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1.
Osami Shoji Christian Wiese Takashi Fujishiro Chikako Shirataki Bernhard Wünsch Yoshihito Watanabe 《Journal of biological inorganic chemistry》2010,15(7):1171-1115
Aromatic C–H bond hydroxylation of 1-methoxynaphthalene was efficiently catalyzed by the substrate misrecognition system of
the hydrogen peroxide dependent cytochrome P450BSβ (CYP152A1), which usually catalyzes hydroxylation of long-alkyl-chain fatty acids. Very importantly, the hydroxylation of
1-methoxynaphthalene can be monitored by a color change since the formation of 4-methoxy-1-naphthol was immediately followed
by its further oxidation to yield Russig’s blue. Russig’s blue formation allows us to estimate the peroxygenation activity
of enzymes without the use of high performance liquid chromatography, gas chromatography, and nuclear magnetic resonance measurements. 相似文献
2.
The monooxygenese activity of cytochrome P450 is successfully introduced into myoglobin by rational design of its active site. Introduction of an aromatic ring, tryptophan, near the heme by site-directed mutagenesis resulted in the hydroxylation of tryptophan at the C6 position by using an almost stoichiometric amount of H(2)O(2). We also altered the substrate specificity of H(2)O(2)-dependent P450 by employing a simple substrate trick. Although P450(BSβ) exclusively catalyzes peroxygenation of long-alkyl-chain fatty acids, oxidation of non-natural substrates such as styrene, ethylbenzene, and 1-methoxynaphthalen are catalyzed by P450(BSβ) in the presence of decoy molecules having a carboxyl group. Advantageously, the substrate specificity of P450(BSβ) can be altered by simply adding the decoy molecule without replacing any amino acid residues. Moreover, the stereoselectivity can be controlled by changing the structure of the decoy molecule. The crystal structure analysis of the decoy molecule bound-form of P450(BSβ) shows that P450(BSβ) accepts the decoy molecule, whose carboxylate is located at the same position to that of long-alkyl-chain fatty acid. 相似文献
3.
Fujishiro T Shoji O Nagano S Sugimoto H Shiro Y Watanabe Y 《The Journal of biological chemistry》2011,286(34):29941-29950
Cytochrome P450SPα (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H2O2-dependent P450. P450SPα hydroxylates fatty acids with high α-regioselectivity. Herein we report the crystal structure of P450SPα with palmitic acid as a substrate at a resolution of 1.65 Å. The structure revealed that the Cα of the bound palmitic acid in one of the alternative conformations is 4.5 Å from the heme iron. This conformation explains the highly selective α-hydroxylation of fatty acid observed in P450SPα. Mutations at the active site and the F–G loop of P450SPα did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of cytochrome P450BSβ (CYP152A1), which shows β-regioselectivity. This implies that the high regioselectivity of P450SPα is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450BSβ. 相似文献
4.
James Belcher Kirsty J. McLean Sarah Matthews Laura S. Woodward Karl Fisher Stephen E. J. Rigby David R. Nelson Donna Potts Michael T. Baynham David A. Parker David Leys Andrew W. Munro 《The Journal of biological chemistry》2014,289(10):6535-6550
The production of hydrocarbons in nature has been documented for only a limited set of organisms, with many of the molecular components underpinning these processes only recently identified. There is an obvious scope for application of these catalysts and engineered variants thereof in the future production of biofuels. Here we present biochemical characterization and crystal structures of a cytochrome P450 fatty acid peroxygenase: the terminal alkene forming OleTJE (CYP152L1) from Jeotgalicoccus sp. 8456. OleTJE is stabilized at high ionic strength, but aggregation and precipitation of OleTJE in low salt buffer can be turned to advantage for purification, because resolubilized OleTJE is fully active and extensively dissociated from lipids. OleTJE binds avidly to a range of long chain fatty acids, and structures of both ligand-free and arachidic acid-bound OleTJE reveal that the P450 active site is preformed for fatty acid binding. OleTJE heme iron has an unusually positive redox potential (−103 mV versus normal hydrogen electrode), which is not significantly affected by substrate binding, despite extensive conversion of the heme iron to a high spin ferric state. Terminal alkenes are produced from a range of saturated fatty acids (C12–C20), and stopped-flow spectroscopy indicates a rapid reaction between peroxide and fatty acid-bound OleTJE (167 s−1 at 200 μm H2O2). Surprisingly, the active site is highly similar in structure to the related P450BSβ, which catalyzes hydroxylation of fatty acids as opposed to decarboxylation. Our data provide new insights into structural and mechanistic properties of a robust P450 with potential industrial applications. 相似文献
5.
Pseudomonas saccharophila NRRL B-628 is capable of utilizing agricultural lipids for growth. The organism exhibited good growth with triacylglycerol
substrates that contained saturated fatty acyl moieties such as coconut oil (CO; C10–12 fatty acids) and tallow (T; C16–18 fatty acids). Electron micrographs of the triacylglycerol-grown cells showed the presence of intracellular granules indicative
of poly(β-hydroxyalkanoate) (PHA) production. Cells grown in a 250-ml CO-containing medium produced ca. 0.2 g of medium-chain-length
(mcl)-PHA. Gas chromatographic analysis showed that β-hydroxyoctanoic acid (30%), β-hydroxydecanoic acid (40%), and β-hydroxydodecanoic
acid (16%) were the major monomer repeat-units of the CO-derived polymer. The estimated mean molecular mass of the CO-derived
mcl-PHA as determined by gel permeation chromatography was 13.1 × 104 g/mol with a polydispersity of 3.16.
Received: 15 August 1998 / Accepted: 25 September 1998 相似文献
6.
Fong-Chin Huang Alyssa Peter Wilfried Schwab 《Applied and environmental microbiology》2014,80(2):766-776
Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C16 to C20 fatty acids preferentially. It converted oleic acid (C18:1) more efficiently than stearic acid (C18:0) and linoleic acid (C18:2) and much more effectively than α-linolenic acid (C18:3). No products were detected when C10 to C12 fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C14 to C20 saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps. 相似文献
7.
Kitazume T Yamazaki Y Matsuyama S Shoun H Takaya N 《Applied microbiology and biotechnology》2008,79(6):981-988
Cytochrome P450foxy (P450foxy) is a fatty acid (FA) monooxygenase that is characterized by self-sufficient catalysis and high turnover numbers due to the fused structure of cytochrome P450 and its reductase. Here we found that resting recombinant Escherichia coli cells producing P450foxy converted saturated FA with a chain length of 7-16 carbon atoms to their omega-1 to omega-3 hydroxy derivatives. Most products were recovered from the culture supernatant. Decanoic acid was most efficiently converted to omega-1 to omega-3 hydroxy decanoic acids in the order of omega-1>omega-2>omega-3, with a total product yield of 47%. We also found that P450foxy was more active against physiological fatty acyl esters such as monopalmitoyl glycerol, monopalmitoyl phospholipid, and palmitoyl CoA than free palmitic acid. The bacteria producing P450foxy were applicable as biocatalysts in the production of omega-1 hydroxy palmitic acid from lard, vegetable, and soy sauce oil wastes from the food industry. 相似文献
8.
Fatty acid and carotenoid composition ofRhodotorula strains 总被引:4,自引:0,他引:4
Lipid content and composition of fatty acids with 6–25 carbon atoms were studied on strains of the 13 pink or red yeast species
belonging to the genus Rhodotorula. The total amount of lipid represented an average of 13% of the dry weight. The neutral and polar lipid fractions were analyzed
separately. For all the strains studied, the major fatty acids in both fractions were oleic, linoleic and palmitic acids,
which formed 80% of the total number of fatty acids. A notable amount of arachidonic acid, a precursor of eicosanoid hormones,
was found in R. acheniorum, R. aurantiaca and R. bacarum. Depending on the strain, 1–10 carotenoid pigments were detected; β-carotene was always the major carotenoid present.
Received: 13 December 1994 / Accepted: 18 May 1995 相似文献
9.
Newly isolated Acinetobacter (NRRL B-14920, B-14921, B-14923) and coryneform (NRRL B-14922) strains accumulated oleyl oleate and homologous liquid wax
esters (C30:2–C36:2) in culture broths. Diunsaturated oleyl oleate preponderated in 75 mg liquid wax esters (280 mg lipid extract) recovered
from 100-ml cultures of Acinetobacter B-14920 supplemented with 810 mg oleic acid–oleyl alcohol. With soybean oil instead of oleic acid, wax esters (260 mg) were
increased to approximately 50% of the lipid extract. Production of wax esters by cultures supplemented with combined fatty
(C8–C18) alcohols and acids suggests a coordinated synthesis whereby the exogenous alcohol remains unaltered, and the fatty acid
is partially oxidized with removal of C2 units before esterification. Consequently, C8–C18 primary alcohols control chain lengths of the wax esters. Exogenous fatty acids are presumed to enter an intracellular oxidation
pool from which is produced a homologous series of liquid wax esters. 相似文献
10.
Merja Suutari 《Archives of microbiology》1995,164(3):212-216
The effect of growth temperature on the lipid fatty acid composition was studied over a temperature range from 35 to 10°
C with 5° C intervals in four exponentially growing fungi: Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, and Trichoderma reesei. Fatty acid unsaturation increased in A. niger, P. chrysogenum, and T. reesei when the temperature was lowered to 20–15, 20, and 26–20° C, respectively. In A. niger and T. reesei, this was due to the increase in linolenic acid content. In P. chrysogenum, the linolenic acid content increased concomitantly with a more pronounced decrease in the less-unsaturated fatty acid, oleic
acid, and in palmitic and linoleic acids; consequently, the fatty acid content decreased as the temperature was lowered to
20° C. In T. reesei, when the growth temperature was reduced below 26–20° C, fatty acid unsaturation decreased since the mycelial linolenic acid
content decreased. In A. niger and P. chrysogenum, the mycelial fatty acid content increased greatly at temperatures below 20–15° C. In contrast, in N. crassa, fatty acid unsaturation was nearly temperature-independent, although palmitic and linoleic acid contents clearly decreased
when the temperature was lowered between 26 and 20° C; concomitantly, the growth rate decreased. Therefore, large differences
in the effects of growth temperature on mycelial fatty acids were observed among various fungal species. However, the similarities
found may indicate common regulatory mechanisms causing the responses.
Received: 1 March 1995 / Accepted: 8 May 1995 相似文献
11.
Gilmour KM Perry SF Esbaugh AJ Genz J Taylor JR Grosell M 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2012,182(2):259-274
In seawater-acclimated rainbow trout (Oncorhynchus mykiss), base secretion into the intestine is a key component of the intestinal water absorption that offsets osmotic water loss
to the marine environment. Acid–base balance is maintained by the matched excretion of acid equivalents via other routes,
presumably the gill and/or kidney. The goal of the present study was to examine acid–base balance in rainbow trout upon transfer
to more dilute environments, conditions under which base excretion into the intestine is predicted to fall, requiring compensatory
adjustments of acid excretion at the gill and/or kidney if acid–base balance is to be maintained. Net acid excretion via the
gill/kidney and rectal fluid, and blood acid–base status were monitored in seawater-acclimated rainbow trout maintained in
seawater or transferred to iso-osmotic conditions. As predicted, transfer to iso-osmotic conditions significantly reduced
base excretion into the rectal fluid (by ~48%). Transfer to iso-osmotic conditions also significantly reduced the excretion
of titratable acidity via extra-intestinal routes from 183.4 ± 71.3 to −217.5 ± 42.7 μmol kg−1 h−1 (N = 7). At the same time, however, ammonia excretion increased significantly during iso-osmotic transfer (by ~72%) so that
the apparent overall reduction in net acid excretion (from 419.7 ± 92.9 to 189.2 ± 76.5 μmol kg−1 h−1; N = 7) was not significant. Trout maintained blood acid–base status during iso-osmotic transfer, although arterial pH was significantly
higher in transferred fish than in those maintained in seawater. To explore the mechanisms underlying these adjustments of
acid–base regulation, the relative mRNA expression and where possible, activity of a suite of proteins involved in acid–base
balance were examined in intestine, gill and kidney. At the kidney, reduced mRNA expression of carbonic anhydrase (CA; cytosolic
and membrane-associated CA IV), V-type H+-ATPase, and Na+/HCO3
− co-transporter were consistent with a reduced role in net acid excretion following iso-osmotic transfer. Changes in relative
mRNA expression and/or activity at the intestine and gill were consistent with the roles of these organs in osmotic rather
than acid–base regulation. Overall, the data emphasize the coordination of acid–base, osmoregulatory and ionoregulatory processes
that occur with salinity transfer in a euryhaline fish. 相似文献
12.
Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis 总被引:1,自引:0,他引:1
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William H. Eschenfeldt Yeyan Zhang Hend Samaha Lucy Stols L. Dudley Eirich C. Ronald Wilson Mark I. Donnelly 《Applied microbiology》2003,69(10):5992-5999
Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C14:0) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development. 相似文献
13.
Eduardo Forján Inés Garbayo Marta Henriques Jorge Rocha José M. Vega Carlos Vílchez 《Marine biotechnology (New York, N.Y.)》2011,13(3):366-375
Nannochloropsis, a green microalga, is a source for commercially valuable compounds as extensively described and, in particular, is recognised
as a good potential source of eicosapentaenoic acid (20:5ϖ3), an important polyunsaturated fatty acid for human consumption
for prevention of several diseases. Climate change might include variation in the ultraviolet (UV) levels as one of the consequences
derived from the anthropogenic activity. This paper shows the response of Nannochloropsis cultures exposed for 7 days to UV-A (320–400 nm) added to photosynthetically active radiation (PAR; 400–700 nm). Growth rates
and photosynthetic activity were assessed to determine the impact of UV-A increased levels on the cell growth and basic metabolism
activity. Xanthophyll pigments (zeaxanthin and violaxanthin), carotenoids (canthaxanthin and β-carotene) and polyunsaturated
fatty acids (myristic, palmitic, palmitoleic, arachidonic and eicosapentaenoic acids) were measured for assessing the antioxidant
response of the microalgae to added UV-A radiation to PAR. The results show that the modulated use of UV-A radiations can
lead to increased growth rates, which are sustained in time by an increased light transduction activity. The expected antioxidant
response to the incident UV-A radiation consisted of increases in zeaxanthin and β-carotene contents—synthesis of antioxidant
carotenoids—and increases in the saturated fatty acids to polyunsaturated fatty acids ratio. The results suggest that modulated
UV-A radiation can be used as a tool to stimulate value molecules accumulation in microalgae through an enhanced both light
transduction process and antioxidant response, while sustaining cell growth. 相似文献
14.
Magdalena A. Gutowska F. Melzner M. Langenbuch C. Bock G. Claireaux H. O. Pörtner 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2010,180(3):323-335
Acidification of ocean surface waters by anthropogenic carbon dioxide (CO2) emissions is a currently developing scenario that warrants a broadening of research foci in the study of acid–base physiology.
Recent studies working with environmentally relevant CO2 levels, indicate that some echinoderms and molluscs reduce metabolic rates, soft tissue growth and calcification during hypercapnic
exposure. In contrast to all prior invertebrate species studied so far, growth trials with the cuttlefish Sepia officinalis found no indication of reduced growth or calcification performance during long-term exposure to 0.6 kPa CO2. It is hypothesized that the differing sensitivities to elevated seawater pCO2 could be explained by taxa specific differences in acid–base regulatory capacity. In this study, we examined the acid–base
regulatory ability of S. officinalis in vivo, using a specially modified cannulation technique as well as 31P NMR spectroscopy. During acute exposure to 0.6 kPa CO2, S. officinalis rapidly increased its blood [HCO3
−] to 10.4 mM through active ion-transport processes, and partially compensated the hypercapnia induced respiratory acidosis.
A minor decrease in intracellular pH (pHi) and stable intracellular phosphagen levels indicated efficient pHi regulation. We conclude that S. officinalis is not only an efficient acid–base regulator, but is also able to do so without disturbing metabolic equilibria in characteristic
tissues or compromising aerobic capacities. The cuttlefish did not exhibit acute intolerance to hypercapnia that has been
hypothesized for more active cephalopod species (squid). Even though blood pH (pHe) remained 0.18 pH units below control values,
arterial O2 saturation was not compromised in S. officinalis because of the comparatively lower pH sensitivity of oxygen binding to its blood pigment. This raises questions concerning
the potentially broad range of sensitivity to changes in acid–base status amongst invertebrates, as well as to the underlying
mechanistic origins. Further studies are needed to better characterize the connection between acid–base status and animal
fitness in various marine species. 相似文献
15.
Interactions of cytochrome P450 2B1 were probed using 8-anilino-1-naphthalenesulfonic acid (ANS), a well known and frequently used reporter group for hydrophobic interactions. Titration of cytochrome P450 2B1 with ANS revealed 6.6 ± 0.2 ANS binding sites per molecule of P450 2B1 with a Kd value of 42 ± 2 M. In our evaluation of the consequences of the binding of ANS to cytochrome P450 2B1, we found that the binding of ANS to P450 2B1 increased benzphetamine demethylation activity by 1.5-fold, indicating a role for ANS as an effector in addition to its role as a reporter group. Kinetic analysis of the effects of ANS on P450 2B1-dependent demethylation activity revealed that ANS increased both the Vmax and Km of the benzphetamine demethylation reaction. ANS stimulation of activity appears not to be due to the replacement or augmentation of the role of lipid since studies of binding and catalytic activities in the presence and absence of added lipid gave the same array of effects. These results demonstrate that ANS can bind to cytochrome P450 2B1 as would be expected of a reporter group probe but show in addition that this probe also acts as an effector molecule stimulating catalytic activity. Thus, results of ANS studies should be viewed with caution since the molecule may play more than one role in its reaction with a protein.Abbreviations ANS
8-anilino-1-naphthalenesulfonic acid
- bis-ANS
1,1-bis(4-anilino-5-naphthalenesulfonic acid
- DTT
dithiothreitol
- P450
cytochrome P450 相似文献
16.
Marco Girhard Florian Tieves Evelyne Weber Martha Sophia Smit Vlada B. Urlacher 《Applied microbiology and biotechnology》2013,97(4):1625-1635
Candida apicola belongs to a group of yeasts producing surface-active glycolipids consisting of sophorose and long-chain (ω)- or (ω-1)-hydroxy fatty acids. Hydroxylation of the fatty acids in this strain is likely catalyzed by cytochrome P450 monooxygenases (P450), which require reducing equivalents delivered via a cytochrome P450-diflavin reductase (CPR). We herein report cloning and characterization of the cpr gene from C. apicola ATCC 96134. The gene encoding a protein of 687 amino acids was cloned in Escherichia coli and the enzyme was expressed in functional form after truncation of its N-terminal putative membrane anchor. The truncated recombinant protein showed cytochrome c reducing activity (K M of 13.8 μM and k cat of 1,915 per minute). Furthermore, we herein demonstrate to our best knowledge for the first time the use of a eukaryotic CPR to transfer electrons to bacterial P450s (namely CYP109B1 and CYP154E1). Cloning and characterization of this CPR therefore is not only an important step in the study of the P450 systems of C. apicola, but also provides a versatile redox partner for the characterization of other bacterial P450s with appealing biotechnological potential. The GenBank accession number of the sequence described in this article is JQ015264. 相似文献
17.
Human cytochrome P450 (P450) 4F11 is still considered an “orphan” because its function is not well characterized. A bacterial expression system was developed for human P450 4F11, producing ∼230 nmol P450 from a 3-l culture of Escherichia coli. P450 4F11 was purified and utilized for untargeted substrate searches in human liver extract using a liquid chromatography/mass spectrometry-based metabolomic and isotopic labeling approach (Tang et al., 2009 [19]). Four fatty acids—palmitic, oleic, arachidonic, and docosahexaenoic—were identified in human liver and verified as substrates of P450 4F11. The products were characterized as ω-hydroxylated fatty acids by gas chromatography-mass spectrometry analysis of their trimethylsilyl derivatives. Kinetic analysis of the oxidation products confirmed that the fatty acids are substrates oxidized by P450 4F11. P450 4F11 also exhibited low activity for some drug N-demethylation reactions but none for activation of several pro-carcinogens. 相似文献
18.
19.
The cytochrome P-450K containing monooxygenase system of rat kidney cortex microsomes catalyzes the hydroxylation of various saturated fatty acids of medium chain length to the corresponding ω- and (ω-1)-hydroxy derivatives. The hydroxylation activity, as well as the ratio between the two hydroxylated products, vary with the carbon chain length of the fatty acid. Optimal hydroxylation activity is observed with myristic acid which yields the 13- and 14-hydroxylated products at a ratio of about 1. The ω/(ω-1)-hydroxylation ratio decreases with increasing carbon chain length of the fatty acid. On the other hand, with lauric acid as a substrate the ratio between ω- and (ω-1)-hydroxylation does not change significantly with varying time of incubation or substrate concentration, or incubation in a medium containing D2O or after induction of enhanced hydroxylation activity by starvation of the animals. Furthermore, 12-hydroxylauric acid and capric acid—which is almost exclusively ω-hydroxylated by rat kidney cortex microsomes—inhibit both 11- and 12-hydroxylation of lauric acid to a similar extent whereas 11-hydroxylauric acid does not seem to inhibit either 11- or 12-hydroxylation.C10-C16 fatty acids produce the type I spectral change upon addition to rat kidney cortex microsomes and seem to interact with similar amounts of the cytochrome P-450K present in these particles. In agreement with the metabolic studies, 12-hydroxylauric acid interacts with cytochrome P-450K giving rise to a reverse type I spectral change, whereas 11-hydroxylauric acid does not produce an observable spectral change. Finally, results of binding experiments with a series of derivatives of dodecane suggest that type I binding to cytochrome P-450K requires, besides a proper chain length, the presence of a carbonyl group together with an electron pair on a neighboring atom at the end of the carbon chain. A chain length of 14 carbon atoms seems to be optimal and it is suggested that this chain length may correspond to the distance between a possible binding site and the catalytic site of cytochrome P-450K 相似文献
20.
Cytochrome P450 (CYP) 147F1 from Streptomyces peucetius is a new CYP subfamily of that has been identified as ω-fatty acid hydroxylase. We describe the identification of CYP147F1 as a fatty acid hydroxylase by screening for the substrate using a substrate binding assay. Screening of substrates resulted in the identification of fatty acid groups of compounds as potential hits for CYP147F1 substrates. Fatty acids from C10:0 to C18:0 all showed type I shift spectra indicating their potential as substrates. Among several fatty acids tested, lauric acid, myrsitic acid, and palmitic acid were used to characterize CYP147F1. CYP147F1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida as surrogate electron transfer partners. Kinetic parameters, including the dissociation constant, Km, NADH consumption assay, production formation rate, and coupling efficiency for CYP147F1 were also determined. 相似文献