Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis
Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis
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DOI：
10.1074/jbc.m900719200
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发表时间：
2009-04-10
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.;D'Angelo, Igor;Eltis, Lindsay D.
KshAB（3-酮类固醇9 α-羟化酶）是结核分枝杆菌胆固醇分解代谢途径中的双组分Rieske加氧酶（RO）。虽然这种酶与发病机制有关，但生物信息学和分子遗传学对其进行了大量研究。纯化的还原酶组分KshB是含有植物型[2Fe-2S]簇和FAD的单体蛋白。加氧酶KshA是一个含有Rieske [2Fe-2S]簇和单核亚铁离子的同源三聚体。在两种潜在底物中，重组KshAB对1，4-雄甾二烯-3，17-二酮的特异性是对4-雄甾烯-3，17-二酮的两倍。两种底物的转化与O-2的消耗耦合良好。然而，在1，4-雄甾二烯-3，17-二酮存在下，KshAB与O-2的反应性较低，k（cat）/K-mO 2为2450 +/- 80 M-1 s（-1）。KshA的晶体结构，确定为2.3埃，揭示了一个整体的折叠和一个典型的RO头到尾亚基的安排。催化结构域的中心折叠缺乏在表征的RO中发现的所有插入，这与最小的并且可能是原型的RO催化结构域一致。KshA的结构进一步通过C-末端螺旋区分，其稳定功能三聚体中的亚基相互作用。最后，底物结合口袋延伸到KshA比其他RO，与大类固醇底物一致，和漏斗访问的活性位点是不同的取向。本研究为进一步研究具有生物技术和医学重要性的关键类固醇转化酶提供了坚实的基础。
KshAB (3-Ketosteroid 9 alpha-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O-2. Nevertheless, the reactivity of KshAB with O-2 was low in the presence of 1,4- androstadiene-3,17-dione, with a k(cat)/K-mO2 of 2450 +/- 80 M-1 s(-1). The crystallographic structure of KshA, determined to 2.3 angstrom, revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid transforming enzyme of biotechnological and medical importance.