Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases

Abstract

Background: KshA is a bacterial steroid-transforming oxygenase of biocatalytic interest and a virulence determinant in Mycobacterium tuberculosis. Results: Structures of KshA⅐steroid complexes were obtained for three homologs of different substrate specificity. Conclusion: Considerable structural flexibility contributes to the respective specificities of the different KshAs. Significance: This study provides insight into steroid catabolism and the conformational flexibility of Rieske oxygenases.

Molecular characterization of a new gene cluster for steroid degradation in Mycobacterium smegmatis

“…i.e. compounds that still retain 3 or more carbons of the sterol sidechain (Capyk et al, 2011;Penfield et al, 2014). In fact, by inhibiting the B-ring opening of cholesterol, it is possible to observe the accumulation in the culture media of C-22 and C-24 intermediates (Szentirmai, 1990;Wilbrink et al, 2011;Yeh et al, 2014;Gal an et al, 2017).…”

“…Thereafter, the catabolism of AD, ADD and 9OH‐AD has been postulated to proceed for aerobic bacteria through a common catabolic route called 9,10‐seco pathway involving two key activities Δ 1 ‐ketosteroid dehydrogenase (KSTD) and 3‐ketosteroid 9α‐hydroxylase (KSH) (van der Geize et al ., ; Knol et al ., ; Wei et al ., ; Fernández de las Heras et al ., ; Rohman et al ., ; Petrusma et al ., ; Yao et al ., ). KSH is a two component class IA monooxygenase comprised of the terminal oxygenase KshA and the reductase component KshB (Andor et al ., ; van der Geize et al ., ; Petrusma et al ., ; Capyk et al ., ; Petrusma et al ., ; Penfield et al ., ) This enzyme introduces a 9α‐hydroxyl group fundamental for steroid B‐ring opening and thus, for its further degradation (García et al ., ; Petrusma et al ., ). The expression of the genes encoding the enzymes involved in the side‐chain and the A‐B rings degradation as well as those encoding the sterol uptake system, is controlled by the KstR repressor, a TetR‐like transcriptional regulator (Kendall et al ., ; Uhía et al ., ), while the expression of the genes responsible for the catabolism of C‐D rings is regulated by a second TetR‐like transcriptional regulator, the KstR2 repressor (Kendall et al ., ; Casabon et al ., ; Crowe et al ., ; García‐Fernández et al ., ).…”

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

“…The 3ketosteroid 9a-hydroxylase (Ksh) has been proposed as the key enzyme for ring-B opening, and therefore, the removal of this activity should render ADD as end-product. The Ksh enzymes of Rhodococcus and Mycobacterium have been characterized as two-component monooxygenases, composed of an oxygenase (KshA) and a ferredoxin reductase (KshB) (van der Geize et al, 2002a;Capyk et al, 2009Capyk et al, , 2011Petrusma et al, 2009Petrusma et al, , 2012Petrusma et al, , 2014Hu et al, 2010;Bragin et al, 2013;Penfield et al, 2014). Therefore, theoretically, a deletion mutant of one of the Ksh encoding genes, i.e.…”

The competition between chemistry and biology in assembling iron–sulfur derivatives. Molecular structures and electrochemistry. Part III. {[Fe2S2](Cys)3(X)} (X=Asp, Arg, His) and {[Fe2S2](Cys)2(His)2} proteins

“…The X-ray structure of its [2Fe-2S] R centre (which has a total negative charge [105a]) is known [107], and the reduction potential of the one-electron reduction of such a Rieske centre (hereafter DMO) is reported in Table 13. As shown, the redox [112,113], but currently no redox data are available for such a Rieske centres.…”