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Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria.

ABSTRACT: 1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.-) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with epsilon(mM) 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg(2+) and corresponded to a previously described ;external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg(2+). Of these two ;internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.-), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do the two internal acyl-CoA synthetases.

SUBMITTER: Garland PB

PROVIDER: S-EPMC1179387 | biostudies-other | 1970 Sep

REPOSITORIES: biostudies-other

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Project description:Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for ?-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 10(5) ± 0.03 × 10(5) M(-1) s(-1)) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1?(2'-propanoate)-3a?-H-4?(3?-propanoate)-7a?-methylhexahydro-5-indanone (kcat/Km = 2.4 × 10(5) ± 0.1 × 10(5) M(-1) s(-1) and 3.2 × 10(5) ± 0.3 × 10(5) M(-1) s(-1), respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and a phylogenetic classification enabling prediction of specific functions of related enzymes.

Project description:The denitrifying betaproteobacterium Sterolibacterium denitrificans Chol1S catabolizes steroids such as cholesterol via an oxygen-independent pathway. It involves enzyme reaction sequences described for aerobic cholesterol and bile acid degradation as well as enzymes uniquely found in anaerobic steroid-degrading bacteria. Recent studies provided evidence that in S. denitrificans, the cholest-4-en-3-one intermediate is oxygen-independently oxidized to Δ4-dafachronic acid (C26-oic acid), which is subsequently activated by a substrate-specific acyl-coenzyme A (acyl-CoA) synthetase (ACS). Further degradation was suggested to proceed via unconventional β-oxidation, where aldolases, aldehyde dehydrogenases, and additional ACSs substitute for classical β-hydroxyacyl-CoA dehydrogenases and thiolases. Here, we heterologously expressed three cholesterol-induced genes that putatively code for AMP-forming ACSs and characterized two of the products as specific 3β-hydroxy-Δ5-cholenoyl-CoA (C24-oic acid)- and pregn-4-en-3-one-22-oyl-CoA (C22-oic acid)-forming ACSs, respectively. A third heterologously produced ATP-dependent ACS was inactive with C26-, C24-, or C22-oic-acids but activated 3aα-H-4α-(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) to HIP-CoA, a rather late intermediate of aerobic cholesterol degradation that still contains the CD rings of the sterane skeleton. This work provides experimental evidence that anaerobic steroid degradation proceeds via numerous alternate CoA-ester-dependent or -independent enzymatic reaction sequences as a result of aldolytic side chain and hydrolytic sterane ring C-C bond cleavages. The aldolytic side chain degradation pathway comprising highly exergonic ACSs and aldehyde dehydrogenases is considered to be essential for driving the unfavorable oxygen-independent C26 hydroxylation forward.IMPORTANCE The biological degradation of ubiquitously abundant steroids is hampered by their low solubility and the presence of two quaternary carbon atoms. The degradation of cholesterol by aerobic Actinobacteria has been studied in detail for more than 30 years and involves a number of oxygenase-dependent reactions. In contrast, much less is known about the oxygen-independent degradation of steroids in denitrifying bacteria. In the cholesterol-degrading anaerobic model organism Sterolibacterium denitrificans Chol1S, initial evidence has been obtained that steroid degradation proceeds via numerous alternate coenzyme A (CoA)-ester-dependent/independent reaction sequences. Here, we describe the heterologous expression of three highly specific and characteristic acyl-CoA synthetases, two of which play key roles in the degradation of the side chain, whereas a third one is specifically involved in the B ring degradation. The results obtained shed light into oxygen-independent steroid degradation comprising more than 40 enzymatic reactions.

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Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria.

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