Steroid degradation by Comamonas testosteroni and Nocardia restrictus have been intensively studied for the purpose of obtaining materials for steroid drug synthesis. C. testosteroni degrades side chains and converts single/double bonds of certain steroid compounds to produce androsta-1,4-diene 3,17-dione or the derivative. Following 9-hydroxylation leads to aromatization of the A-ring accompanied by cleavage of the B-ring, and aromatized A-ring is hydroxylated at C-4 position, cleaved at ∆4 by meta-cleavage, and divided into 2-hydroxyhexa-2,4-dienoic acid (A-ring) and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (B,C,D-ring) by hydrolysis.Reactions and the genes involved in the cleavage and the following degradation of the A-ring are similar to those for bacterial biphenyl degradation, and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid degradation is suggested to be mainly -oxidation. Genes involved in A-ring aromatization and degradation form a gene cluster, and the genes involved in -oxidation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid also comprise a large cluster of more than 10 genes. The DNA region between these two main steroid degradation gene clusters contain 3-hydroxysteroid dehydrogenase gene, ∆5,3-ketosteroid isomerase gene, genes for inversion of an -oriented-hydroxyl group to a -oriented-hydroxyl group at C-12 position of cholic acid, and genes possibly involved in the degradation of a side chain at C-17 position of cholic acid, indicating this DNA region of more than 100kb to be a steroid degradation gene hot spot of C. testosteroni.

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...

Comamonas testosteroni metabolizes testosterone as the sole carbon source via a meta-cleavage reaction. A meta-cleavage enzyme gene, tesB, was cloned from C. testosteroni TA441. The deduced N-terminal amino acid sequence of tesB matched that of the purified meta-cleavage enzyme which is induced in TA441 during growth on testosterone as the sole carbon source. The tesBdisrupted mutant did not show growth on testosterone, suggesting that tesB is necessary for TA441 to grow on testosterone. Downstream from tesB, three putative ORFs which encode products also necessary for growth of TA441 on testosterone were identified. The usual substrate of TesB is probably 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione. Although this compound was not identified in the gene disrupted mutants, accumulation of upstream metabolites of testosterone degradation, 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione, was shown by TLC analysis.

No 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...

No abstract

Microbial dimethyl sulfide (DMS) conversion is thought to be involved in the global sulfur cycle. We isolated Pseudomonas putida strain DS1 from soil as a bacterium utilizing DMS as a sole sulfur source, and tried to elucidate the DMS conversion mechanism of strain DS1 at biochemical and genetic level. Strain DS1 oxidized DMS to dimethyl sulfone (DMSO(2)) via dimethyl sulfoxide, whereas the oxidation was repressed in the presence of sulfate, suggesting that a sulfate starvation response is involved in DMS utilization by strain DS1. Two of the five DMS-utilization-defective mutants isolated by transposon 5 (Tn 5) mutagenesis had a Tn 5 insertion in the ssuEADCBF operon, which has been reported to encode a two-component monooxygenase system (SsuED), an ABC-type transporter (SsuABC), and a small protein (SsuF), and also to play a key role in utilization of sulfonates and sulfate esters in another bacterium, P. putida strain S-313. Disruption of ssuD and SsuD enzymatic activity demonstrated that methanesulfonate is a metabolic intermediate of DMS and desulfonated by SsuD. Disruption of ssuC or ssuF also led to a DMS-utilization-defective phenotype. Another two mutants had a defect in a gene homologous to pa2354 from P. aeruginosa PAO1, which encodes a putative transcriptional regulator, while the remaining mutant had a defect in cysM encoding O-acetylserine (thiol)-lyase B.

Comamonas testosteroni TA441 degrades steroids such as testosterone via aromatization of the A ring, followed by meta-cleavage of the ring. In the DNA region upstream of the meta-cleavage enzyme gene tesB, two genes required during cholic acid degradation for the inversion of an ␣-oriented hydroxyl group on C-12 were identified. A dehydrogenase, SteA, converts 7␣,12␣-dihydroxyandrosta-1,4-diene-3,17-dione to 7␣-hydroxyandrosta-1,4-diene-3,12,17-trione, and a hydrogenase, SteB, converts the latter to 7␣,12␤-dihydroxyandrosta-1,4-diene-3,17-dione. Both enzymes are members of the short-chain dehydrogenase/reductase superfamily. The transformation of 7␣,12␣-dihydroxyandrosta-1,4-diene-3,17-dione to 7␣,12␤-dihydroxyandrosta-1,4-diene-3,17-dione is carried out far more effectively when both SteA and SteB are involved together. These two enzymes are encoded by two adjacent genes and are presumed to be expressed together. Inversion of the hydroxyl group at C-12 is indispensable for the subsequent effective B-ring cleavage of the androstane compound. In addition to the compounds already mentioned, 12␣-hydroxyandrosta-1,4,6-triene-3,17-dione and 12␤-hydroxyandrosta-1,4,6-triene-3,17-dione were identified as minor intermediate compounds in cholic acid degradation by C. testosteroni TA441. Rhodococcus equi and Comamonas testosteroni (formerlyNocardia restrictus and Pseudomonas testosteroni) are known for the ability to utilize testosterone and various other steroids, such as the cholic acid analogs. In the 1950s and 1960s, the mechanism by which testosterone is degraded in these bacteria was extensively studied, and the main intermediate compounds in the degradation pathway were determined (2-6, 16-19). In our previous work, we simultaneously identified the genes and the intermediate compounds that are accumulated by gene disruption mutants and revealed the testosterone degradation pathway and degradation genes of C. testosteroni TA441 (7-13). Two steroid degradation gene clusters were identified in TA441; one contains the meta-cleavage enzyme gene tesB, 16 ORFs, and the positive regulator of the steroid degradation gene tesR, and the other consists of ORF18, ORF17, and tesIHA2A1DEFG. TA441 degrades testosterone via aromatization of the A ring, followed by cleavage of the ring by enzymes encoded by these genes. Most of the enzymes involved in the transformation of testosterone to 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid are encoded by the ORF18-tesG gene cluster. ORF18 has been reported to encode a CoA transferase which adds CoA to 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, and most of the putative ORFs in the region downstream of tesB have been suggested to be involved in 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid degradation, which is thought to be similar to the ␤-oxidation pathway of fatty acids. However, the region upstream of tesB was not clear. In this report, we describe newly isolated genes essential for the degradation of 12␣-hydroxylated ...