developed in the reduced +CO minus reduced difference spectrum of the 40,000 xg supernatant of both the uninduced and the induced cultures. The 40,000 xg supernatant obtained after lysing protoplasts of B. megaterium did not contain cytochrome a1 and always gave a good measure of cytochrome P-450.

Bacillus megaterium↗
PubMed1983

Nonsubstrate induction of a soluble bacterial cytochrome P-450 monooxygenase by phenobarbital and its analogs.

A soluble, cytochrome P-450-dependent fatty acid hydroxylase--epoxidase complex from Bacillus megaterium ATCC 14581 can be induced more than 100-fold by the addition of phenobarbital or one of its analogs (hexobarbital) to the growth medium. These barbiturate inducers are apparently not substrates for the enzyme nor do they activate the monooxygenase in the cell-free system. The induction efficiency of both phenobarbital and hexobarbital can be significantly increased with respect to monooxygenase activity by autoclaving the inducer in the growth medium rather than by adding it to the medium after autoclaving. Turnover numbers of about 3 000 nmoles of substrate oxygenated per min per nmole of P-450 were obtained in crude cell-free preparations obtained from maximally induced cultures. Our data indicate that products formed by heating phenobarbital or hexobarbital in the growth medium are significantly better inducers of monooxygenase activity than are the unaltered drugs.

Bacillus megaterium↗
PubMed1982

Phenobarbital induction of a soluble cytochrome P-450-dependent fatty acid monooxygenase in Bacillus megaterium.

A soluble, cytochrome P-450-dependent fatty acid hydroxylase-epoxidase isolated from Bacillus megaterium ATCC 14581 can be induced about 28-fold by the addition of phenobarbital (8 mM) to the growth medium. Phenobarbital is not a substrate for the enzyme nor does it activate the monooxygenase in the cell-free system. The level of the P-450-dependent monooxygenase activity in cultures harvested during the early stationary phase of growth increased linearly with phenobarbital concentration up to its solubility limit (8 mM) at 35 degrees C. The time course of induction during culture growth in the presence of 4 mM phenobarbital showed an interesting dichotomy. The specific content of cytochrome P-450 increased until the early stationary phase of growth and then leveled off. P-450-dependent monooxygenase activity, however, continued to increase rapidly to midstationary phase and then decreased just as rapidly after this time. At maximum specific activity, a turnover number of about 2,450 was obtained for palmitoleate hydroxylation-epoxidation by the cytochrome P-450 system.

Bacillus megaterium↗
PubMed1981

Epoxidation of unsaturated fatty acids by a soluble cytochrome P-450-dependent system from Bacillus megaterium.

In previous publications from this laboratory we have described a soluble, partially purified cytochrome P-450-dependent monooxygenase complex that, in the presence of NADPH and O2, catalyzes the monohydroxylation of long chain fatty acids, alcohols, and amides at the omega -1, omega -2, and omega -3 positions. We have now found that this preparation catalyzes the epoxidation as well as the hydroxylation of palmitoleic acid and a variety of other monounsaturated fatty acids. The experimental results reported here strongly support the concept that both hydroxylation and epoxidation are catalyzed by an identical cytochrome P-450 complex utilizing the same active and binding sites. Furthermore, for saturating levels of these substrates, the rate-limiting step in oxygenation does not appear to involve substrate structure. Thus, although the position and geometry of the double bond may dramatically affect the rate of epoxidation relative to hydroxylation, the combined rate of substrate oxygenation is essentially a constant independent of this ratio. Finally, we propose and present evidence for an enzyme-substrate binding model that involves polar binding of the carboxyl terminus and strong hydrophobic binding and sequestering of the terminal methyl group of the fatty acid. The three methylene carbons adjacent to the methyl group are positioned in a set geometry around the active site but the midchain region of a monounsaturated fatty acid is relatively free to interact or bind loosely with the enzyme surface in a variety of conformations. Depending on fatty acid structure, one or more of these conformations can bring the unsaturated center close enough to the active site to permit epoxidation of the double bond.

Bacillus megaterium↗
PubMed1980

Temperature-mediated hyperinduction of fatty acid desaturation in pre-existing and newly formed fatty acids synthesized endogenously in Bacillus megaterium.

3H-labedeled fatty acids synthesized endogenously by Bacillus megaterium ATCC 14581 growing at 35 degrees C in the presence of L-[G-3H]valine exhibited the same time-course of hyperinduced desaturation following a temperature decrease to 20 degrees C as was observed previously with exogenously supplied 14C-labeled fatty acids. Radioactive fatty acids synthesized in the presence of [U-14C]glucose during hyperinduction at 20 degrees C following a shift-down from 35 degrees C were desaturated at the same relative rate as 14C-labeled fatty acids synthesized previously at 35 degrees C, suggesting that the newly synthesized fatty acids equilibrate with a large portion of the preexisting moieties before becoming susceptible to desaturation.

Bacillus megaterium↗
PubMed1980

Two distinct pools of membrane phosphatidylglycerol in Bacillus megaterium.

The predominant membrane lipid in Bacillus megaterium ATCC 14581, phosphatidylglycerol (PG), is present in two distinct pools, as shown by [32P]phosphate incorporation and chase experiments. One pool (PGt) undergoes rapid turnover of the phosphate moiety, whereas the second pool (PGs) exhibits metabolic stability in this group. The phosphate moiety of the other major phospholipid, phosphatidylethanolamine, is stable to turnover. [32P]phosphate- and [2-3H]glycerol-equilibrated cultures yielded the following glycerolipid composition: 56 mol% PG (34 mol% PGt and 22 mol% PGs), 21 mol% phosphatidylethanolamine, 1 to 2 mol% phosphatidylserine, 20 mol% diglycerides, less than 0.5 mol% cardiolipin, and 0.2 to 0.4 mol% lysophosphatidylglycerol. Accumulation of PG was halted immediately after the addition of cerulenin, an inhibitor of de novo fatty acid synthesis, whereas phosphatidylethanolamine accumulation continued at the expense of the diglyceride and PG pools. Strikingly, initial rates of [32P]phosphate incorporation into PG were unaffected by cerulenin. In control cultures at 35 degrees C, incorporation of [32P]phosphate into PG exhibited a biphasic time course, whereas incorporation into phosphatidylethanolamine was concave upward and lagged behind that of PG during the initial rapid phase of PG incorporation. Finally, levels of lysophosphatidylglycerol expanded rapidly after cerulenin addition at 20 degrees C, but not at 35 degrees C. Moreover, incorporation of [32P]phosphate into lysophosphatidylglycerol lagged behind incorporation into PG in both the presence and absence of cerulenin at 20 and 35 degrees C.

Bacillus megaterium↗