Degradation of the Acyl Side Chain of the Steroid Compound Cholate in Pseudomonas sp Strain Chol1 Proceeds via an Aldehyde Intermediate
Degradation of the Acyl Side Chain of the Steroid Compound Cholate in Pseudomonas sp Strain Chol1 Proceeds via an Aldehyde Intermediate
复制标题
DOI：
10.1128/jb.01961-12
复制
发表时间：
2013-02-01
影响因子：
3.2
通讯作者：
Philipp, Bodo
中科院分区：
文献类型：
--
作者：
Holert, Johannes;Kulic, Zarko;Philipp, Bodo
细菌对类固醇的降解很普遍，但其代谢途径很少被探索。假单胞菌的研究进展。菌株Chol1和C-24类固醇胆酸盐表明，胆酸的降解是通过A环的氧化，然后C-17上连接的C-5酰基侧链的裂解进行的，产物为7α，12β-二羟基-1，4-二烯-3，17-二酮(12β-DHADD)。在本研究中，我们用Chol1菌株的细胞提取物在体外研究了胆酸酰基侧链的降解途径。为此，菌株Chol1产生了降解胆酸的中间产物，并以辅酶A(CoA)、三磷酸腺苷(ATP)和NAD(+)为辅底物进行了酶活性测定。当以C-24甾体(22E)-7α，12 alpha-dihydroxy-3-oxochola-1，4，22-triene-24-oate(DHOCTO)为底物时，完全转化为12α-DHADD和7 alpha-hydroxy-androsta-1，4-diene-3，12，17-trione(HADT)作为最终产物，表明酰基侧链被完全去除。以C-22甾体7α，12 alpha-dihydroxy-3-oxopregna-1，4-diene-20-carboxylate(DHOPDC)为底物形成相同的产物。在NADPH依赖的反应中，12-酮化合物HADT转化为12-β-DHADD。当DHOCTO分析中省略NAD(+)时，生成一种新的产物，鉴定为7α，12 alpha-dihydroxy-3-oxopregna1，4-diene-20S-carbaldehyde(DHOPDCA)。该醛在NAD(+)、CoA和ATP存在下转化为DHOPDC和DHOPDC-CoA。这些结果表明，胆酸盐的C-5酰基侧链的降解不是通过经典的β-氧化进行的，而是通过被氧化成相应酸的游离醛进行的。导致醛的反应可能是由基因skt编码的醛缩酶催化的，之前预测它是一种β-酮硫醇酶。
Bacterial degradation of steroids is widespread, but the metabolic pathways have rarely been explored. Previous studies with Pseudomonas sp. strain Chol1 and the C-24 steroid cholate have shown that cholate degradation proceeds via oxidation of the A ring, followed by cleavage of the C-5 acyl side chain attached to C-17, with 7 alpha,12 beta-dihydroxy-androsta-1,4-diene-3,17-dione (12 beta-DHADD) as the product. In this study, the pathway for degradation of the acyl side chain of cholate was investigated in vitro with cell extracts of strain Chol1. For this, intermediates of cholate degradation were produced with mutants of strain Chol1 and submitted to enzymatic assays containing coenzyme A (CoA), ATP, and NAD(+) as cosubstrates. When the C-24 steroid (22E)-7 alpha,12 alpha-dihydroxy-3-oxochola-1,4,22-triene-24-oate(DHOCTO) was used as the substrate, it was completely transformed to 12 alpha-DHADD and 7 alpha-hydroxy-androsta-1,4-diene-3,12,17-trione (HADT) as end products, indicating complete removal of the acyl side chain. The same products were formed with the C-22 steroid 7 alpha,12 alpha-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) as the substrate. The 12-keto compound HADT was transformed into 12 beta-DHADD in an NADPH-dependent reaction. When NAD(+) was omitted from assays with DHOCTO, a new product, identified as 7 alpha,12 alpha-dihydroxy-3-oxopregna1,4-diene-20S-carbaldehyde (DHOPDCA), was formed. This aldehyde was transformed to DHOPDC and DHOPDC-CoA in the presence of NAD(+), CoA, and ATP. These results revealed that degradation of the C-5 acyl side chain of cholate does not proceed via classical beta-oxidation but via a free aldehyde that is oxidized to the corresponding acid. The reaction leading to the aldehyde is presumably catalyzed by an aldolase encoded by the gene skt, which was previously predicted to be a beta-ketothiolase.