Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-018-9239-3 MINI-REVIEW Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms Maciej Szaleniec1 & Agnieszka M. Wojtkiewicz1 & Rita Bernhard2 & Tomasz Borowski1 & Marina Donova3 Received: 25 May 2018 /Revised: 10 July 2018 /Accepted: 10 July 2018 # The Author(s) 2018 Abstract The steroid superfamily includes a wide range of compounds that are essential for living organisms of the animal and plant kingdoms. Structural modifications of steroids highly affect their biological activity. In this review, we focus on hydroxylation of steroids by bacterial hydroxylases, which take part in steroid catabolic pathways and play an important role in steroid degrada- tion. We compare three distinct classes of metalloenzymes responsible for aerobic or anaerobic hydroxylation of steroids, namely: cytochrome P450, Rieske-type monooxygenase 3-ketosteroid 9α-hydroxylase, and molybdenum-containing steroid C25 dehy- drogenases. We analyze the available literature data on reactivity, regioselectivity, and potential application of these enzymes in organic synthesis of hydroxysteroids. Moreover, we describe mechanistic hypotheses proposed for all three classes of enzymes along with experimental and theoretical evidences, which have provided grounds for their formulation. In case of the 3- ketosteroid 9α-hydroxylase, such a mechanistic hypothesis is formulated for the first time in the literature based on studies conducted for other Rieske monooxygenases. Finally, we provide comparative analysis of similarities and differences in the reaction mechanisms utilized by bacterial steroid hydroxylases. Keywords Steroid hydroxylation . Cytochrome P450 . 3-ketosteroid 9α-hydroxylase . Steroid C25 dehydrogenase Introduction D; and insect molting hormones. Multiple functions of ste- roids are essential for living organisms of the animal and plant Role of hydroxylations in aerobic and anaerobic kingdoms (Baker 2011). steroid metabolism Structural modifications of steroids highly affect their bio- logical activity. Hydroxylation results in increase of polarity Steroids (Greek, stereos = solids) represent a specific class of of the hydrophobic steroid molecules, affects their toxicity, terpenoid lipids that contain a gonane core of four fused translocation through the cell envelope, and greatly influences cycloalkane rings (A–D). The steroid superfamily includes their biological effects. Positions of the hydroxyl groups and various structures such as sterols (e.g., cholesterol, sitosterol, stereochemistry around carbons to which they are attached in ergosterol); bile acids; corticoids; cardiac aglycones; vitamin the cycloalkane rings, as well as in the side chain of steroids, are of importance. For example, the presence of hydroxyl function in position 11β is essential for anti-inflammatory activity (e.g., cortisol, prednisolone) (Fegan et al. 2008), the α * Maciej Szaleniec 16 -hydroxyl function is of importance for synthetic gluco- [email protected] corticoids such as triamcinolone and dexamethasone (Berrie et al. 1999), the 14β-hydroxyl group is typically found in 1 cardioactive steroids (Ali Shah et al. 2013), the 7- Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Kraków, Poland hydroxylated derivatives of dehydroepiandrosterone 2 (DHEA) and epiandrosterone (EpiA) have neuroprotective Lehrstuhl für Biochemie, Universität des Saarlandes, Campus B2 2, 66123 Saarbrücken, Germany effects (Milecka-Tronina et al. 2014; Wojtal et al. 2006), and 3 the 1α-and25α-hydroxyl functions are of significance for the G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, vitamin D3 (VD3) activity (Bikle 2014; Prosser and Jones Pushchino, Moscow Oblast 142290, Russia 2004). 25-hydroxycholesterol was shown to exhibit antiviral Appl Microbiol Biotechnol activity towards a broad spectrum of viruses by activating Galán et al. 2017 for a review). At least three steps of this interferons, immune cells, and increasing the production of reaction cascade are catalyzed by cytochromes P450s, such immune mediators (Blanc et al. 2013;Goldetal.2014;Liu as CYP125, CYP142, and CYP124 and they are an initial et al. 2013). reaction, oxyfunctionalization at the ω-position at C26 (27) Hydroxylation of steroids by diverse bacteria is considered to form the terminal alcohol and sequential oxidations to al- mainly as a prelude to their catabolism as carbon and energy dehyde and carboxylic acid (Frank et al. 2015;Johnstonetal. sources. Recent data confirmed that steroid-degrading bacteria 2010; Ouellet et al. 2011). Orthologs of these enzymes have are globally distributed and prevalent in wastewater treatment been identified in all sterol-transforming Actinobacteria, plants, soil, plant rhizospheres, and the marine environment whose genomes are available in the databases, e.g., (Holert et al. 2018). Two hundred sixty-five putative steroid Mycobacterium tuberculosis (Mtb) H37Rv; Mycobacterium degraders have been identified only within Actinobacteria and neoaurum NRRL 3805B; M. neoaurum VKM Ac-1815D, Proteobacteria, whose genomes are available in NCBI (NCBI 1817D; Mycobacterium smegmatis mc2 155; Rhodococcus Rf_Seq) (Bergstrand et al. 2016). Except for Actinobacteria jostii RHA1; Gordonia neofelifaecis NRRL B-59395; and and Proteobacteria, a more recent metagenomics investiga- Nocardioides simplex VKM Ac-2033D (Capyk et al. 2009b; tion suggested that steroid degraders may be present in other Garcia-Fernandez et al. 2013;McLeanetal.2009; Ouellet et bacterial groups; several alpha- and gammaproteobacterial lin- al. 2011; Rosloniec et al. 2009; Shtratnikova et al. 2016). It is eages not previously known to degrade steroids (Holert et al. of importance that these P450 enzymes selectively and 2018). regiospecifically oxidize the relatively unreactive terminal Bacterial hydroxylases, being a part of steroid catabolic methyl group, but not the more reactive tertiary carbon pathways, play an important role in steroid degradation. (C25) of the sidechain (Ouellet et al. 2011). They participate in both cholesterol and cholate degradation Aerobic hydroxylation of the C25-tertiary carbon atom was pathways by initiating the degradation of the aliphatic side reported for Pseudonocardia autotrophica, Streptomyces chain or the opening of the sterane ring. Depending on the griseolus,andSebekia benihana. The reaction results in 25- oxic or anoxic environment, different catalytic strategies are hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 (Ban et realized (Fig. 1). The aerobic bacterial degradation of the side al. 2014; Sawada et al. 2004). The VD3-specific hydroxy- chain of cholesterol and other sterols generally occurs through lases, Vdh in P. autotrophica, CYP105A1 in S. griseolus, the mechanism which is similar to the β-oxidation of fatty and CYP-sb3a, a member of the bacterial CYP107 family in acids and proceeds via CoA thioester intermediates (see e.g., S. benihana, were shown to perform double hydroxylations at Fig. 1 Hydroxylation of steroids by bacterial enzymes. a Reaction al. 2016), CYP154C5 N. farcinica IFM 10152 (Bracco et al. 2013), schemes for CYP, KSH, and S25DH. b Regioselectivity of CYP260A1, CYP260B1 from Sorangium cellulosum So ce56 hydroxylations reported for aerobic cytochrome P450 family (Litzenburger and Bernhardt 2016) CYP125, CYP142, CYP124 from represented by: CYP106A1 Bacillus megaterium DSM319, CYP106A2 M. tuberculosis CDC1551 or H37Rv (Johnston et al. 2010), non-heme B. megaterium ATCC13368 (Kiss et al. 2015c), CYP109B1 B. subtilis KSH from Actinobacteria (Petrusma et al. 2014), and anaerobic S25DH 168 (Agematu et al. 2006), CYP109E1 B. megaterium DSM19 (Jozwik et from Sterolibacterium denitricans (Dermer and Fuchs 2012) Appl Microbiol Biotechnol both positions 25 and 1α. These reactions are of great impor- 2017; Hu et al. 2010; Penfield et al. 2014; Petrusma et al. tance for the production of physiologically active forms of 2011; Petrusma et al. 2012; Petrusma et al. 2014). Along with VD3. Hydroxylation at the C25-tertiary atom of the diosgenin 3-ketosteroid-1(2)-dehydrogenases (KstD), KSH is responsi- F-ring to form the spirosteroid isonuatigenone has been re- ble for the cleavage of the 9(10)-C-C bond to form 9(10)- ported for the soil-dwelling actinomycete strain secosteroids with an aromatized A-ring. Deletion mutations Streptomyces virginiae IBL-14 (Wang et al. 2007; Wang et of KstD or KshA/KshB resulted in the production of valuable al. 2009). androstane steroids such as 9α-hydroxyandrostenedione (9- Under anaerobic conditions, the hydroxylation of steroids OH-AD), or ADD, respectively, as major products from phy- seems to be less frequent. Ten years ago, a new class of tosterol (Galan et al. 2017;Garcia-Fernandezetal.2017;Yao molybdopterin containing steroid hydroxylases has been dis- et al. 2014). Deletion of both, KstD and KSH, enables pro- covered in a denitrifying β-proteobacterium Sterolibacterium duction of AD (Galan et al. 2017). These approaches are denitrificans Chol-1S that is capable of anaerobic mineraliza- widely exploited for the generation of industrial whole cell tion of cholesterol (Chiang et al. 2008b). After initial oxida- biocatalysts for the pharmaceutical industry, since AD, ADD tion of cholesterol to cholest-4-en-3-one by the and 9-OH-AD are the key starting molecules for multiple dehydrogenase/isomerase anaerobic cholesterol