3-酮类固醇-9α-羟化酶,也称为KshAB[androsta-1,4-二烯-3,17-二酮,NADH:氧氧化还原酶(9α-羟基化);EC1.14.13.142)],是细菌类固醇分解代谢与3-酮类固醇-Δ1-脱氢酶活性(KstD)组合的一般方案中的关键酶,两者都是类固醇核(环A/B)断裂的原因。KshAB通过4-烯-3-氧代类固醇(例如AD)或1,4-二烯-3-氧代类固醇(例如ADD)的C9碳的9α-羟基化引发类固醇环的开放,将它们转化为9α-羟基-4-雄蕊-3,17-二酮(9OHAD)或9α-羟基-1,4-雄蕊-3,17-二酮(9OHADD),分别。这些酶在放线菌基因组中的冗余导致代谢工程化该分解代谢途径以获得工业感兴趣的中间体的严重困难。在这项工作中,我们已经在R.ruber菌株Chol-4的不同基因组区域中鉴定了三个同源kshA基因和一个kshB基因。我们提供了一组数据,这些数据有助于了解它们在该菌株中的特定作用,包括:i)KshAB酶的描述ii)ΔkshB和单,R.ruber中的双重和三重ΔkshA突变体iii)上述菌株在不同底物上的生长研究,以及iv)这些菌株的遗传互补和生物转化测定。我们的结果表明,KshA2同工型是降解短侧链类固醇底物所需要的,而KshA3作用于那些具有较长侧链的分子。KshA1是一种与胆酸分解代谢有关的更通用的酶,尽管它在类固醇的分解代谢中也与KshA2或KshA3活性合作。根据对其他红球菌菌株的描述,我们的结果还表明,侧链降解是KshAB非依赖性的。
The 3-Ketosteroid-9α-Hydroxylase, also known as KshAB [androsta-1,4-diene-3,17-dione, NADH:oxygen oxidoreductase (9α-hydroxylating); EC 1.14.13.142)], is a key enzyme in the general scheme of the bacterial steroid catabolism in combination with a 3-ketosteroid-Δ1-dehydrogenase activity (KstD), being both responsible of the steroid nucleus (rings A/B) breakage. KshAB initiates the opening of the steroid ring by the 9α-hydroxylation of the C9 carbon of 4-ene-3-oxosteroids (e.g. AD) or 1,4-diene-3-oxosteroids (e.g. ADD), transforming them into 9α-hydroxy-4-androsten-3,17-dione (9OHAD) or 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD), respectively. The redundancy of these enzymes in the actinobacterial genomes results in a serious difficulty for metabolic engineering this catabolic pathway to obtain intermediates of industrial interest. In this work, we have identified three homologous kshA genes and one kshB gen in different genomic regions of R. ruber strain Chol-4. We present a set of data that helps to understand their specific roles in this strain, including: i) description of the KshAB enzymes ii) construction and characterization of ΔkshB and single, double and triple ΔkshA mutants in R. ruber iii) growth studies of the above strains on different substrates and iv) genetic complementation and biotransformation assays with those strains. Our results show that KshA2 isoform is needed for the degradation of steroid substrates with short side chain, while KshA3 works on those molecules with longer side chains. KshA1 is a more versatile enzyme related to the cholic acid catabolism, although it also collaborates with KshA2 or KshA3 activities in the catabolism of steroids. Accordingly to what it is described for other Rhodococcus strains, our results also suggest that the side chain degradation is KshAB-independent.