PMC

Compounds investigated as KshAB substrates. Shown are 4-AD (A), ADD (B), 4-BNC (C), 1,4-BNC (D), and 1,4-BNC-CoA (E). Not shown is 4-BNC-CoA, which differs from E by a 1–2 saturated bond on ring A.

Docking of 1,4-BNC-CoA in active site of KshA. 1,4-BNC-CoA is shown as a ball and stick representation. KshA (PDB ID 2ZYL ()) is shown as a green, semitransparent surface. KshA amino acids within 4 Å of the CoA group of 1,4-BNC-CoA are shown as sticks and labeled. Carbon atoms of amino acid residues and the substrate are colored green and yellow, respectively. Oxygen, phosphorus, and nitrogen atoms are shown in red, orange, and blue, respectively. The catalytic iron is shown as a rust-colored sphere. Residues mentioned in the text are labeled.

Steady-state kinetic analyses of KshAB with CoA thioesters. Shown is the dependence of the initial velocity of O₂ consumption on 1,4-BNC-CoA (A) and 4-BNC-CoA (B) concentrations in air-saturated buffer with fitted parameters K_m = 17 ± 3 and 6.8 ± 0.1 μm, and V_max = 0.56 ± 0.04 and 0.146 ± 0.007 μm s⁻¹, respectively. 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.

Schematic of the proposed cholesterol degradation pathway of Mtb. Key carbons of cholesterol are numbered using the steroid numbering convention. The processes of cholesterol side chain and partial ring degradation are shown as parallel pathways. Enzymes known to catalyze specific reactions in Mtb are indicated. Non-enzymatic reaction is shown as a dashed arrow. Compounds 1, 2, and 3 represent 4-AD, ADD, and 3-hydroxy-9,10-seconandrost-1,3,5(10)-trien-9,17-dione (3-HSA) (R = ketone); 4-BNC, 1,4-BNC, and 3-HSBNC, (R = isopropionate); 4-BNC-CoA, 1,4-BNC-CoA, and 3-HSBNC-CoA, (R = isopropionyl-CoA).

Transformation of 4-BNC to 4-BNC-CoA by CasI. HPLC chromatograms are shown of 50 μl of a CasI reaction (2 μm, dashed line) and no-enzyme control (solid line) containing 0.9 mm 4-BNC, 1.1 mm CoASH, 5 mm MgCl₂, and 2.5 mm ATP after 7 h. Peaks were resolved using a linear gradient of 0 to 90% methanol in 0.1 m ammonium acetate, pH 4.5, over 20 min. Substrate peaks are numbered: 1, ATP; 3, CoASH; 5, 4-BNC. Product peaks are numbered: 2, AMP; 4, 4-BNC-CoA.