Characterization of 3-ketosteroid 9{alpha}-hydroxylase, a Rieske oxygenase in the cholesterol degradation pathway of Mycobacterium tuberculosis

Abstract

KshAB (3-Ketosteroid 9alpha-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O(2). Nevertheless, the reactivity of KshAB with O(2) was low in the presence of 1,4-androstadiene-3,17-dione, with a k(cat)/K(m)(O(2)) of 2450 +/- 80 m(-1) s(-1). The crystallographic structure of KshA, determined to 2.3A(,) revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid-transforming enzyme of biotechnological and medical importance.

FIGURE 1.

Reactions catalyzed by KshAB in the cholesterol catabolic pathway. Cholesterol is transformed to AD via previous enzymatic steps. KshA catalyzes the 9α-hydroxylation of AD and ADD, yielding 9-OHAD and 9-OHADD, respectively. KstD catalyzes the desaturation of ring A of AD and 9-OHAD to form ADD and 9-OHADD, respectively; the physiological electron acceptor of KstD has yet to be identified. 9-OHADD undergoes a nonenzymatic ring cleavage and aromatization to form HSA.

FIGURE 2.

Steady-state kinetic analyses of KshAB-catalyzed transformations. Shown is the dependence of the initial velocity of O₂ consumption on ADD (A) and AD (B) concentrations in air-saturated buffer with fitted parameters K_m = 110 ± 20 μm and 24 ± 16 μm, and V_max = 0.32 ± 0.02 μm s^–1 and 0.032 ± 0.006 μm s^–1, respectively. C, dependence of the initial velocity of O₂ consumption on O₂ concentration in the presence of 380 μm ADD. The best fit of the Michaelis-Menten equation to the data using the least squares dynamic weighting options of LEONORA is represented as a solid line.

FIGURE 3.

Crystal structure of KshA from M. tuberculosis. A, ribbon representation of the KshA trimer viewed along the 3-fold symmetry axis. Monomers are colored in different shades of green. Iron ions and acid-labile sulfur atoms are shown as orange and yellow spheres, respectively. B, ribbon representation of the KshA monomer. The Rieske domain is labeled and represented in light brown, and the catalytic domain is labeled and shown in green. The location and position of the β-strand β19 and terminal helix α9 are indicated. C, mononuclear iron coordination sphere. The ribbon representation of KshA is shown as a semitransparent background. The residues ligating the iron are shown as stick representations. The mononuclear iron and solvent molecule S1 are shown as orange and red spheres, respectively. 2F_o – F_c electron density (blue mesh) is shown at 1.7 σ. Residual F_o – F_c density green mesh is shown at 3.5 σ. D, active site and adjacent Rieske center of KshA. The distance between atoms involved in proposed electron transfer is indicated. Residues from the adjacent molecule are indicated with an asterisk. Metal centers are shown as spheres, and side chains are labeled and shown as stick diagrams.

FIGURE 4.

Structural superposition of KshA (green) with catalytic domains of OMO-O₈₆ (beige, rmsd = 2.8 Å) (A) and NDO-O_9816-4 (magenta, rmsd = 3.0 Å) (B). Directionality of the active site channel of KshA is indicated with a solid arrow; that of OMO-O₈₆ and NDO-O_9816–4 is indicated with a dashed arrow. Selected secondary structural features of KshA are labeled. C termini are indicated. rmsd values were calculated for superimposed catalytic domains using the SSM algorithm as implemented in COOT.

FIGURE 5.

The secondary structure of KshA. α-Helices and β-strands are represented to scale by cylinders and arrows, respectively. β-Strands and α-helices are numbered. Elements not conserved in all structurally characterized ROs are shown in yellow. The positions of mononuclear iron ligands and Asp¹⁷⁸ are indicated with asterisks. Points of inserted elements found in previous structures are indicated as follows. Insertion 1, α-helix in α₃β₃ ROs or α-helix and 1–2 β-strands in α₃ ROs; Insertion 2, 1–2 α-helices in α₃β₃ROs or β-hairpin and β-strand α₃ ROs; Insertion 3, β-strand pairing with N terminus and 1–2 β-strands on central sheet in α₃β₃ ROs; Insertion 4, C-terminal α-helices in α₃ ROs.

FIGURE 6.

Stereo image of ADD docking simulation in the KshA active site. The model shown represents the best ranking solution (predicted free energy of binding =–7.04 kcal/mol) of the top rmsd cluster. The distance between the mononuclear iron and the C9 atom of ADD is 3.8 Å. The shown residues are conserved in KshAs and are labeled, except for Val¹⁷⁶ and Gln²⁰⁴. Protein carbons are shown in green, ADD carbons in yellow, oxygen atoms in red, nitrogen in blue, and sulfur in yellow. The mononuclear iron is represented as an orange sphere.