Rhodococcus rhodochrous DSM 43269 3-Ketosteroid 9α-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity

Abstract

This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9␣-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in Escherichia coli. E. coli cell cultures, expressing both KshA and KshB, converted 4-androstene-3,17-dione (AD) into 9␣-hydroxy-4-AD (9OHAD) with a >60% molar yield over 48 h of incubation. Coexpression and copurification were critical to successfully obtain pure and active KSH. Biochemical analysis revealed that the flavoprotein KshB is an NADH-dependent reductase using flavin adenine dinucleotide as a cofactor. Reconstitution experiments confirmed that KshA, KshB, and NADH are essential for KSH activity with steroid substrates. KSH hydroxylation activity was inhibited by several divalent metal ions, especially by zinc. The reconstituted KSH displayed subtle steroid substrate specificity; a range of 3-ketosteroids, i.e., 5␣-⌯, 5␤-⌯, ⌬1, and ⌬4 steroids, could act as KSH substrates, provided that they had a short side chain. The formation of 9OHAD from AD by KSH was confirmed by liquid chromatography-mass spectrometry analysis and by the specific enzymatic conversion of 9OHAD into 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione using 3-ketosteroid ⌬1-dehydrogenase. Only a single KSH is encoded in the genome of the human pathogen Mycobacterium tuberculosis H37Rv, shown to be important for survival in macrophages. Since no human KSH homolog exists, the M. tuberculosis enzyme may provide a novel target for treatment of tuberculosis. Detailed knowledge about the biochemical properties of KSH thus is highly relevant in the research fields of biotechnology and medicine.Hydroxylated steroids are pharmaceutically very interesting bioactive compounds. 9␣-Hydroxylated steroids are of particular importance for the synthesis of corticoids such as 9␣-fluorohydrocortisone. Microorganisms are widely used for the stereo-specific hydroxylation of steroids, but little is known about the enzymes involved, and current processes suffer from low conversion rates and yields (12,18,23).Rhodococcus species are well known for their broad catabolic potential and ability to degrade sterols and steroids (14,21,25,39). In this paper, we focus on 3-ketosteroid 9␣-hydroxylase (KSH), which is essential for the growth of Rhodococcus strains on steroids (38). KSH acts on the B-ring of 3-keto-⌬4 steroids, e.g., 4-androstene-3,17-dione (AD), introducing a 9␣-hydroxyl moiety (Fig. 1). Subsequent ⌬1-dehydrogenation of 9␣-hydroxy-AD (9OHAD) by 3-ketosteroid ⌬1-dehydrogenase (⌬1-KSTD) initiates the opening of the B-ring through formation of a chemically unstable intermediate that spontaneously hydrolyzes, forming 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione (3-HSA). KSH activity has been observed in various actinobacterial genera, e.g., Mycobacterium (1,3,6), Nocardia (35), Arthrobacter (11), and Rhodococcus (38). In view of their amino acid sequences, KSH ...

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp

“…Finally, HsaA RHA1 and its more numerous orthologs among diverse mycolic acid bacteria comprise a distinct clade whose members are all encoded by genes in putative cholesterol catabolism gene clusters. The above pattern is also found in the phylogeny of KshA (22). We tested a number of strains, some containing the cholate catabolism gene cluster, for the ability to grow on cholate.…”

Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9 -Hydroxylase Enzymes

“…The chimeric kshA genes were cloned into expression vector pET15b (Novagen) and then subcloned into pET15b containing kshB DSM43269 , as described previously (21), to obtain constructs for coexpression of a kshA chimer with kshB. Details of plasmid vectors used are given in Table S1 in the supplemental material.…”

“…Each of these five kshA genes displayed a unique steroid induction pattern, coding for KshA enzymes with an identical reaction selectivity (they all introduce a hydroxyl moiety at the C-9 position of 3-ketosteroids), but interesting differences in their substrate preferences were observed (21,22). Empowered with multiple kshA genes, R. rhodochrous DSM43269 may deal in an effective manner with the various sterol/steroid substrates present in its natural habitat (soil), with C9␣-hydroxylation occurring at different levels during microbial steroid degradation (22).…”

“…9␣-Hydroxylated steroids are of particular interest for the synthesis of corticosteroids (14). KSH is a class IA monooxygenase, a two-component enzyme system comprised of the terminal oxygenase KshA, containing a Rieske Fe 2 S 2 cluster and a nonheme Fe 2ϩ located at the active site, and the reductase KshB, containing a plant-type Fe 2 S 2 cluster and the flavin cofactor flavin adenine dinucleotide (2,21,25). Interestingly, KshA and KshB both have been identified to be essential factors in the pathogenicity of Mycobacterium tuberculosis H37Rv (11).…”

From Soybean Phytosterols to Steroid Hormones

“…Kshs and KstDs have attracted much attention for their special roles in initiating cleavage of the steroid nucleus. One enzyme must be inactivated to block the decomposition of the steroid nucleus for the development of steroid-producing strains (van der Geize, et al, 2000(van der Geize, et al, , 2002aPetrusma, et al, 2009). As shown in Figures 2 and 4, the inactivation of Kshs, KstDs, or both can result in the production of C19 steroids such as AD, ADD, and 9 -OHAD.…”