Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis
Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis
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DOI：
10.1074/jbc.m111.289975
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发表时间：
2011-11-25
影响因子：
4.8
通讯作者：
Eltis, Lindsay D.
中科院分区：
文献类型：
--
作者：
Capyk, Jenna K.;Casabon, Israeel;Eltis, Lindsay D.
结核分枝杆菌(Mtb)是一种重要的全球性致病菌，含有胆固醇分解代谢途径。尽管胆固醇分解代谢在结核分枝杆菌中的确切作用尚不清楚，但该途径中的Rieske单加氧酶-3-酮类固醇9α-羟基酶(KshAB)已被确定为毒力因子。为了研究KshAB的生理底物，利用罗氏球菌酰基辅酶A合成酶合成了两种胆固醇衍生物的辅酶A硫酸酯：3-氧代-23，24-二降冰醇-4-烯-22-甲酸(形成4-BNC-CoA)和3-氧代-23，24-二去甲胆酸-1，4-二烯-22-甲酸(形成1，4-BNC-CoA)。KshAB对辅酶A硫酸酯底物的表观特异性常数(k(Cat)/K-m)是先前提出的作为生理底物的17-酮类化合物的20-30倍。在1，4-BNC-CoA存在下，表观K-mO2为90+/-10µM，与另外两种胆固醇分解代谢加氧酶的值一致。Delta(1)酮类固醇脱氢酶KstD与KshAB共同作用，切割类固醇环B，对CoA硫代酯的比活性是相应的酮的8倍。最后，将1，4-BNC-CoA模拟到KshA晶体结构中，表明CoA部分结合在活性部位通道口部的口袋中，可能有助于底物专一性。这些结果表明，KshAB的生理底物是胆固醇侧链降解的辅酶A硫酯中间体，在Mtb中侧链和环的降解同时发生。这一发现对病原体在感染过程中可能释放的类固醇代谢产物以及胆固醇降解酶抑制剂的设计具有重要意义。用于生成硫代酯的方法学和罗氏球菌酶将有助于进一步研究胆固醇分解代谢。
Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9 alpha-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (k(cat)/K-m) of KshAB for the CoA thioester substrates was 20-30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent K-mO2 was 90 +/- 10 mu M in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Delta(1) ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety, binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism.