A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism
A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism
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DOI：
10.1074/jbc.m109.099028
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发表时间：
2010-07-16
影响因子：
4.8
通讯作者：
Eltis, Lindsay D.
中科院分区：
文献类型：
--
作者：
Dresen, Carola;Lin, Leo Y. -C.;Eltis, Lindsay D.
结核分枝杆菌（Mtb）和约氏红球菌RHA 1具有相似的胆固醇分解代谢途径。该途径有助于Mtb的致病性。已经预测hsaAB胆固醇分解代谢基因分别编码黄素依赖性单加氧酶的加氧酶和还原酶，所述黄素依赖性单加氧酶将3-羟基-9，10-二雄甾-1，3，5（10）-三烯-9，17-二酮（3-HSA）羟基化为儿茶酚。RHA 1的hsaA缺失突变体不能在胆固醇上生长，但将后者转化为3-HSA和相关代谢物，其中两个酮基中的每一个都被还原：3，9-二羟基-9，10-二雄甾-1，3，5（10）-三烯-17-酮（3，9-DHSA）和3，17-二羟基-9，10-二雄甾-1，3，5（10）-三烯-9-酮（3，17-DHSA）。从结核分枝杆菌纯化的3-羟基-9，10-二雄甾-1，3，5（10）-三烯-9，17-二酮4-羟化酶（HsaAB）对3-HSA的特异性高于对3，17-DHSA的特异性（表观k（cat）/K-m =1000 +/-100 M-1 s（-1）对700 +/- 100 M-1 s（-1））。然而，3，9-DHSA是比3-羟基联苯差的底物（表观k（cat）/K-m = 80 +/- 40 M-1 s（-1））。在3-HSA存在的情况下，O-2的K-mapp为100 +/- 10 μ M。2.5埃分辨率的HsaA的晶体结构显示，该酶具有相同的折叠，黄素结合位点，和催化残基作为对羟基苯基乙酸羟化酶。然而，HsaA具有更大的酚结合位点，与酶的底物特异性一致。此外，HsaA的第二种晶体形式揭示了C-末端瓣（瓦尔（367）-瓦尔（394））可以采用两种构象，其不同之处在于围绕Arg（366）的25度刚体旋转。这种旋转似乎门可能黄素入口的活性位点。在与3-HSA和黄素的对接研究中，闭合构象为酶的底物特异性提供了理论基础。总的来说，结构和功能数据建立了HsaAB的生理作用，并提供了基础，以进一步研究一类重要的单加氧酶以及细菌的类固醇催化剂。
Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17- dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene- 17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5( 10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10- seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k(cat)/K-m =1000 +/- 100 M-1 s(-1) versus 700 +/- 100 M-1 s(-1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k(cat)/K-m = 80 +/- 40 M-1 s(-1)). In the presence of 3-HSA the K-mapp for O-2 was 100 +/- 10 mu M. The crystal structure of HsaA to 2.5-angstrom resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val(367)-Val(394)) could adopt two conformations differing by a rigid body rotation of 25 degrees around Arg(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.