Steroid-1-dehydrogenase of Mycobacterium sp. VKM Ac-1817D strain producing 9α-hydroxy-androst-4-ene-3,17-dione from sitosterol

Abstract

The strain of Mycobacterium sp. VKM Ac-1817D forms 9alpha-hydroxy-androst-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol. The formation of 9-OH-AD was accompanied with its partial destruction due to residual steroid-1-dehydrogenase (St1DH) activity. The activity was found to be induced by androst-4-ene-3,17-dione (AD), while other intermediates of sitosterol oxidation did not influence 1(2)-dehydrogenation. The enzyme is located mainly in the cytosolic fraction. The cytosolic St1DH (dimer, M (r) approximately 58 kDa) was partially purified by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose, and gel filtration on Bio-Gel A-0.5M. It expressed the St1DH activity toward both AD and 9-OH-AD.

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

“…The microbial transformation of steroids has long prevailed in the pharmaceutical industry since the 1950s [2,3]. Degradation of steroids can yield much valuable steroidal derivatives, such as 4-androstene-3,17-dione (AD), androst-1,4-diene-3,17-dione (ADD), 9α-OH-AD, and 9α-OH-ADD [4,5,6,7,8]. ADD has been (FAD)-binding site was coincided with the sequence G-S-G-(A/G)-(A/ G)-(A/G)-X 17 -E [18,19].…”

Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates

“…1 a), has been found in several steroid-degrading bacteria, including A. simplex [ 22 ], Rhodococcus rhodochrous [ 23 ], Pseudomonas testosteroni [ 24 ], Nocardia coralline [ 25 ], and Mycobacterium sp. [ 26 ]. The enzyme KsdD is a flavoprotein dehydrogenating a wide variety of 3-ketosteroids.…”

Biochemical and structural characterization of 3‐ketosteroid‐Δ1‐dehydrogenase, a candidate enzyme for efficient bioconversion of steroids

“…3‐ketosteroid‐Δ 1 ‐dehydrogenase (KsdD, EC 1.3.99.4) was found in several steroid‐degrading bacteria, including bacteria from Arthrobacter , Mycobacterium , Rhodococcus , Comamonas and Nocardia . The KsdD enzyme is a flavoprotein with the ability to dehydrogenate a wide variety of 3‐ketosteroids, with a preference for substrates unsaturated at the C4‐C5 position, such as AD, 9α‐OH‐AD, or progesterone . The catalytic mechanism of dehydrogenation on C1‐C2 of 3‐ketosteroids involves the direct elimination of two hydrogen atoms on the respective C1 and C2 atoms of substrate without the formation of a hydroxylated intermediate or H 2 O .…”

“…a C1-C2 double bond is introduced into the ring A of 3-ketosteroid substrates by 3-ketosteroid-Δ 1 -dehydrogenase. 15, 16 3-ketosteroid-Δ 1 -dehydrogenase (KsdD, EC 1.3.99.4) was found in several steroid-degrading bacteria, including bacteria from Arthrobacter, 17 Mycobacterium, 18,19 Rhodococcus, 7,[20][21][22][23][24] Comamonas 25 and Nocardia. 26,27 The KsdD enzyme is a flavoprotein with the ability to dehydrogenate a wide variety of 3-ketosteroids, with a preference for substrates unsaturated at the C4-C5 position, such as AD, 9 -OH-AD, or progesterone.…”

“…26,27 The KsdD enzyme is a flavoprotein with the ability to dehydrogenate a wide variety of 3-ketosteroids, with a preference for substrates unsaturated at the C4-C5 position, such as AD, 9 -OH-AD, or progesterone. 19,27,28 The catalytic mechanism of dehydrogenation on C1-C2 of 3-ketosteroids involves the direct elimination of two hydrogen atoms on the respective C1 and C2 atoms of substrate without the formation of a hydroxylated intermediate or H 2 O. [27][28][29] An isotopic exchange experiment was performed to illustrate the catalytic process showing the preferential process of trans-diaxial elimination of the 1 , 2 H atoms from 3-ketosteroid substrates, and the author proposed a two-step mechanism: keto-enol tautomerization, and a proton is simultaneously removed from C2.…”