Identification of constitutive and herbicide inducible cytochromes P-450 in Streptomyces griseolus

O’Keefe, Daniel P.; Romesser, James A.; Leto, Kenneth J.

doi:10.1007/bf00425579

Cited by 51 publications

(33 citation statements)

References 10 publications

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“…4) compared to authentic heme protein standards (myoglobin, catalase). The specific content of heme from the pyridine hemochromogen assay (15.9 nmol/mg protein), or from the extinction coefficient (22) for Cyt P-450 chromophores (17.9 nmol/mg protein; see Table I), are close to the expected value of 21 nmol/mg for a 47 kD protein with one heme per monomer. Further, these values are high enough to make it unlikely that the native form of either ARP-1 or ARP-2 is a chromophore free protein, although we cannot rule out the unprecedented possibility that one ofthem contains 2 hemes/ polypeptide.…”

Section: Properties Of Native Avocado Cyt P-450supporting

confidence: 56%

“…7A), indicating that binding is accompanied by a shift in the heme spin equilibrium toward the high spin state. Figure 7D In contrast to the microsomal protein, the unusual difference spectrum from the RTX-100 containing enzyme most closely resembles a 'reverse type I' spectrum (15) indicating a (15,21,27). C, Purified Cyt P450 which has had RTX-100 exchanged for CHAPS; in 0.1 M Mops-NaOH (pH 7.0), 0.8% CHAPS.…”

Section: Substrate Binding and Enzymatic Activitymentioning

confidence: 99%

“…The difficulty in separating the proteins suggests that they are very similar. They were not resolved by high performance anion exchange chromatography, although this technique can resolve a variety of soluble (20,21) and membrane bound (9, 20) Cyt P-450 in extracts from other organisms. ARP-1 and ARP-2 can be separated by reverse phase HPLC, which is capable of resolving smaller proteins differing by as little as a single amino acid (14,25).…”

Section: Substrate Binding and Enzymatic Activitymentioning

confidence: 99%

“…Hass) were purchased locally, and ripened at room temperature. All HPLC was carried out at room temperature on a system previously described (21).…”

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confidence: 99%

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Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

O’Keefe

Leto²

1989

Plant Physiol.

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The microsomal fraction from the mesocarp of avocado (Persea amerkana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest pattems, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarites with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450.Cytochrome P450 dependent monooxygenases are widespread in nature. They are involved in a variety of catabolic and biosynthetic pathways and in the metabolism of drugs and xenobiotics. In plants, Cyt P-450 has been identified, for example, in the trans-cinnamic acid and kaurene hydroxylases (10,11). Others are suspected participants in a number of reactions associated with xenobiotic metabolism (6, 8) (for reviews see Refs. 4 and 20). Within these monooxygenase systems, Cyt P-450 serves as a terminal oxidase, responsible for substrate recognition, binding, and oxygen redox chemistry (5, 29).The mechanistic and structural details of Cyt P450 dependent monooxygenases have been developed largely from studies of the bacterial (Pseudomonas putida) camphor hydroxylase system (23,29). Because of their importance in xenobiotic and drug metabolism, these enzymes have also been thoroughly studied in mammalian liver (3,5,31). In contrast, Cyt P-450 of higher plant origin has not been the subject of extensive biochemical characterization, primarily because of the low content in many plant tissues, difficulties with identification in Chl containing tissues, and supposed lability in homogenized plant extracts. ' (6,10,12,13,19,20). Cyt P-450 has been purified from tulip (Tulipa gesneriana) bulbs (12), and from tubers of Jerusalem artichoke (Helianthus tuberosus) (10). Additionally, an NADPH:Cyt P450 reductase has been purified from Jerusalem artichoke (2), and the related Cyt b5 and NADH:Cyt b5 reductase have been purified from Pisum sativum (16). Unfortunately, the tulip bulb Cyt P450 has not been associated with any enzymic activity, and polyclonal antibodies raised against it were not cross reactive with proteins from other species (1 1). The Jerusalem artichoke preparation can be reconstituted into its trans-cinnamate hydroxylase activity, but it has a low heme specific content and is not homogeneous (10). No primary sequence information has been reported for either o...

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Section: Properties Of Native Avocado Cyt P-450supporting

confidence: 56%

Section: Substrate Binding and Enzymatic Activitymentioning

confidence: 99%

Section: Substrate Binding and Enzymatic Activitymentioning

confidence: 99%

“…Hass) were purchased locally, and ripened at room temperature. All HPLC was carried out at room temperature on a system previously described (21).…”

mentioning

confidence: 99%

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Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

O’Keefe

Leto²

1989

Plant Physiol.

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“…In the former organism, a constitutive P-450 exists in addition to two cytochromes, P-450su1 and P-450SU2, that are induced by and catalyze various oxidations of sulfonylurea herbicides (27,33). In B. megaterium, at least three P-450s are induced by phenobarbital.…”

Section: Methodsmentioning

confidence: 99%

Two independently regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzoate

Karlson¹,

Dwyer²,

Hooper³

et al. 1993

J Bacteriol

127

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A red-pigmented coryneform bacterium, identified as Rhodococcus rhodochrous strain 116, that grew on 2-ethoxyphenol and 4-methoxybenzoate as sole carbon and energy sources was isolated. Phylogenetic analysis based on the 16S rDNA sequences indicates that the strain clusters more closely to other rhodococci than to other gram-positive organisms with a high G+C content. Each of the abovementioned growth substrates was shown to induce a distinct cytochrome P450: cytochrome P-450O1 was induced by 2-ethoxyphenol, and cytochrome P-450R= was induced by 4-methoxybenzoate. A type I difference spectrum typical of substrate binding was induced in cytochrome P-450RR1 by both 2-ethoxyphenol (K, = 4.2 0.3 pM) and 2-methoxyphenol (K, = 2.0 + 0.1 pM), but not 4-methoxybenzoate or 4-ethoxybenzoate. Similarly, a type I difference spectrum was induced in cytochrome P-450Rm by both 4-methoxybenzoate (K, = 2.1 0.1 FM) and 4-ethoxybenzoate (K, = 1.6 0.1 RM), but not 2-methoxyphenol or 2-ethoxyphenol. A purified polyclonal antiserum prepared against cytochrome P-450RR1 did not cross-react with cytochrome P-45Rm, indicating that the proteins are immunologically distinct. The cytochromes appear to catalyze the 0-dealkylation of their respective substrates. The respective products of the 0-dealkylation are further metabolized via ortho cleavage enzymes, whose expression is also regulated by the respective aromatic ethers.The design of novel efficient microbial catabolic pathways to degrade otherwise recalcitrant environmental compounds involves not only the recruitment of enzymes with desirable catalytic activities from different organisms but also the modification of such enzymes to improve their catalytic abilities. Cytochromes P-450 are good candidates for such recruitment, as this class of heme monooxygenases catalyze a remarkably broad range of chemical reactions on an equally broad range of substrates (29). In bacterial systems, P-450s generally occur as part of a soluble three-component system, consisting of a flavin-containing reductase and an iron-sulfur electron transfer protein in addition to the cytochrome (37). Bacterial P-450s often occur in catabolic pathways, frequently catalyzing an initial hydroxylation reaction of the growth substrate, as in the case of P-450cam (21) and P-4501in (42). P-450's have also been reported to catalyze the O-dealkylation of para-substituted benzoates (3), veratrole (41), and ortho-substituted alkoxyphenols (5). The characteristic absorbance peak at approximately 450 nm of the CO-difference spectrum of the reduced P-450 provides a ready means to screen for the presence of these enzymes in whole cells and crude extracts (31 interest in recruiting P-450's into both biotransformation and biodegradation processes has increased rapidly (6, 29, 37).A structural element that contributes to both the recalcitrance and the toxicity of such pollutants as 2,3,7,8-tetrachlorodibenzo-p-dioxin is the ether bond. To investigate the mechanism of ether bond cleavage in aromatic compounds, microorganisms were sc...

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Mode of action, crop selectivity, and soil relations of the sulfonylurea herbicides

1990

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The sulfonylurea herbicides are characterized by broad-spectrum weed control at very low use rates (c. 2-75 g ha-'), good crop selectivity, and very low acute and chronic animal toxicity. This class of herbicides acts through inhibition of acetolactate synthase (EC 4.1.3.18; also known as acetohydroxyacid synthase), thereby blocking the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. This inhibition leads to the rapid cessation of plant cell division and growth. Crop-selective sulfonylurea herbicides have been commercialized for use in wheat, barley, rice, corn, soybeans and oilseed rape, with additional crop-selective compounds in cotton, potatoes, and sugarbeet having been noted. Crop selectivity results @om rapid metabolic inactivation of the herbicide in the tolerant crop. Under growth-room conditions, metabolic half-lives in tolerant crop plants range fiom 1-5 h, while sensitive plant species metabolize these herbicides much more slowly, with half-lives >20 h. Pathways by which sulfonylurea herbicides are inactivated among these plants include aryl and aliphatic hydroxylation followed by glucose conjugation, sulfonylurea bridge hydrolysis and sulfonamide bond cleavage, oxidative 0-demethylation and direct conjugation with (homo)glutathione. Sulfonylurea herbicides degrade in soil through a combinatiori of bridge hydrolysis and microbial degradation. Hydrolysis is signijicantly faster under acidic (PH 5 ) than alkaline (PH 8 ) conditions, allowing the use of soil pH as a predictor of soil residual activity. Chemical and microbial processes combine to give typical field dissipation half-lives of I d weeks, depending on the soil type, location and compound. Very short residual sulfonylurea herbicides with enhanced susceptibility to hydrolysis (DPX-L5300) and microbial degradation (thifensuljiuron-methyl) have been developed.

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Identification of constitutive and herbicide inducible cytochromes P-450 in Streptomyces griseolus

Cited by 51 publications

References 10 publications

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

Two independently regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzoate

Mode of action, crop selectivity, and soil relations of the sulfonylurea herbicides

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