Department of Microbiology and Immunology, University of British Columbia, Vancouver V6T 1Z3, Canada.
J Biol Chem. 2010 Jul 16;285(29):22264-75. doi: 10.1074/jbc.M109.099028. Epub 2010 May 6.
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(m)(app) for O(2) was 100 +/- 10 microM. The crystal structure of HsaA to 2.5-A 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.
结核分枝杆菌（Mtb）和罗氏红红球菌 RHA1 具有相似的胆固醇代谢途径。该途径有助于结核分枝杆菌的致病性。hsaAB 胆固醇代谢基因被预测分别编码黄素依赖性单加氧酶的加氧酶和还原酶，该酶将 3-羟基-9,10-降二氢胆甾-1,3,5(10)-三烯-9,17-二酮（3-HSA）羟基化为儿茶酚。RHA1 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，其中两个酮基均被还原：3,9-二羟基-9,10-降二氢胆甾-1,3,5(10)-三烯-17-酮（3,9-DHSA）和 3,17-二羟基-9,10-降二氢胆甾-1,3,5(10)-三烯-9-酮（3,17-DHSA）。从结核分枝杆菌中纯化的 3-羟基-9,10-降二氢胆甾-1,3,5(10)-三烯-9,17-二酮 4-羟化酶（HsaAB）对 3-HSA 的特异性高于 3,17-DHSA（表观 kcat/Km = 1000 ± 100 M-1 s-1 对 700 ± 100 M-1 s-1）。然而，3,9-DHSA 是比 3-羟基联苯更差的底物（表观 kcat/Km = 80 ± 40 M-1 s-1）。在存在 3-HSA 的情况下，O2 的 K(m)(app)为 100 ± 10 microM。HsaA 的 2.5-A 分辨率晶体结构表明，该酶具有相同的折叠、黄素结合位点和催化残基，与 p-羟基苯乙酸羟化酶相同。然而，HsaA 具有更大的苯酚结合位点，与酶的底物特异性一致。此外，HsaA 的第二种晶体形式表明，C 末端瓣（Val367-Val394）可以通过围绕 Arg366 的刚性体旋转 25 度来采用两种构象。这种旋转似乎是活性位点中黄素入口的门。在与 3-HSA 和黄素的对接研究中，封闭构象为酶的底物特异性提供了依据。总的来说，结构和功能数据确立了 HsaAB 的生理作用，并为进一步研究一类重要的单加氧酶以及细菌对类固醇的代谢提供了基础。
