Characterization of a Second Rhodococcus erythropolis SQ1 3-Ketosteroid 9α-Hydroxylase Activity Comprising a Terminal Oxygenase Homologue, KshA2, Active with Oxygenase-Reductase Component KshB

Abstract

Previously we have characterized 3-ketosteroid 9␣-hydroxylase (KSH), a key enzyme in microbial steroid degradation in Rhodococcus erythropolis strain SQ1, as a two-component iron-sulfur monooxygenase, comprised of the terminal oxygenase component KshA1 and the oxygenase-reductase component KshB. Deletion of the kshA1 gene resulted in the loss of the ability of mutant strain RG2 to grow on the steroid substrate 4-androstene-3,17-dione (AD). Here we report characteristics of a close KshA1 homologue, KshA2 of strain SQ1, sharing 60% identity at the amino acid level. Expression of the kshA2 gene in mutant strain RG2 restored growth on AD and ADD, indicating that kshA2 also encodes KSH activity. The functional complementation was shown to be dependent on the presence of kshB. Transcriptional analysis showed that expression of kshA2 is induced in parent strain R. erythropolis SQ1 in the presence of AD. However, promoter activity studies, using ␤-lactamase of Escherichia coli as a convenient transcription reporter protein for Rhodococcus, revealed that the kshA2 promoter in fact is highly induced in the presence of 9␣-hydroxy-4-androstene-3,17-dione (9OHAD) or a metabolite thereof. Inactivation of kshA2 in parent strain SQ1 by unmarked gene deletion did not affect growth on 9OHAD, cholesterol, or cholic acid. We speculate that KshA2 plays a role in preventing accumulation of toxic intracellular concentrations of ADD during steroid catabolism. A third kshA homologue was additionally identified in a kshA1 kshA2 double gene deletion mutant strain of R. erythropolis SQ1. The developed degenerate PCR primers for kshA may be useful for isolation of kshA homologues from other (actino) bacteria.Rhodococcus species display a wide range of metabolic capabilities, degrading a variety of environmental pollutants and transforming or synthesizing compounds with possible useful applications (3,26,30). A well-known feature of rhodococci is their ability to degrade a range of naturally occurring steroids, including cholesterol and phytosterols, e.g., ␤-sitosterol (26).3-Ketosteroid 9␣-hydroxylase (KSH) is a key enzymatic step in the microbial steroid catabolic pathway, acting on 4-androstene-3,17-dione (AD) or 1,4-androstadiene-3,17-dione (ADD) (Fig. 1). Introduction of the 9␣-hydroxyl moiety into the steroid polyheterocyclic ring structure, combined with steroid ⌬ 1 -dehydrogenation by 3-ketosteroid ⌬ 1 -dehydrogenase (KSTD), initiates steroid B-ring opening due to the formation of chemically unstable 9␣-hydroxy-1,4-androstadiene-3,17-dione (9OHADD) (Fig. 1). KSH of Rhodococcus erythropolis SQ1 is a two-component class IA monooxygenase comprised of the terminal oxygenase KshA1 and the oxygenase reductase component KshB (23). Classification of nonheme oxygenases is based on the number of constituent components and the nature of their redox centers (2,6,(8)(9)(10)13) In previous work we have shown that gene inactivation of either kshA1 (mutant strain RG2) or kshB (mutant strain RG4) in parent strain R. erythropolis SQ1 results in loss o...

Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis

“…The ability of KshAB to transform both ADD and AD is consistent with studies based on gene deletion and recombinant strains that have indicated that both compounds are physiological substrates of KshAB from R. erythropolis SQ1 (6,57,58). In contrast to what was proposed in R. erythropolis SQ1 based on the moderate toxicity of ADD (58), the clear preference of KshAB from M. tuberculosis for ADD suggests that, at least in the pathogen, KstD precedes KshAB in the steroid catabolic pathway.…”

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

“…Nevertheless, using the sequence of the annotated kshA and kshB genes from Rhodococcus (van der Geize et al ., 2002a, 2008) and M. tuberculosis (Capyk et al ., 2009) as probes, we have localized the corresponding orthologues in M. smegmatis (Table 1). This analysis revealed that there are at least two genes encoding putative oxygenase components, named kshA1 and kshA2 , and two genes encoding putative reductase components, named kshB1 and kshB2 .…”

“…the best characterized cholesterol‐degrading organism) to produce AD, ADD and 9α‐hydroxy‐4‐androstene‐3,17‐dione (9OH‐AD) from natural sterols (e.g. cholesterol or phytosterols), but these mutants have not been used at industrial scale yet (van der Geize et al ., 2000, 2001a,b, 2002a,b, 2008; Wilbrink et al ., 2011; Yeh et al ., 2014). …”

“…(2002a, 2008) have demonstrated that Ksh mutants of R. erythropolis SQ1 deleted in kshA or kshB genes can produce ADD from AD, but surprisingly they do not accumulate AD, ADD or other metabolites during sterol conversion. Interestingly, the degradation of phytosterols was not impaired in a kshA ‐ mutant and rates of degradation were comparable to those of the parent strain, suggesting that Rhodococcus has alternative enzymes to catabolize phytosterols.…”

Multiplicity of 3-Ketosteroid-9α-Hydroxylase Enzymes in Rhodococcus rhodochrous DSM43269 for Specific Degradation of Different Classes of Steroids

“…The kshA and kshB genes, encoding KshA and KshB, respectively, were first identified in Rhodococcus erythropolis SQ1, a strain possessing at least 3 kshA homologues (31,34). A crystal structure of KshA of M. tuberculosis H37Rv was recently elucidated and revealed that KshA of H37Rv likely functions as a trimer (␣3) (4).…”