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Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6673-6677, August 1992 Biochemistry NADPH-dependent a-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms (5-enoyl-CoA/A3,A2-enoyl-CoA isomerase/A3',52'4-dienoyl-CoA isomerase/2,4-dienoyl-CoA reductase) TOR E. SMELAND*, MOHAMED NADA*, DEAN CUEBASt, AND HORST SCHULZ* *Department of Chemistry, City College of the City University of New York, New York, NY 10031; and tJoined Departments of Chemistry, Manhattan College/College of Mount Saint Vincent, Riverdale, NY 10471 Communicated by Salih J. Wakil, April 13, 1992 ABSTRACT The mitochondrial metabolism of 5-enoyl- a recent observation of Tserng and Jin (5) who reported that CoAs, which are formed during the (3-oxidation of unsaturated the mitochondrial -oxidation of 5-enoyl-CoAs is dependent fatty acids with double bonds extending from odd-numbered on NADPH. Their analysis of metabolites by gas chroma- carbon atoms, was studied with mitochondrial extracts and tography/mass spectrometry led them to propose that the purified enzymes of (3-oxidation. Metabolites were identified double bond of 5-enoyl-CoAs is reduced by NADPH to yield spectrophotometrically and by high performance liquid chro- the corresponding saturated fatty acyl-CoAs, which are then matography. 5-cis-Octenoyl-CoA, a putative metabolite of further degraded by P-oxidation. linolenic acid, was efficiently dehydrogenated by medium- This report addresses the question of how 5-enoyl-CoAs chain acyl-CoA dehydrogenase (EC 1.3.99.3) to 2-trans-5-cis- are chain-shortened by P-oxidation. We demonstrate that octadienoyl-CoA, which was isomerized to 3,5-octadienoyl- 5-enoyl-CoAs, after dehydrogenation to 2,5-dienoyl-CoAs, CoA either by mitochondrial A3,A2-enoyl-CoA isomerase (EC can be isomerized to 2,4-dienoyl-CoAs, which are reduced by 5.3.3.8) or by peroxisomal trifunctional enzyme. Further the NADPH-dependent 2,4-dienoyl-CoA reductase. Thus, isomerization of 3,5-octadienoyl-CoA to 2-trans-4-trans- odd-numbered double bonds, like even-numbered double octadienoyl-CoA in the presence ofsoluble extracts ofeither rat bonds, can be reductively removed during p-oxidation. liver or rat heart mitochondria was observed and attributed to a A3',542'4-dienoyl-CoA isomerase. Qualitatively similar re- sults were obtained with 2-trans-5-trans-octadienoyl-CoA MATERIALS AND METHODS formed by dehydrogenation of 5-trans-octenoyl-CoA. 2-trans- Materials. Acetyl-CoA, n-butyryl-CoA, n-hexanoyl-CoA, 4-trans-Octadienoyl-CoA was a substrate for NADPH- n-octanoyl-CoA, CoA, NADPH, NADI, bovine serum al- dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34). A soluble bumin, pig heart L-3-hydroxyacyl-CoA dehydrogenase (EC extract of rat liver mitochondria catalyzed the isomerization of 1.1.1.35), acyl-CoA oxidase from Candida sp., and all stan- 2-trans-5-cis-octadienoyl-CoA to 2-trans-4-trans-octadienoyl- dard biochemicals were obtained from Sigma. 2-trans-4- CoA, which upon addition of NADPH, NAD+, and CoA was trans-Octadienal and 3-trans-octadecenoic acid were pur- chain-shortened to hexanoyl-CoA, butyryl-CoA, and acetyl- chased from Bedoukian Research (Danbury, CT) and Pfaltz CoA. Thus we conclude that odd-numbered double bonds, like & Bauer, respectively. 2-trans-4-trans-Octadienoic acid was even-numbered double bonds, can be reductively removed prepared from 2-trans-4-trans-octadienal by oxidation with during the (3-oxidation of polyunsaturated fatty acids. Ag20 according to a general procedure for the oxidation of aldehydes to acids that are sensitive to strong oxidizing The degradation of unsaturated fatty acids by 8-oxidation agents (6). 2-trans-4-trans-Octadienoic acid after crystalliza- involves at least two auxiliary enzymes in addition to the tion from hexane had a melting point of 75-760C [literature enzymes required for the breakdown of saturated fatty acids melting point, 760C (7)]. The methyl esters of 5-cis-octenoic (1). The auxiliary enzymes acting on double bonds are acid and 5-trans-octenoic acid were generously provided by 2,4-dienoyl-CoA reductase or 4-enoyl-CoA reductase (EC Howard Sprecher (Ohio State University). The purities ofthe 1.3.1.34) and A3,A2-enoyl-CoA isomerase (EC 5.3.3.8) (2). cis- and trans-isomers were 98% and 96%, respectively. The Chain shortening of unsaturated fatty acids with double methyl esters were saponified with a 3-fold molar excess of bonds extending from even-numbered carbon atoms leads to aqueous 0.4 M KOH until the system became monophasic. the formation of4-enoyl-CoAs, which are dehydrogenated by The resultant acids were obtained after acidification and acyl-CoA dehydrogenase (EC 1.3.99.3) to 2,4-dienoyl-CoAs. extraction with ether. The CoA derivatives of 5-cis-octenoic An NADPH-dependent 2,4-dienoyl-CoA reductase, origi- acid, 5-trans-octenoic acid, 3-trans-octenoic acid, and nally described by Kunau and Dommes (3), catalyzes the 2-trans-4-trans-octadienoic acid were synthesized according reduction of 2,4-dienoyl-CoAs to 3-enoyl-CoAs, which, after to the procedure of Goldman and Vagelos (8). All synthetic isomerization by A3,A2-enoyl-CoA isomerase to 2-enoyl- acyl-CoAs used in HPLC or spectrophotometric experiments CoAs, can be completely degraded via the P-oxidation spiral. were purified by HPLC. Concentrations of acyl-CoAs were Unsaturated fatty acids with double bonds extending from determined by the method of Ellman (9) after cleaving the odd-numbered carbon atoms are, according to Stoffel and thioester bond with NH20H at pH 7. 2-trans-5-cis- Caesar (4), chain-shortened to 3-enoyl-CoAs, which, after Octadienoyl-CoA and 2-trans-5-trans-octadienoyl-CoA were isomerization to 2-enoyl-CoAs by A3,A2-enoyl-CoA synthesized from the corresponding 5-octenoyl-CoAs by isomerase, reenter the p-oxidation spiral. Ifso, 5-enoyl-CoAs allowing them to react with oxygen in the presence of are intermediates that would pass once more through the acyl-CoA oxidase from Candida sp. as described (10). Bovine ,8-oxidation spiral before being acted upon by A3,A2-enoyl- liver enoyl-CoA hydratase (EC 4.2.1.17) or crotonase (11), CoA isomerase. This prediction, however, is contradicted by the trifunctional enzyme from rat liver peroxisomes (12, 13), and pig heart 3-ketoacyl-CoA thiolase (EC 2.3.1.16) (14) were The publication costs of this article were defrayed in part by page charge purified as described. Mitochondrial A3,A2-enoyl-CoA payment. This article must therefore be hereby marked "advertisement" isomerase (EC 5.3.3.8) was partially purified by chromatog- in accordance with 18 U.S.C. §1734 solely to indicate this fact. raphy of a soluble extract of rat liver mitochondria on 6673 Downloaded by guest on September 29, 2021 6674 Biochemistry: Smeland et al. Proc. Natl. Acad Sci. USA 89 (1992) hydroxylapatite as described by Kilponen et al. (15). 2,4- double bonds extending from odd-numbered carbon atoms, Dienoyl-CoA reductase (EC 1.3.1.34) was partially purified was studied with 5-cis-octenoyl-CoA and 5-trans-octenoyl- by chromatography of a soluble extract of rat liver mitochon- CoA. The suggested reduction of 5-cis-enoyl-CoAs to acyl- dria on agarose-heptane-adenosine-2',5'-diphosphate as de- CoAs by NADPH (5) was investigated. When 5-cis-octenoyl- scribed by Wang and Schulz (16). Medium-chain acyl-CoA CoA was incubated with NADPH in the presence of rat liver dehydrogenase was partially purified from bovine liver mi- mitochondria, no oxidation of NADPH was observed. Thus, tochondria as described (17). Mitochondria, isolated from a it seems that 5-enoyl-CoAs are not directly converted to their single rat liver (18), were suspended in 0.1 M potassium saturated analogs by a hypothetical NADPH-dependent phosphate (pH 8), sonicated for ten 20-s bursts at 0C with an 5-enoyl-CoA reductase. Ultrasonic sonifier (model W-385) equipped with a microtip, Purified 5-cis-octenoyl-CoA, which gave a single peak on and centrifuged at 100,000 x g for 1 hr. The soluble mito- HPLC (Fig. 1 A), was incubated with acyl-CoA oxidase chondrial extract (5.9 mg/ml) was stored at -70'C. Protein either in the presence or absence ofcatalase. The same major concentrations were determined by the method of Bradford reaction product was obtained under both conditions and was (19). purified by HPLC to remove unreacted starting material as Enzyme Assays. Acyl-CoA dehydrogenase was assayed well as a more polar reaction product. The product of this spectrophotometrically as described (17). A3,A2-Enoyl-CoA enzymatic reaction was assumed to be 2-trans-5-cis- isomerase was assayed spectrophotometrically at 340 nm in octadienoyl-CoA (Fig. 1B) since acyl-CoA oxidases are a coupled assay (20) with 3-trans-octenoyl-CoA as substrate. known to dehydrogenate acyl-CoAs to 2-trans-enoyl-CoAs 2,4-Dienoyl-CoA reductase was assayed spectrophotometri- while reducing oxygen to H202 (12). The dehydrogenation cally at 340 nm with 2-trans-4-trans-octadienoyl-CoA as a product 2-trans-5-cis-octadienoyl-CoA was clearly separated substrate as described (3). from the starting material 5-cis-octenoyl-CoA by HPLC (Fig. Metabolic Studies. The hydration of 2,5-octadienoyl-CoA 1C). The absorbance spectrum of 2-trans-5-cis-octadienoyl- by crotonase and its isomerization to 3,5-octadienoyl-CoA CoA (Fig. 2A) is characteristic of an acyl-CoA with a max- were followed spectrophotometrically at 263 nm and 238 nm, imum close to 260 nm due to the adenine moiety of CoA. respectively. Incubation mixtures contained 50 gM 2,5- When 2-trans-5-cis-octadienoyl-CoA was incubated with octadienoyl-CoA in 0.1 M potassium phosphate (pH 8) and crotonase, the absorbance around 260 nm decreased as then purified bovine liver crotonase, purified trifunctional expected for a 2-enoyl-CoA compound that is hydrated to enzyme from rat liver peroxisomes, or partially purified 3-hydroxyacyl-CoA (Fig.
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  • Enzyme Preparations: Recommendations for Submission of Chemical and Technological Data for Food Additive Petitions and GRAS Notices

    Enzyme Preparations: Recommendations for Submission of Chemical and Technological Data for Food Additive Petitions and GRAS Notices

    Contains Nonbinding Recommendations Guidance for Industry Enzyme Preparations: Recommendations for Submission of Chemical and Technological Data for Food Additive Petitions and GRAS Notices Additional copies are available from: Office of Food Additive Safety Division of Biotechnology and GRAS Notice Review, HFS-255 Center for Food Safety and Applied Nutrition Food and Drug Administration 5001 Campus Drive College Park, MD 20740 (Tel) 301-436-1200 http://www.fda.gov/FoodGuidances You may submit written comments regarding this guidance at any time. Submit written comments on the guidance to the Division of Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852. All comments should be identified with the title of the guidance document. U.S. Department of Health and Human Services Food and Drug Administration Center for Food Safety and Applied Nutrition January 1993; Revised July 2010 Contains Nonbinding Recommendations Table of Contents I. Introduction II. Background III. Discussion A. Petitions for Enzyme Preparations B. GRAS Notices for Enzyme Preparations IV. Recommendations for Petition or GRAS Notice Preparation A. Identity B. Manufacturing Process C. Specifications for Identity and Purity D. Intended Technical Effects and Use E. Intake Estimate V. Additional Information A. Enzyme Preparations Used in Meat and Poultry Processing B. Enzyme Preparations Containing Allergenic Ingredients 2 Contains Nonbinding Recommendations Guidance for Industry1 Enzyme Preparations: Recommendations for Submission of Chemical and Technological Data for Food Additive Petitions and GRAS Notices This guidance represents the Food and Drug Administration’s current thinking on this topic. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public.