Gilbert Stephanie, Hood LaChae, Seah Stephen Y K
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Canada.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Canada
J Bacteriol. 2017 Dec 20;200(2). doi: 10.1128/JB.00512-17. Print 2018 Jan 15.
The heteromeric acyl coenzyme A (acyl-CoA) dehydrogenase FadE28-FadE29 and the enoyl-CoA hydratase ChsH1-ChsH2, encoded by genes within the intracellular growth () operon of , catalyze the dehydrogenation of the cholesterol metabolite 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA), with a 3-carbon side chain, and subsequent hydration of the product 3-oxo-4,17-pregnadiene-20-carboxyl-CoA (3-OPDC-CoA) to form 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). The gene downstream of , i.e., , was expressed in recombinant RHA1 in combination with other genes within the operon. His-tagged Ltp2 copurified with untagged ChsH1-ChsH2, ChsH2, or the C-terminal domain of ChsH2, which contains a domain of unknown function (DUF35). Ltp2 in association with ChsH1-ChsH2 or just the DUF35 domain of ChsH2 was shown to catalyze the retroaldol cleavage of 17-HOPC-CoA to form androst-4-ene-3,17-dione and propionyl-CoA. Steady-state kinetic analysis using the Ltp2-DUF35 complex showed that the aldolase had optimal activity at pH 7.5, with a of 6.54 ± 0.90 μM and a of 159 ± 8.50 s ChsH1-ChsH2 could hydrate only about 30% of 3-OPDC-CoA, but this unfavorable equilibrium could be overcome when the aldolase was present to remove the hydrated product, providing a rationale for the close association of the aldolase with the hydratase. Homologs of ChsH1, ChsH2, and Ltp2 are found in steroid-degrading Gram-positive and Gram-negative bacteria, suggesting that side chains of diverse steroids may be cleaved by aldolases in the bacteria. The C-C bond cleavage of the D-ring side chain of cholesterol was shown to be catalyzed by an aldolase. The aldolase associates with the hydratase that catalyzes the preceding reaction in the cholesterol side chain degradation pathway. These enzymes are encoded by genes within the intracellular growth () operon of , and the operon was demonstrated previously to be linked to the pathogenicity and persistence of the bacteria in macrophages and in mice.
由结核分枝杆菌细胞内生长（）操纵子中的基因编码的异源二聚体酰基辅酶A（酰基-CoA）脱氢酶FadE28-FadE29和烯酰-CoA水合酶ChsH1-ChsH2，催化具有3碳侧链的胆固醇代谢物3-氧代-4-孕烯-20-羧基-CoA（3-OPC-CoA）的脱氢反应，以及产物3-氧代-4,17-孕二烯-20-羧基-CoA（3-OPDC-CoA）随后的水合反应，形成17-羟基-3-氧代-4-孕烯-20-羧基-CoA（17-HOPC-CoA）。结核分枝杆菌细胞内生长（）操纵子中，即下游的基因，与操纵子中的其他基因一起在重组结核分枝杆菌RHA1中表达。带有His标签的Ltp2与未标记的ChsH1-ChsH2、ChsH2或ChsH2的C末端结构域共纯化，ChsH2的C末端结构域包含一个功能未知的结构域（DUF35）。已证明Ltp2与ChsH1-ChsH2或仅与ChsH2的DUF35结构域结合时，可催化17-HOPC-CoA的逆羟醛裂解反应，形成雄甾-4-烯-3,17-二酮和丙酰-CoA。使用Ltp2-DUF35复合物进行的稳态动力学分析表明，醛缩酶在pH 7.5时具有最佳活性，Km为6.54±0.90μM，kcat为159±8.50 s。ChsH1-ChsH2仅能使约30%的3-OPDC-CoA水合，但当存在醛缩酶以去除水合产物时，这种不利的平衡可以被克服，这为醛缩酶与水合酶紧密结合提供了理论依据。在降解类固醇的革兰氏阳性和革兰氏阴性细菌中发现了ChsH1、ChsH2和Ltp2的同源物，这表明不同类固醇的侧链可能在这些细菌中被醛缩酶裂解。已证明胆固醇D环侧链的C-C键裂解是由一种醛缩酶催化的。醛缩酶与水合酶结合，水合酶催化胆固醇侧链降解途径中的前一步反应。这些酶由结核分枝杆菌细胞内生长（）操纵子中的基因编码，并且先前已证明该操纵子与细菌在巨噬细胞和小鼠中的致病性及持续性有关。
