A New Bacterial Steroid Degradation Gene Cluster in Comamonas testosteroni TA441 Which Consists of Aromatic-Compound Degradation Genes for Seco-Steroids and 3-Ketosteroid Dehydrogenase Genes

Abstract

In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-⌬ 1 -dehydrogenase and 3-ketosteroid-⌬ 4 (5␣)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9␣-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.Comamonas testosteroni is able to utilize certain C 19 and C 21 steroids, as well as a number of aromatic compounds, as sole carbon and energy sources via a complex metabolic pathway involving many steroid-inducible enzymes. The mechanism by which testosterone is degraded in Nocardia restrictus and C. testosteroni was proposed in the 1960s (4,8,14). However, the genes involved in this degradation pathway in C. testosteroni have yet to be identified, except for the initial 17␤-dehydrogenation (1, 3, 7) and the following ⌬ 1 -dehydrogenation (13). In previous studies, we identified the testosterone degradation genes tesB and tesDEFG among some open reading frames (ORFs) which are involved in testosterone degradation in C. testosteroni TA441 (9, 10). tesB encodes the meta-cleavage enzyme for 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,4-DHSA) (10), and tesD encodes the hydrolase for 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid (4,9-DSHA) (9). TesEFG are probably the enzymes for degradation of 2-oxohex-4-enoic acid, which is one of the two products of the hydrolysis of 4,9-DSHA by TesD.Studies on these genes have shown the probable testosterone degradation pathway of C. testosteroni TA441 to be that presented in Fig. 1. The process is initiated by dehydrogenation of the 17␤-hydroxyl group on testosterone to 4-androstene-3,17-dione (4-AD) (Fig. 1, reaction 1), which then undergoes ⌬ 1 -dehydrogenation to 1,4-androstadiene-3,17-dione (ADD) (reaction 2), followed by 9␣-hydroxylation to produce 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3-HSA) (reaction 3 and the following spontaneous cleavage). The C-4 of 3-HSA is hydroxylated to yield 3,4-DHSA (Fig. 1, reaction 4), which is cleaved between C-4 and C-5 via a meta-cleavage re...

Steroid degradation in Comamonas testosteroni

“…[1,2,[4][5][6][7][8][9][10][11][12]. The degradation pathway was confirmed with detail using gene-disrupted mutants in early 2000s, with further studies revealing that A-ring is degraded with the pathway similar to a common bacterial aromatic compound degradation pathway and B,C,D-rings is degraded mainly by -oxidation [13][14][15][16][17][18]. Investigation of the detail of degradation pathway of B,C,D-ring and other side chains of steroid compounds are still in progress.…”

“…Similar ∆1-DH (TesH) and ∆4-DH (TesI) genes were identified in strain TA441 with genes involved in A-ring degradation to reveal that ∆1-DH and ∆4-DH are adjacent each other and are contained in the same steroid degradation gene cluster, ORF18, ORF17, and tesIHA2A1DEFG. Most of the genes in this cluster are involved in aromatization and following degradation of the A-ring in steroid degradation [14] (Figure 11). testosteroni [17,[67][68][69].…”

“…Investigation of steroid degradation genes of in C. testosteroni launched fully in 1990s when 3-dehydrogenase gene, 3-dehydrogenase gene, and ∆5,3-ketosteroid isomerase gene were isolated consistently, then degradation genes for basic steroidal structure were revealed with detailed degradation pathway in the next decade (Figure 1., table 1) [13][14][15][16][17][18][22][23][24][25][26][27][28]. The aromatized A-ring is cleaved and completely degraded by meta-cleavage pathway, a common bacterial degradation pathway for aromatic compounds, and the genes involved in the A-ring degradation show significant homology to the corresponding genes in bacterial aromatic compound degradation.…”

Steroid degradation and two steroid-inducible enzymes in the marine bacterium H5

“…These two genes code for 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase, respectively. 3-Ketosteroid-delta-1-dehydrogenase is an enzyme that is involved in the breakdown of the steroid nucleus and is also one of the first enzymes to initiate steroid degradation [26]. In mammals, steroids like estrogens are mainly excreted in their conjugated form [27].…”

Purification, crystallization and preliminary X-ray crystallographic analysis of 3-ketosteroid Δ1-dehydrogenase fromRhodococcus erythropolisSQ1

“…Through their biotransformation, a large variety of physiologically active steroid intermediates are produced (Horinouchi et al, 2003;Sedlaczek, 1988). These intermediates and their derivatives are utilized extensively as drugs and hormones because of their anti-inflammatory, diuretic, anabolic, contraceptive, anti-androgenic, progestational and anticancer properties (Donova, 2007;Mahato & Garai, 1997).…”