Gene Encoding the Hydrolase for the Product of the meta -Cleavage Reaction in Testosterone Degradation by Comamonas testosteroni

Abstract

In a previous study we isolated the meta-cleavage enzyme gene, tesB, that encodes an enzyme that carries out a meta-cleavage reaction in the breakdown of testosterone by Comamonas testeroni TA441 (M. Horinouchi et al., Microbiology 147:3367-3375, 2001). Here we report the isolation of a gene, tesD, that encodes a hydrolase which acts on the product of the meta-cleavage reaction. We isolated tesD by using a Tn5 mutant of TA441 that showed limited growth on testosterone. TesD exhibited ca. 40% identity in amino acid sequence with BphDs, known hydrolases of biphenyl degradation in Pseudomonas spp. The TesD-disrupted mutant showed limited growth on testosterone, and the culture shows an intense yellow color. High-pressure liquid chromatography analysis of the culture of TesD-disrupted mutant incubated with testosterone detected five major intermediate compounds, one of which, showing yellow color under neutral conditions, was considered to be the product of the meta-cleavage reaction. The methylation product was analyzed and identified as methyl-4,5-9,10-diseco-3-methoxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oate, indicating that the substrate of TesD in testosterone degradation is 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid. 4,5-9,10-Diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid was transformed by Escherichia coli-expressed TesD. Downstream of tesD, we identified tesE, F, and G, which encode for enzymes that degrade one of the products of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid converted by TesD.Via a complex metabolic pathway involving many steroidinducible enzymes, 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. The mechanism by which testosterone is degraded in Nocardia restrictus and C. testosteroni was proposed more than 30 years ago ( Fig. 1) (4,7,16). The process is initiated by dehydrogenation of the 17␤-hydroxyl group on testosterone to 4-androstene-3,17-dione (4AD) (reaction 1 in Fig. 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) (16). The C-4 of 3-HSA is hydroxylated to yield 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,4-DHSA) (reaction 4), which is cleaved between C-4 and C-5 via a meta-cleavage reaction (reaction 5) (7). The product of the meta-cleavage reaction of 3,4-DHSA, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid (4,9-DSHA), is dark yellow in color and exists as a mixture of keto-enol forms in the culture (7). According to the speculation of Gibson et al. (7), 4,9-DSHA would be degraded in two ways: degraded into two compounds by hydrolysis (reaction 6) or hydroxylated at the 1-position before hydrolysis (reaction 7). 4,9-DSHA could not be isolated successfully from the r...

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.…”

“…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.…”

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

“…The enzyme can dehydrogenate a wide variety of 3-ketosteroids, but not 3-hydroxysteroids, and has a preference for substrates unsaturated at the C4-C5 position, such as 4-androstene-3,17-dione (4-AD), 9␣-hydroxy-4-androstene-3,17-dione, or 4-pregnene-17␣,21-diol-3,11,20-trione (cortisone) (1,2). ⌬ 1 -KSTDs are found in many steroid-degrading bacteria, particularly in those belonging to the genera Arthrobacter, Comamonas, Mycobacterium, and Rhodococcus (formerly Nocardia) (3,4). They are, together with 3-ketosteroid 9␣-hydroxylase, essential for the opening of the steroid B-ring of the key intermediate 4-AD ( Fig.…”

“…4) 3 is a FADdependent enzyme that catalyzes the introduction of a double bond into the C1-C2 position of the 3-ketosteroid A-ring (Fig. 1).…”

Testosterone-inducible Regulator Is a Kinase That Drives Steroid Sensing and Metabolism in Comamonas testosteroni

“…However, the organization and regulation of their corresponding genes is largely unknown, although two steroid degradation gene clusters in C. testosteroni TA441 were identified (12,14,15,26).…”