Abstract
An aldolase from the bile acid-degrading actinobacterium Thermomonospora curvata catalyzes the C-C bond cleavage of an isopropyl-CoA side chain from the D-ring of the steroid metabolite 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). Like its homolog from Mycobacterium tuberculosis, the T. curvata aldolase is a protein complex of Ltp2 with a DUF35 domain derived from the C-terminal domain of a hydratase (ChsH2DUF35) that catalyzes the preceding step in the pathway. We determined the structure of the Ltp2-ChsH2DUF35 complex at 1.7 Å resolution using zinc-single anomalous diffraction. The enzyme adopts an αββα organization, with the two Ltp2 protomers forming a central dimer, and the two ChsH2DUF35 protomers being at the periphery. Docking experiments suggested that Ltp2 forms a tight complex with the hydratase but that each enzyme retains an independent CoA-binding site. Ltp2 adopted a fold similar to those in thiolases; however, instead of forming a deep tunnel, the Ltp2 active site formed an elongated cleft large enough to accommodate 17-HOPC-CoA. The active site lacked the two cysteines that served as the nucleophile and general base in thiolases and replaced a pair of oxyanion-hole histidine residues with Tyr-246 and Tyr-344. Phenylalanine replacement of either of these residues decreased aldolase catalytic activity at least 400-fold. On the basis of a 17-HOPC-CoA -docked model, we propose a catalytic mechanism where Tyr-294 acts as the general base abstracting a proton from the D-ring hydroxyl of 17-HOPC-CoA and Tyr-344 as the general acid that protonates the propionyl-CoA anion following C-C bond cleavage.
Highlights
An aldolase from the bile acid-degrading actinobacterium Thermomonospora curvata catalyzes the C–C bond cleavage of an isopropyl-CoA side chain from the D-ring of the steroid metabolite 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA)
In the last cycle of -oxidation of the side chains of cholesterol or bile acids, the hydroxyl substituent on the tertiary D-ring carbon cannot be oxidized to form a 3-ketoacyl-CoA; C–C bond cleavage cannot be performed by a thiolase
It has been determined recently that in M. tuberculosis a protein named Ltp2 is responsible for catalyzing a retro-aldol cleavage of the isopropyl side chain from the hydroxyl-substituted cholesterol metabolite, 17-HOPC-CoA5 (Fig. 1) [8]
Summary
A cholesterol-degradation pathway has been found to be important for the persistence of Mycobacterium tuberculosis in host macrophages. In a typical -oxidation cycle, a thiolase is responsible for a C–C bond cleavage, releasing a molecule of acetyl-CoA or propionyl-CoA from a 3-ketoacyl-CoA intermediate, resulting in the shortening of the side chain by 2–3 carbon atoms. In the last cycle of -oxidation of the side chains of cholesterol or bile acids, the hydroxyl substituent on the tertiary D-ring carbon cannot be oxidized to form a 3-ketoacyl-CoA; C–C bond cleavage cannot be performed by a thiolase. It has been determined recently that in M. tuberculosis a protein named Ltp is responsible for catalyzing a retro-aldol cleavage of the isopropyl side chain from the hydroxyl-substituted cholesterol metabolite, 17-HOPC-CoA5 (Fig. 1) [8].
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