Functional characterization of 3-ketosteroid 9α-hydroxylases in Rhodococcus ruber strain chol-4

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.