Pseudomonas 3β‐hydroxysteroid dehydrogenase

Abstract

The 3B-hydroxysteroid dehydrogenase of Pseudomonas testosteroni commercially available was purified by an FPLC step and submitted to sequence determination by peptide analysis. The structure obtained reveals a 253-residue polypeptide chain, with an N-terminal, free a-amino group, and a low cysteine content. Comparisons with other hydroxysteroid dehydrogenases recently characterized reveal distant similarities with prokaryotic and, to some extent, also eukaryotic forms of separate specificities. Residue identities with a Streptomyces 20g-hydroxysteroid dehydrogenase are 35% and distributed over the entire molecule, whereas residue identities with the mammalian 17P-hydroxysteroid dehydrogenase only constitute 20%, and are essentially limited to the Nterminal and central parts, Nevertheless, all these enzymes exhibit a conserved tyrosine residue (position 151 in the present enzyme) noted as possibly having a functional role in some members of this protein family. Combined, the results establish the prokaryotic 3B-hydroxysteroid dehydrogenase as belonging to the family of shortchain alcohol dehydrogenases, reveal that the hydroxysteroid dehydrogenases are no more closely related than dehydrogenases with other enzyme activities within the family (e. g. glucose, ribitol, hydroxyprostaglandin dehydrogenases), show several of the mammalian hydroxysteroid dehydrogenases to have subunits of longer size with different patterns of similarity than those of the prokaryotic family members characterized, and define important segments of the coenzyme-binding region for this enzyme group.

Engineering Mycobacterium smegmatis for testosterone production

“…As the genes encoding 17β‐HSD enzymes from mycobacterial species have not been identified and these proteins have been only partially purified and characterized (Goren et al ., 1983; Egorova et al ., 2002a, 2005), we initially selected as enzyme candidates for metabolic engineering the well‐described 17β‐HSDs from the bacterium C. testosteroni (Schultz et al ., 1977; Lefebvre et al .,1979; Minard et al ., 1985 ; Genti‐Raimondi et al ., 1991; Yin et al ., 1991; Abalain et al ., 1993; Benach et al ., 1996, 2002; Oppermann et al ., 1997; Cabrera et al ., 2000) and the fungus C. lunatus (Plemenitas et al ., 1988; Rižner et al ., 1996, 1999, 2000, 2001a,b; Rižner and Zakelj‐Mavric, 2000; Zorko et al ., 2000; Kristan et al ., 2003, 2005, 2007a,b; Cassetta et al ., 2005; Ulrih and Lanisnik Rižner, 2006; Brunskole et al ., 2009; Svegelj et al ., 2012), because both enzymes present some relevant differences. Although they catalyse a reversible reaction and display similar reaction mechanisms, the reaction equilibrium of the fungal 17β‐HSD is shifted towards reduction, whereas the bacterial enzyme is shifted towards oxidation, as this enzyme is mainly involved into the TS catabolism in C. testosteroni (Genti‐Raimondi et al., 1990; Cabrera et al ., 2000).…”

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

“…They are 3p-hydroxysteroid dehydrogenase from P. testosteroni (50), estradiol 173-dehydrogenase from humans (32), 20f-hydroxysteroid dehydrogenase from Streptomyces hydrogenans (27), and corticosteroid 11,-dehydrogenase from rats (1). These enzymes are soluble proteins and members of a short-chain alcohol dehydrogenase family.…”

Steroid degradation in Comamonas testosteroni

“…The enzyme was initially isolated as 3-hsd and the cloned enzyme was revealed to act on both 3-hydroxyl group and 17-hydroxyl group of steroid compounds ( Figure 2.) [22,[30][31][32]. In 2002, crystallographic analysis at 1.2 A resolution revealed the enzyme to have nearly identical subunits that form a tetramer with the active site containing a Ser-Tyr-Lys triad, typical for short-chain dehydrogenases/reductases (SDR) [33].…”