Functional Characterization of Three Specific Acyl-Coenzyme A Synthetases Involved in Anaerobic Cholesterol Degradation in Sterolibacterium denitrificans Chol1S
Functional Characterization of Three Specific Acyl-Coenzyme A Synthetases Involved in Anaerobic Cholesterol Degradation in Sterolibacterium denitrificans Chol1S
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DOI：
10.1128/aem.02721-17
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发表时间：
2018-04-01
影响因子：
4.4
通讯作者：
Boll, Matthias
中科院分区：
文献类型：
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作者：
Warnke, Markus;Jung, Tobias;Boll, Matthias
分解β-变形杆菌Sterolibacterium acetylficans Chol 1 S通过非氧依赖性途径分解代谢类固醇，如胆固醇。它涉及有氧胆固醇和胆汁酸降解所描述的酶反应序列，以及厌氧类固醇降解细菌中特有的酶。最近的研究表明，在S。胆甾-4-烯-3-酮中间体胆甾-4-烯-3-酮被氧非依赖性氧化为δ（4）-达法膦酸（C-26-oic acid），其随后被底物特异性酰基辅酶A（酰基辅酶A）合成酶（ACS）激活。进一步的降解被认为是通过非常规的β-氧化进行的，其中醛缩酶、醛脱氢酶和另外的ACS取代经典的β-羟酰基-CoA脱氢酶和硫解酶。在这里，我们异源表达了三种胆固醇诱导的基因，这些基因可编码AMP形成的ACS，并将其中两种产物分别表征为特异性3 β-羟基-δ 5-胆烯酰-CoA（C-24-oic acid）-和孕甾-4-烯-3-酮-22-酰-CoA（C-22-oic acid）-形成的ACS。第三种异源产生的ATP依赖性ACS对C-26-、C-24-或C-22-油酸无活性，但将3a α-H-4 α-（3'丙酸酯）-7a β-甲基六氢-1，5-茚二酮（HIP）活化为HIP-CoA，HIP-CoA是有氧胆固醇降解的晚期中间体，仍含有甾烷骨架的CD环。这项工作提供了实验证据表明，厌氧类固醇降解通过许多交替CoA-酯依赖性或非依赖性酶促反应序列作为aldolytic侧链和水解甾环COC键裂解的结果。aldolytic侧链降解途径，包括高度放能ACS和醛脱氢酶被认为是必不可少的驱动不利的氧不依赖性C-26羟基化forward.IMPORTANCE的生物降解无处不在丰富的类固醇是阻碍了他们的低溶解度和存在的两个季碳原子。好氧放线菌对胆固醇的降解已经被详细研究了30多年，涉及许多依赖于加氧酶的反应。相比之下，很少有人知道类固醇的氧不依赖性降解细菌。在胆固醇降解的厌氧模式生物Sterolibacterium plastificans Chol 1 S中，已经获得了初步证据，即类固醇降解通过许多交替的辅酶A（CoA）酯依赖/独立反应序列进行。在这里，我们描述了异源表达的三个高度特异性和特性的酰基辅酶A合成酶，其中两个发挥关键作用的侧链的降解，而第三个是专门参与的B环降解。所获得的结果揭示了包括40多个酶促反应的不依赖于氧的类固醇降解。
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 Delta(4)-dafachronic acid (C-26-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 beta-oxidation, where aldolases, aldehyde dehydrogenases, and additional ACSs substitute for classical beta-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 3 beta-hydroxy-Delta 5-cholenoyl-CoA (C-24-oic acid)- and pregn-4-en-3-one-22-oyl-CoA (C-22-oic acid)-forming ACSs, respectively. A third heterologously produced ATP-dependent ACS was inactive with C-26-, C-24-, or C-22-oic-acids but activated 3a alpha-H-4 alpha-(3' propanoate)-7a beta-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 COC 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 C-26 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.