The steroid side-chain-cleaving aldolase Ltp2-ChsH2DUF35 is a thiolase superfamily member with a radically repurposed active site

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 (ChsH2_DUF35) that catalyzes the preceding step in the pathway. We determined the structure of the Ltp2-ChsH2_DUF35 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 ChsH2_DUF35 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.

Keywords: C–C bond cleavage; actinobacteria; aldolase; bile acid; biodegradation; cholesterol; protein evolution; steroid; thiolase; β-oxidation.

Figure 1.

Reactions catalyzed by the hydratase and aldolase in the last round of the β-oxidation of a steroid side chain. The metabolite 3-OPDC-CoA thioester is hydrated to 17-HOPC-CoA thioester that subsequently undergoes C–C bond cleavage catalyzed by an aldolase.

Figure 2.

Structure of Ltp2–ChsH2_DUF35. A, secondary structure organization of Ltp2, with secondary structure labeled. B, secondary structural organization of the ChsH2_DUF35 domain. The zinc ion is depicted as a blue sphere, with the coordinating cysteine residues shown as sticks. C, organization of Ltp2–ChsH2_DUF35 heterotetramer. The Ltp2 chains are colored dark orange and blue, and the DUF35 domains of ChsH2 are colored light orange and light blue. A faint gray silhouette depicts the extent of the protein surface. D, Ltp2–ChsH2_DUF35 complex depiction is 90° from C.

Figure 3.

Modeling the Ltp2 multiprotein and substrate complexes. A, Rosettadock model of the Ltp2–ChsH2_DUF35 complex (colored as in Fig. 2) docked onto the M. tuberculosis ChsH1–ChsH2_MaoC complex (ChsH2 in pale cyan and pale orange, and ChsH1 in gray shades). Docking is constrained by the high probability of the proteins docking with their 2-fold axis aligned, plus the limited length of the linker joining the C terminus of the ChsH2_MaoC and N terminus of the ChsH2_DUF35 (marked with spheres). Both proteins have a distinctly saddle-like shape, drastically restricting the plausible conformations. Note that this complex omits two extended loops (joining the ChsH2 domains and covering the Ltp2-active site). B, electrostatic surface (generated using APBS) for Ltp2 with 17-HOPC-CoA manually docked. C, close-up of the model, showing details of 17-HOPC-CoA binding (yellow sticks) and key proposed binding and catalytic residues (white sticks; note: protein residues are shown in their experimentally observed positions and were not shifted during docking).

Figure 4.

Comparison of the active site of Ltp2 aldolase with an SCP-2 thiolase. A, active-site structure of the SCP-2 thiolase from L. mexicana (PDB code 3zbg). Larger panel shows key active-site residues, and the smaller inset panel shows a surface view of the same region. CoA (yellow) was positioned from 3zbn. Colored circles denote equivalent residues in A, B, and C. B, equivalent active-site region in Ltp2. C, multiple sequence alignment of Ltp2 from T. curvata and its homologs. The first six sequences (yellow bar) are Ltp2 aldolases; the following two (blue bar) are thiolase-like proteins of unknown activity; and the last two sequences are thiolases. Amino acid sequences of proteins are as follows: T. curvata Ltp2 (Ltp2_Tcur); M. tuberculosis Ltp2 (Ltp2_Mtb); R. jostii RHA1 cholesterol Ltp2 (Ltp2C_Rjost); bile acid Ltp2 (Ltp2B_Rjost); Comamonas testosteroni KF1 Ltp2 (Ltp2_Ctest); Pseudomonas sp. Chol1 Ltp2 (Ltp2_Pchol); PhlB from P. protogens (PhlB_Pprotegens); thiolase-type SCP-2-like from T. brucei (SCP2like_Tbrucei); M. thermolithotrophicus acetoacetyl-CoA thiolase-type SCP-2 (Thiolase_Mthermo); and L. mexicana thiolase-type SCP-2 (SCP2_Lm). Sequences are aligned using Clustal Omega and displayed using ESPRIPT (41, 42).

Figure 5.

Proposed retro-aldol cleavage mechanism of Ltp2–ChsH2_DUF35. This prediction was made based on the modeling of the 17-HOPC-CoA into the active site and the results obtained from site-directed mutagenesis studies. Tyr-294 deprotonates the C17 hydroxyl group of 17-HOPC-CoA leading to C–C bond cleavage. The second tyrosine residue, Tyr-344, acts as an acid, donating a proton to the enolate anion of the CoA thioester, forming propionyl-CoA and androst-4-ene-3,17-dione. Either Tyr-294 or Tyr-344 could be activated by the nearby histidine residues.