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...