Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid delta 1-dehydrogenase

Abstract

Pseudomonas testosteroni ATCC 17410 is able to grow on testosterone. I'his strain was mutagenized by Tn5, and 41 mutants defective in the utilization of testosterone were isolated. One of them, called mutant 06, expressed 3-oxosteroid A'-and 3-oxosteroid A4_5 a-dehydrogenases only at low levels. The DNA region around the TnS insertion in mutant 06 was cloned into pUC19, and the 1-kbp EcoRI-BamHI segment neighbor to the TnS insertion was used to probe DNA from the wild-type strain. The probe hybridized to a 7.8-kbp Sall fragment. Plasmid pTES5, which is a pUC19 derivative containing this 7.8-kbp Sail fragment, was isolated after the screening by the 1-kbp EcoRI-BamHI probe. This plasmid expressed A'-dehydrogenase in Eschenichia coli cells. The 2.2-kbp KpnI-KpnI segment of pTES5 was subcloned into pUC18, and pTEK21 was constructed. In E. coli containing the lacI plasmid pRG1 and pTEK21, the expression of Al-dehydrogenase was induced by isopropyl-I8-D-thiogalactopyranoside (IPTG). The induced level was about 40 times higher than the induced level in P. testosteroni. Al-Dehydrogenase synthesized in E. coli was localized in the inner membrane fraction.The minicell experiments showed that a 59-kDa polypeptide was synthesized from pTEK21, and this polypeptide was located in the inner membrane fraction. The complete nucleotide sequence of the 2.2-kbp KpnI-KpnI segment of pTEK21 was determined. An open reading frame which encodes a 62.4-kDa polypeptide and which is preceded by a Shine-Dalgarno-like sequence was identified. The first 44 amino acids of the putative product exhibited significant sequence similarity to the N-terminal sequences of lipoamide dehydrogenases.Steroids are growth substrates for a wide variety of microorganisms (28). In the steroid catabolic pathways of these organisms, the degradation of the compounds is usually initiated by the introduction of unsaturated bonds into ring A. In Pseudomonas testosteroni, which can grow on testosterone (25), A'-dehydrogenase (EC 1.3.99.4) and two A4-dehydrogenases (A4-5a-dehydrogenase and A4-53-dehydrogenase) which introduce double bonds into ring A at positions C1-C2 and C4-C5, respectively (10,26), are induced by steroids. Unlike A4-5p-dehydrogenase, which is a soluble protein, the A'-and A4-5a-enzymes are membrane bound (26). A'-Dehydrogenases from other steroid-degrading bacteria are also membrane bound (33,38,47). The analysis of A'-dehydrogenase in P. testosteroni has suggested that the major fraction of this enzyme is firmly attached to the inner membrane (44). Partial purification of A'-and A4-5a-dehydrogenases has revealed that these two activities are determined by different proteins (26). By analogy with isofunctional steroid dehydrogenase of Bacillus sphaericus (35), the reaction catalyzed by Al-dehydrogenase of P. testosteroni is thought to occur by the direct and irreversible trans-diaxial removal of the la,2, hydrogens.Very little genetic information is available on the steroid catabolic pathway in P. testosteroni (8, 25). The catabolic enzymes includ...

Meta-cleavage enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni TA441

“…1 ; 1-androstene-3,17-dione is not indicated in Fig. 1) (Florin et al, 1996 ;Plesiat et al, 1991). In addition to these dehydrogenases, the genes for 3α-hydroxysteroid hydrogenase and ∆& -$ ketosteroid isomerase from C. testosteroni have also been cloned and characterized (Abalain et al, 1995 ;Mo$ bus & Maser, 1998 ;Kuliopulos et al, 1987 ;Choi & Benisek, 1988).…”

Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid Δ1-dehydrogenase isoenzyme b bThe GenBank accession number for the sequence reported in this paper is AY078169

“…r et al, 1995 ;Morii et al, 1998 ;Plesiat et al, 1991 ;Sih & Bennet, 1962 ;Wagner et al, 1992 ;Wovcha et al, 1979). Functional studies of the steroid ∆"-dehydrogenation system in the micro-organism itself and analysis of the metabolic effects of their inactivation, however, are limited (van der Geize et al, 2000(van der Geize et al, , 2001, probably due to the paucity of genetic tools for actinomycetes able to perform the KSTD reaction (e.g.…”

Steroid degradation in Comamonas testosteroni

“…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.…”

“…∆1-Dehydrogenase (∆1-DH) gene was cloned from C. testosteroni strain ATCC17410 in 1991 [26], and ∆4-dehydrogenase (∆4-DH) gene was cloned from the same strain in 1996 [27]. 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.…”